Kota Mochizuki

Daniel Ken Inaoka
email: danielken@nagasaki-u.ac.jp

Muriel Mazet

Tomoo Shiba
email: tomooshiba@kit.ac.jp

Keisuke Fukuda

Hana Kurasawa

Yoann Millerioux

Michael Boshart

Emmanuel O Balogun

Shigeharu Harada

Kenji Hirayama

Frédéric Bringaud

Kiyoshi Kita

The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei

Keywords: Mitochondria, Energy metabolism, Substrate-level phosphorylation, Acetate:succinate CoA transferase, Fatty acid biosynthesis, Trypanosoma brucei, Succinyl-CoA:3-ketoacid CoA transferase

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Energy metabolism of parasites is quite diverse and essential for their survival in the host [START_REF] Kita | Role of complex II in anaerobic respiration of the parasite mitochondria from Ascaris suum and Plasmodium falciparum[END_REF][START_REF] Harada | Diversity of parasite complex II[END_REF]. Human African trypanosomiasis (HAT) is a parasitic disease transmitted by the tsetse fly. HAT includes two forms: Trypanosoma brucei gambiense, which causes a slow-progressing form of the disease and is endemic in western and central Africa, and T. b. rhodesiense, which causes a rapidly progressing form endemic in eastern Africa [START_REF] Buscher | Human African trypanosomiasis[END_REF].

Although five drugs are available for treating HAT, they are suboptimal in terms of safety and efficacy [START_REF] Baker | The Long Wait for a New Drug for Human African Trypanosomiasis[END_REF]. T. brucei also infects livestock animals such as cattle and sheep (Nagana), causing considerable economic losses in endemic countries [START_REF] Yaro | Combatting African Animal Trypanosomiasis (AAT) in livestock: The potential role of trypanotolerance[END_REF][START_REF] Shaw | Assessing the economics of animal trypanosomosis in Africa--history and current perspectives[END_REF]. Although efforts to control African trypanosomiasis have been ongoing for a century, the disease persists, and the outlook for eradication is poor [START_REF] Yaro | Combatting African Animal Trypanosomiasis (AAT) in livestock: The potential role of trypanotolerance[END_REF][START_REF] Nakayima | Molecular epidemiological studies on animal trypanosomiases in Ghana[END_REF][START_REF] Aksoy | Human African trypanosomiasis control: Achievements and challenges[END_REF].

During its life cycle, T. brucei undergoes developmental changes including metabolic reprogramming dependent on the host environment (Fig. S1). When the bloodstream form (BSF) in mammalian host, which includes long slender (LS) and short stumpy (SS) forms, is ingested by the tsetse fly, the replicative LS form dies and the SS form differentiates into a procyclic form (PCF) in the midgut [START_REF] Lee | A fatty-acid synthesis mechanism specialized for parasitism[END_REF]. The parasite then migrates to the insect salivary glands, where it differentiates into infective metacyclic trypomastigotes, which are injected into mammalian hosts during blood meals [START_REF] Lee | A fatty-acid synthesis mechanism specialized for parasitism[END_REF]. In the tsetse fly, the parasite switches from glucose to amino acid metabolism in order to adapt to its new environment. In the PCF in the insect vector, the end products of energy metabolism are, acetate, succinate, alanine, pyruvate and glycine [START_REF] Millerioux | The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control[END_REF][START_REF] Van Weelden | Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation[END_REF][START_REF] Coustou | Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei[END_REF][START_REF] Smith | Metabolic reprogramming during the Trypanosoma brucei life cycle[END_REF], whereas pyruvate followed by acetate and succinate are the end products in the LS and SS forms in mammalian host [START_REF] Smith | Metabolic reprogramming during the Trypanosoma brucei life cycle[END_REF][START_REF] Van Grinsven | Adaptations in the glucose metabolism of procyclic Trypanosoma brucei isolates from tsetse flies and during differentiation of bloodstream forms[END_REF][START_REF] Mazet | Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for parasite viability[END_REF]. In both of the BSFs, enzymes associated with the mitochondrial electron transport chain (ETC) are downregulated [START_REF] Schnaufer | The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function[END_REF][START_REF] Brown | ATP synthase is responsible for maintaining mitochondrial membrane potential in bloodstream form Trypanosoma brucei[END_REF], whereas cyanide-insensitive trypanosome alternative oxidase (TAO) is upregulated and functions as the sole terminal oxidase. TAO is required to re-oxidize NADH produced in the glycosomes via the glycerol-3-phosphate/dihydroxyacetone phosphate shuttle in order to maintain the glycolytic flux and ATP production [START_REF] Shiba | Structure of the trypanosome cyanide-insensitive alternative oxidase[END_REF]. Due to the downregulation of mitochondrial proton pump enzymes (complexes III and IV), BSFs maintain the electrochemical gradient by the reverse reaction of F o /F 1 -ATP synthase hydrolyzing ATP, which is produced by substrate-level phosphorylation (SLPHOS) [START_REF] Schnaufer | The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function[END_REF][START_REF] Brown | ATP synthase is responsible for maintaining mitochondrial membrane potential in bloodstream form Trypanosoma brucei[END_REF]. A potential candidate for ATP supply is the acetate:succinate CoA-transferase/succinyl-CoA synthetase (ASCT/SCS) cycle due to its ability to produce ATP independent of oxygen and an electrochemical gradient [START_REF] Van Hellemond | Trypanosomatidae produce acetate via a mitochondrial acetate:succinate CoA transferase[END_REF]. In this cycle, ASCT transfers the CoA moiety from acetyl-CoA to succinate, producing acetate and succinyl-CoA, which is used by SCS to produce ATP, CoA, and succinate [START_REF] Van Hellemond | Trypanosomatidae produce acetate via a mitochondrial acetate:succinate CoA transferase[END_REF] (Fig. 1A). The ASCT/SCS cycle was initially investigated in T. brucei PCF, and is thought to have two functions: i) ATP and ii) acetate production [START_REF] Riviere | Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism[END_REF][START_REF] Tielens | Acetate formation in the energy metabolism of parasitic helminths and protists[END_REF]. The role of the ASCT/SCS cycle in ATP production was demonstrated indirectly by the hypersensitivity of TbASCT null mutants (~1000-fold) to oligomycin A (a specific F o /F 1 -ATP synthase inhibitor) [START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF], indicating that ATP production by this cycle is essential for ATP synthesis when oxidative phosphorylation (OXPHOS) is impaired. In addition, SLPHOS observed in the presence of pyruvate and succinate was abolished upon RNAi knockdown of SCS β subunit [START_REF] Bochud-Allemann | Mitochondrial substrate level phosphorylation is essential for growth of procyclic Trypanosoma brucei[END_REF], which further support the role of ATP production by ASCT/SCS cycle in PCF. From previous reports, SCS activity was detected in both BSF and PCF [START_REF] Jenkins | Two distinct succinate thiokinases in both bloodstream and procyclic forms of Trypanosoma brucei[END_REF], and shown to be essential by RNAi knockdown [START_REF] Bochud-Allemann | Mitochondrial substrate level phosphorylation is essential for growth of procyclic Trypanosoma brucei[END_REF][START_REF] Zhang | The Trypanosoma brucei MitoCarta and its regulation and splicing pattern during development[END_REF]. Furthermore, the expression of TbASCT and SCS (α and β subunits) were also confirmed by proteome analyses in both BSF and PCF at levels comparable to other mitochondrial enzymes such as pyruvate dehydrogenase complex [START_REF] Butter | Comparative proteomics of two life cycle stages of stable isotope-labeled Trypanosoma brucei reveals novel components of the parasite's host adaptation machinery[END_REF][START_REF] Dejung | Quantitative Proteomics Uncovers Novel Factors Involved in Developmental Differentiation of Trypanosoma brucei[END_REF].

Regarding acetate production catalyzed by the ASCT/SCS cycle, glucose-and L-threonine-derived acetyl-CoA is converted into acetate by TbASCT. Mitochondrial acetate is also produced by acetyl-CoA hydrolase (TbACH) [START_REF] Millerioux | The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control[END_REF][START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF], which hydrolyzes acetyl-CoA to acetate and CoA (Fig. 1B). It is important to note that, in contrast to TbASCT, TbACH has no CoA transferase activity, as previously demonstrated [START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF]. Most of the acetate formed by TbASCT and TbACH is excreted as an end product, but a portion of it is used to supply de novo fatty acid biosynthesis in PCF trypanosomes [START_REF] Riviere | Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes[END_REF] via a process termed the 'acetate shuttle', in which the acetate produced in mitochondria is transported to the cytosol, where it is re-converted to acetyl-CoA by 'AMP-forming' acetyl-CoA synthase (TbACS) at the expense of ATP [START_REF] Mazet | Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for parasite viability[END_REF][START_REF] Riviere | Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes[END_REF]. To date, the acetate shuttle has been demonstrated only in trypanosomes and replaces the ubiquitous citrate shuttle, which does not operate in trypanosomatids [START_REF] Riviere | Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes[END_REF]. This shuttling is essential for de novo biosynthesis of fatty acids, as RNAi silencing of TbACS inhibits the incorporation of acetate into fatty acids and is lethal for the PCF [START_REF] Riviere | Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes[END_REF] and LS [START_REF] Mazet | Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for parasite viability[END_REF] form. Similarly, acetate production catalyzed by TbASCT and TbACH is essential, at least in the PCF, as shown by the lethal phenotype of the ∆ach/ RNAi ASCT double mutant [START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF].

The ASCT/SCS cycle has been identified in mitochondria and hydrogenosomes of microaerophilic organisms with an incomplete ETC (T. brucei, Fasciola hepatica, and Ascaris sp.) or that lack complex V (Trichomonas vaginalis, Tritrichomonas foetus, and Blastocystis sp.) [START_REF] Muller | Biochemistry and evolution of anaerobic energy metabolism in eukaryotes[END_REF]. In helminthes such as Ascaris suum [START_REF] Kita | Role of complex II in anaerobic respiration of the parasite mitochondria from Ascaris suum and Plasmodium falciparum[END_REF], Haemonchus contortus [START_REF] Omura | An anthelmintic compound, nafuredin, shows selective inhibition of complex I in helminth mitochondria[END_REF],

and Echinococcus multilocularis [START_REF] Matsumoto | Anaerobic NADH-fumarate reductase system is predominant in the respiratory chain of Echinococcus multilocularis, providing a novel target for the chemotherapy of alveolar echinococcosis[END_REF], the adult stage live in hypoxic conditions and complexes III and IV are not functional [START_REF] Inaoka | Structural Insights into the Molecular Design of Flutolanil Derivatives Targeted for Fumarate Respiration of Parasite Mitochondria[END_REF]. In this case, complex I is the only active ETC enzyme with proton pump activity capable of maintaining the electrochemical gradient for OXPHOS, which is inefficient; thus, SLPHOS mediated by the ASCT/SCS cycle becomes significant.

Although ASCT activity has been described in T. brucei, F. hepatica, and T. vaginalis, the identified proteins shared no homology [START_REF] Riviere | Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism[END_REF][START_REF] Tielens | Acetate formation in the energy metabolism of parasitic helminths and protists[END_REF][START_REF] Van Grinsven | Acetate:succinate CoA-transferase in the anaerobic mitochondria of Fasciola hepatica[END_REF][START_REF] Van Grinsven | Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization[END_REF]. On the basis of amino acid sequence, ASCTs have been classified into three CoA-transferase subfamilies (IA, IB, and IC) [START_REF] Tielens | Acetate formation in the energy metabolism of parasitic helminths and protists[END_REF]. Although ASCT activity has not been detected in mammals, TbASCT and human succinyl-CoA:3-ketoacid CoA-transferase (HsSCOT) are both annotated as subfamily IA CoA-transferases [START_REF] Tielens | Acetate formation in the energy metabolism of parasitic helminths and protists[END_REF], exhibiting 52% amino acid sequence identity (Fig. S2). HsSCOT catalyzes the production of acetoacetyl-CoA and succinate from succinyl-CoA and acetoacetate (Fig. 1B), the rate-limiting step in ketone body catabolism [START_REF] Kayer | Disorders of ketone production and utilization[END_REF]. Circulating acetoacetate produced by the liver is taken by extrahepatic cells, where acetoacetyl-CoA is produced and metabolized to acetyl-CoA to feed the tricarboxylic acid cycle for ATP synthesis [START_REF] Fukao | Pathways and control of ketone body metabolism: on the fringe of lipid biochemistry[END_REF]. Although HsSCOT and TbASCT are believed to have similar functions [START_REF] Tielens | Acetate formation in the energy metabolism of parasitic helminths and protists[END_REF], neither biochemical evidence nor structural studies supporting this hypothesis have been reported so far. It has been previously demonstrated that a heterodimeric CoA transferase composed by ScoA and ScoB (WP_007410135.1 and WP_160182702.1, respectively) from Bacillus subtilis, which showed considerable high SCOT activity, exhibits 55% amino acid identity to corresponding N-terminal and C-terminal regions of homodimeric HsSCOT (Fig. S2), respectively [START_REF] Stols | New vectors for co-expression of proteins: structure of Bacillus subtilis ScoAB obtained by high-throughput protocols[END_REF]. Since TbASCT share 52% amino acid identity to HsSCOT and currently annotated as SCOT, it is possible that TbASCT may also have SCOT activity and vise versa. Because ASCT and SCOT belong to different metabolic pathways, it is important to characterize and compare both enzymes.

Despite the essential roles of mitochondrial acetate production by TbASCT and TbACH in the PCF, their roles in BSFs have not been investigated. In addition, biochemical and structural biology studies of ASCT family enzymes have been hampered due to the difficulty to purify ASCT [START_REF] Riviere | Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism[END_REF][START_REF] Van Grinsven | Acetate:succinate CoA-transferase in the anaerobic mitochondria of Fasciola hepatica[END_REF][START_REF] Van Grinsven | Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization[END_REF] in its active form. In this study, we report the first detailed biochemical features of purified TbASCT by using the new assay method, which is totally dependent on TbASCT substrates and enables to monitor the initial velocity of the reaction.

Characterization of the in vitro reconstituted ASCT/SCS cycle and site-directed mutagenesis analyses provide insights into the catalytic mechanism of TbASCT and identification of the active site glutamate residue.

Material and methods

Knock-out of the ACH gene

Replacement of the acetyl-CoA hydrolase gene (ACH: Tb927.3.4260) with the phleomycin (BLE) and puromycin (PAC) resistance markers via homologous recombination was performed using DNA fragments containing the resistance marker gene flanked by the ACH UTR sequences, as described previously [START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF]. Briefly, the pGEMt plasmid was used to clone an HpaI DNA fragment containing the BLE and PAC resistance marker genes preceded by the ACH 5'-UTR fragment (537 or 492 bp) and followed by the ACH 3'-UTR fragment (521 or 485 bp) [START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF]. The ACH knock-out was generated in the 427 MITat1.2 13-90 (Hyg-Neo) monomorphic BSF cell line, which constitutively expresses the T7 RNA polymerase gene and the Tet repressor under control of a T7 RNA polymerase promoter for Tet-inducible expression [START_REF] Wirtz | A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei[END_REF]. Transfections were performed as previously reported, using the Nucleofector ® system [START_REF] Burkard | Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei[END_REF], and selection of drug-resistant clones, designated Δach (TetR-HYG T7RNAPOL-NEO Δach::BLE/Δach::PURO), was performed in HMI-9 medium containing hygromycin (5 µg/ml), neomycin (2.5 µg/ml), phleomycin (2.5 µg/ml), and puromycin (0.1 µg/ml). After transfection, the cells were re-suspended in 50 ml of conditioned medium and aliquoted into two 25-well titer plates; antibiotics were added the following day.

Inhibition of gene expression by RNAi

Inhibition of expression of the ASCT gene (Tb927.11.2690) by RNAi in the 427 MITat1.2 13-90 BSF strain was performed by expression of stem-loop "sense/anti-sense" RNA molecules of the targeted sequences introduced in the pHD1336 expression vector.

Construction of the pLew-ASCT-SAS vector was as described previously [START_REF] Riviere | Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism[END_REF]. The ASCT-SAS HindIII-BamHI cassette extracted from the pLew-ASCT-SAS plasmid was inserted into the HindIII-BamHI-digested pHD1336 vector. The resulting pHD-ASCT-SAS plasmid contained a sense and antisense version of the targeted gene fragment (572 bp fragment from nt positions 85 to 657), separated by a 64-bp fragment, under control of the PARP promoter linked to a prokaryotic Tet operator. The RNAi ASCT and Δach/ RNAi ASCT cell lines were produced by transfecting (with the NotI-linearized pHD-ASCT-SAS plasmid) the 427 and Δach cell lines, respectively. Transfected cells were incubated in HMI-9 medium containing hygromycin (5 µg/ml), neomycin (2.5 µg/ml), and blasticidin (10 µg/ml) for selection of RNAi ASCT cells or the same medium plus phleomycin (2.5 µg/ml) and puromycin (0.1 µg/ml) for selection of Δach/ RNAi ASCT cells. Induction of double-stranded RNA expression in the latter mutant was performed by addition of 10 µg/ml tetracycline.

Western blot analysis

Total protein extracts of wild-type or mutant T. brucei BSFs (5×10 6 cells) were size-fractionated by SDS-PAGE (10%) and immunoblotted onto Immobilon-P filters (Millipore) [START_REF] Harlow | Antibodies: A Laboratory Manual[END_REF]. Immunodetection was performed as described elsewhere [START_REF] Harlow | Antibodies: A Laboratory Manual[END_REF][START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF] using rabbit anti-ASCT (diluted 1:1000) [START_REF] Riviere | Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism[END_REF], purified rabbit anti-ACH [START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF], and rabbit anti-GPDH (glycerol-3-phosphate dehydrogenase, EC 1.1.1.8; diluted 1:100) [START_REF] Denise | Affinity chromatography using trypanocidal arsenical drugs identifies a specific interaction between glycerol-3-phosphate dehydrogenase from Trypanosoma brucei and Cymelarsan[END_REF] as primary antibodies and anti-rabbit IgG conjugated to horseradish peroxidase (BioRad, 1:5,000 dilution) as the secondary antibody. Blots were developed using SuperSignal ® West Pico Chemiluminescent Substrate as described by the manufacturer (Thermo Scientific). Images were acquired and analyzed using a KODAK Image Station 4000 MM.

Bacterial strains, plasmids, and reagents for recombinant TbASCT and HsSCOT production

The TbASCT gene lacking the mitochondrial targeting signal (MTS; Δ1-9 residues) was amplified by PCR from genomic DNA (ILtat 1.4 strain [START_REF] Miller | Analysis of antigenic types appearing in first relapse populations of clones of Trypanosoma brucei[END_REF]) and inserted into the pETSUMO vector (Thermo Fisher Scientific) by TA cloning [START_REF] Zhou | Universal TA cloning[END_REF] according to the manufacturer's protocol. The generated plasmid (pETSUMO/TbASCT) was used to transform chemically competent One Shot TM TOP10 (Thermo Fisher Scientific) Escherichia coli. In comparison to the TREU 927 strain, ASCT cloned from ILtat 1.4 had a single substitution of serine to asparagine at position 419, which is located at the protein surface. Next, BL21 star TM (DE3) (Thermo Fisher Scientific) was transformed with pETSUMO/TbASCT. The final expression host, BL21 star TM (DE3)pETSUMO/TbASCT, was stored in 20% (v/v) glycerol at -80°C until use.

The plasmid used for HsSCOT expression was prepared similarly to that for TbASCT expression, with slight modifications. The HsSCOT (GI number: 4557817) gene lacking the MTS (Δ1-39 residues) and fused with a His 10 -SUMO tag at the N-terminus was codon optimized, amplified by PCR, and inserted into the pET101/D-TOPO vector (Thermo Fisher Scientific).

MagExtractor TM -PCR & Gel Clean-up for gel extraction and PCR product purification kits, and MagExtractor TM -plasmid for plasmid purification were purchased from TOYOBO (Japan). Ni-NTA affinity agarose was obtained from QIAGEN (Germany). SCS from prokaryote origin was obtained from Megazyme (Ireland). All other reagents were of the highest grade available and purchased from Sigma (USA), WAKO (Japan), or TCI (Japan).

Optimized large-scale purification of TbASCT and HsSCOT

A glycerol stock of E. coli BL21 star TM (DE3)pETSUMO/TbASCT was streaked onto an LB agar plate containing 50 µg/ml kanamycin and incubated overnight at 37°C. The next day, a single colony was inoculated into 360 ml of Terrific Broth (TB) medium and cultured for with collection of 1-ml fractions. Protein content in the fractions was measured at 280 nm using a Nanodrop 2000 (Thermo Fischer Scientific). Fractions exhibiting an absorbance >2.0 were pooled and concentrated to more than 10 mg/ml using a centrifugal filter device (Amicon Ultra-15, 50-kDa cutoff; Millipore). The concentrated fraction was diluted 10-fold with 50 mM Tris-HCl pH 8.0, 20% (v/v) glycerol, 150 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA (protease buffer) to decrease the imidazole and NaCl concentrations. SUMO protease (Invitrogen) was added at a 1:20 protein ratio and incubated at 4°C overnight.

Pre-equilibrated Ni-NTA agarose (2 ml) was added to the mixture and mixed for 1 hour. SUMO protease and the His 6 -SUMO tag were removed by loading the mixture onto an Econo-Pac ® chromatography column. Pure TbASCT, collected as the flow-through, was concentrated to more than 10 mg/ml using a centrifugal filter device (Amicon Ultra-15, 50-kDa cutoff; Millipore), glycerol was added to a final concentration of 50% (v/v) and kept at -30°C until use. The purification of TbASCT is summarized in Table S1.

HsSCOT was purified by a similar protocol as that used for TbASCT purification, with minor modifications. Specifically, after induction, the cells were cultured for 16.5 hours at 37°C, the column was not washed with wash buffer containing ATP, and 50 ml of wash buffer containing 50 mM imidazole was used for the initial wash.

Protein quantification, denaturation, and BN-PAGE

Protein concentration was determined using a Bio-Rad Protein Assay kit with bovine serum albumin as the standard.

The cytosolic, flow-through, elution, and tag-free fractions from each purification step were subjected to discontinuous SDS-PAGE according to Laemmli [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. The stacking and separating gels were 4% and 12% (w/v) acrylamide, respectively. Samples were mixed 1:3 BN-PAGE was performed as follows. Dilutions of purified TbASCT were prepared in 50 mM Tris-HCl pH 8.0, 8% (v/v) glycerol, 0.01% (w/v) n-Dodecyl-β-D-maltoside, 0.002% (w/v) G-250, and 25 mM NaCl and loaded onto a 4-16% (w/v) Bis-Tris gradient gel (Invitrogen) at final concentration of 1 µg to 1 ng for CBB staining and 0.1 µg to 0.01 ng for in-gel activity staining per lane, respectively. Anode and cathode buffers were prepared according to the manufacturer's protocol using 20 × running buffer (Invitrogen), with the cathode buffer supplemented with 0.002% (w/v) G-250. The gel was run at 4°C under constant voltage of 150 V for 1 hour and 250 V for 1 hour.

(v/v)

In-gel ASCT activity stain

Free thiol groups from CoA can reduce nitroblue tetrazolium salt (NBT) in the presence of phenazine methosulfate (PMS) [START_REF] Rothe | Electrophoresis of Enzymes: Laboratory Methods[END_REF][START_REF] Manchenko | Handbook of Detection of Enzymes on Electrophoretic Gels[END_REF]. To assay TbASCT activity (in-gel ASCT-activity staining), the BN-PAGE gel was washed twice for 5 min each at room temperature with 50 mM Tris-HCl pH 7.2, 5 mM MgCl 2 , and 25 mM sodium phosphate. After washing, the BN-PAGE gel was incubated for 20 minutes in same buffer supplemented with 0.5 mM ADP, 0.5 mM acetyl-CoA, 2.5 mg/ml NBT, and 0.44 U/ml of SCS; thereafter, 50 mM succinate and 0.12 mg/ml PMS were added and incubated static in the dark at room temperature.

In vitro reconstitution of the TbASCT/SCS cycle

The in vitro reconstituted ASCT/SCS cycle was assayed by quantification of released CoA (Fig. 1A) using DTNB (Sigma). In this system, DTNB reacts with one molecule of CoA, forming TNB, which can be followed at 412 nm (ε 412 = 14.0 mM -1 cm -1 ) [START_REF] Watanabe | Method for the Accurate Measurement of Freezing-induced Denaturation of Ovalbumin with 5,5'-Dithiobis-(2-nitrobenzoic acid)[END_REF]. The rate of CoA production was monitored using a V-660 spectrophotometer (JASCO) equipped with a water bath circulator (Taitec).

The dose-response range of TbASCT was determined at 30°C. ASCT activity at different concentrations of purified TbASCT, ranging from 12.5 to 200 ng/ml, was assayed in 1 ml reaction mixture (100 mM Tris-HCl pH 7.0, 10 mM sodium phosphate, 2 mM MgCl 2 , 1 mM ADP, 0.1 mM DTNB, 0.5 mM acetyl-CoA, 0.44 U/ml SCS) using a black quartz cuvette.

After addition of enzyme, the background was recorded for 5 min, and initial velocity was monitored after addition of succinate to a final concentration of 20 mM.

The optimal temperature for the TbASCT assay was also determined. ASCT activity was assayed in 100 mM Tris-HCl pH 6.8, 1 mM sodium phosphate, 2 mM MgCl 2 , 0.1% (v/v) Triton X-100, 1 mM ADP, 0.1 mM DTNB, 0.44 U/ml SCS, 0.5 mM acetyl-CoA, and 10 ng of purified TbASCT at 20, 25, 30, 35, 37, 40, and 45°C in triplicate. The reaction was initiated by the addition of 10 mM succinate from a 1 M stock solution with pH adjusted to 7.0.

The optimal pH for TbASCT activity was determined using a 96-well plate (C96 Maxisorp Nunc-Immuno Plate, Thermo Scientific) and a SpectraMax ® Paradigm ® Multi-Mode Detection Platform (Molecular Devices) at 30°C in triplicate. Background was recorded for 5 min at 412 nm in 195 µl of reaction mix per well (50 mM of each buffer with different pH, 10 mM sodium phosphate, 2 mM MgCl 2 , 1 mM ADP, 0.1 mM DTNB, 0.5 mM acetyl-CoA, 0.44 U/ml SCS, TbASCT 100 ng/ml), and the reaction was initiated by adding 5 µl of 400 mM succinate. ASCT activity was calculated using the end-point method after 10 min, and the highest activity (Tris-HCl pH 7.0) was set as 100%.

Routine TbASCT assays were conducted in 1.0 ml of reaction mixture (100 mM Tris-HCl pH 7.0, 10 mM sodium phosphate, 2 mM MgCl 2 , 1 mM ADP, 0.1 mM DTNB, 0.5 mM acetyl-CoA, 0.44 U/ml SCS) using a black quartz cuvette at 30°C. After addition of enzyme, the background was recorded for 5 min, and the reaction was initiated by addition of succinate to a final concentration of 20 mM. K m and V max values for succinate were determined by measuring the TbASCT activity at varying succinate concentrations ranging from 0.5 to 50 mM at a fixed acetyl-CoA concentration of 0.5 mM. Similarly, the kinetic parameters for acetyl-CoA were determined at concentrations ranging from 0.01 to 1 mM in the presence of 20 mM succinate.

The reaction mechanism was analyzed by varying the concentration of succinate (1, 2, 5, 10, or 20 mM) under a fixed acetyl-CoA concentration of 0.02, 0.05, or 0.1 mM and by varying the acetyl-CoA concentration (0.025, 0.05, 0.1, 0.25, or 0.5 mM) under a fixed succinate concentration of 5, 10, or 20 mM, with all samples in triplicate. All enzyme kinetic parameters were obtained by fitting the data via nonlinear regression analysis using GraphPad Prism 7 software.

CoA-donor specificity of TbASCT

The substrate specificity of TbASCT was determined in 96-well plates in a reaction mix containing 200 ng/ml TbASCT. CoA donors used in this study included acetyl-CoA, n-propionyl-CoA, butyril-CoA, 2-butenoyl-CoA, acetoacetyl-CoA, β-hydroxybutyryl-CoA, malonyl-CoA, hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, and stearoyl-CoA. Two microliters of each CoA donor (10 mM) was transferred to the plate and mixed with 193 µl of reaction mix. After recording the background for 5 min, the reaction was initiated by adding 5 µl of 800 mM succinate. Each condition was assayed in quadruplicate; the ASCT activity in the presence of acetyl-CoA was set to 100%, and relative activity of TbASCT under each condition was then calculated.

Biochemical characterization of HsSCOT

HsSCOT was characterized similarly to TbASCT, as described above. Briefly, the dose-response range of HsSCOT was assayed in 1 ml of reaction mixture containing 100 mM Tris-HCl pH 8.6, 15 mM MgCl 2 , and 1 mM succinyl-CoA in the presence of 58-1860 ng of purified HsSCOT at 37°C. To start the reaction, 7.5 mM acetoacetate was added, and the increase in absorbance at 310 nm, which indicates the formation of acetoacetyl-CoA (ε 310 = 7.8 mM -1 cm -1 ) [START_REF] Howard | Mechanism-based fragmentation of coenzyme A transferase. Comparison of alpha 2-macroglobulin and coenzyme A transferase thiol ester reactions[END_REF], was monitored. Next, HsSCOT activity was assayed under different pH and temperature conditions to optimize the assay. The kinetic parameters (K m and V max ) for acetoacetate and succinyl-CoA were determined under optimal conditions (232 ng purified HsSCOT, 100 mM Tris-HCl pH 8.6, 15 mM MgCl 2 and 1 mM succinyl-CoA at 37°C).

Site-directed mutagenesis

TbASCT mutants were obtained by quick change (QIAGEN) according to the supplier's protocol using pETSUMO/TbASCT as the template. After the desired plasmid was obtained, it was used to transform BL21 star TM (DE3) cells. Each mutant was expressed and purified as described for wild-type TbASCT.

Crystallization of TbASCT

Crystallization conditions were screened using the sitting-drop vapor diffusion method with reservoir solutions supplied in commercially available screening kits (Crystal Screen, Before being flash-cooled in liquid nitrogen, TbASCT crystals were soaked in reservoir solution supplemented with 20% (v/v) glycerol as a cryoprotectant.

Determination of the TbASCT structure

X-ray diffraction data were collected using the synchrotron radiation at Spring-8

(beamline BL44XU, Japan). X-ray diffraction data set at 2.01-Å resolution were obtained after processing and scaling using XDS and XSCALE [START_REF] Kabsch | Integration, scaling, space-group assignment and post-refinement[END_REF], respectively. The structure of TbASCT was solved according to the molecular replacement method with the MOLREP [START_REF] Vagin | MOLREP: an Automated Program for Molecular Replacement[END_REF] program using the structure of HsSCOT (PDB code 3DLX; 52% amino-acid sequence identity with TbASCT) as the initial model and refined using the REFMAC5 [START_REF] Murshudov | Refinement of macromolecular structures by the maximum-likelihood method[END_REF] and COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF] programs. Data collection and refinement statistics are summarized in Supplementary Table 4. All figures of protein structures were prepared using the graphics program PyMol (http://www.pymol.org/). The atomic coordinates and structural factors were deposited in the Protein Data Bank under accession code 6LP1.

Results

Growth phenotype of Δach, RNAi ASCT and Δach/ RNAi ASCT in BSF

In this study, we investigated the roles of TbACH and TbASCT in the monomorphic 427 BSF strain, in which differentiation into the SS form is defective, leading to an almost exclusive composition of LS cells in vitro. Western blot analyses of the BSF show that both TbACH and TbASCT were expressed in the T. brucei wild type 427 strain, although at a much lower level for TbASCT compared to the PCF (Fig. 2A and 2B, bottom panels). This was consistent with the 6-fold higher level of TbASCT expression in PCF trypanosomes compared to LS BSFs quantified in a previous proteome study [START_REF] Gunasekera | Proteome remodelling during development from blood to insect-form Trypanosoma brucei quantified by SILAC and mass spectrometry[END_REF]. Similar result was shown for TbACH in other studies [START_REF] Butter | Comparative proteomics of two life cycle stages of stable isotope-labeled Trypanosoma brucei reveals novel components of the parasite's host adaptation machinery[END_REF][START_REF] Dejung | Quantitative Proteomics Uncovers Novel Factors Involved in Developmental Differentiation of Trypanosoma brucei[END_REF].

To study the role of TbACH and TbASCT in the BSF, we have prepared mutant cell lines of either ACH knock-out, ASCT knockdown, and its combination. We generated the TbACH knock-out mutant (Δach::BLE/Δach::PAC, abbreviated as Δach) by replacing both ACH alleles with phleomycin (BLE) and puromycin (PAC) resistance markers in the 427 BSF background. Deletion of both ACH alleles in the selected Δach cell line was confirmed by PCR (Fig. 2C) and Western blotting (Fig. 2A) using immunopurified anti-ACH antibodies raised against recombinant histidine-tagged TbACH [START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF]. TbASCT expression was down-regulated by RNAi by introducing the pHD-ASCT-SAS plasmid in the same parental BSF cell line to generate the RNAi ASCT mutant. Neither the Δach nor RNAi ASCT mutants exhibited significant change in growth phenotype (Fig. 2A and 2B, top panels).

To generate the Δach/ RNAi ASCT double mutant, the pHD-ASCT-SAS construct was introduced in the Δach cell line. After several transfection attempts, a single cell line was obtained, although all of the transfections of the parental 427 cells with the same plasmid preparation (pHD-ASCT-SAS) each produced approximately a dozen cell lines. Three days after induction by addition of tetracycline, the Δach/ RNAi ASCT.i double mutant exhibited a strong reduction in growth rate, correlated with reduced TbASCT expression indicating that mitochondrial acetate production is essential in BSF as well as PCF [START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF]. Growth of the parental cell line was not affected by addition of 10 µg/ml tetracycline. Cells were maintained in the exponential growth phase (between 10 5 and 2×10 6 cells/ml), and cumulative cell numbers reflect normalization for dilution during cultivation. Induction (.i) refers to addition of 10 µg/ml tetracycline.

Expression and purification of recombinant TbASCT and HsSCOT

Recombinant His 6 -SUMO TbASCT was successfully expressed in E. coli BL21 star TM (DE3) cells and purified to homogeneity. Under optimal conditions, bacterial growth reached an OD 600 of 10, from which 48 g of wet cell pellet per 3.6-L culture of TB medium was obtained. Finally, after adding SUMO protease, 26 mg of tag-free TbASCT was purified with a yield of 18% and specific ASCT activity of 119 µmol/min/mg protein, corresponding to a 20-fold increase compared to the cytosolic fraction (Table S1). Under similar conditions, we purified 0.485 mg of tag-free HsSCOT with a 6% yield and specific SCOT activity of 16.6 µmol/min/mg protein, representing a 325-fold increase compared to the cytosolic fraction (Table S2). According to our protocol, both enzymes were purified to homogeneity as judged by SDS-PAGE (Fig. S3).

The ASCT/SCS cycle coupled assay system

As previously reported assay method of ASCT is not suitable for the detailed kinetic analysis of the enzyme, we established a new system. In the ASCT/SCS cycle, one molecule of acetyl-CoA is consumed by TbASCT, and equimolar amounts of ATP and CoA are produced by SCS (Fig. 1A) [START_REF] Hunger-Glaser | Histidine-phosphorylation of succinyl CoA synthetase from Trypanosoma brucei[END_REF]. We developed a new coupled enzyme assay for ASCT based on this cycle and the reaction of 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) with the thiol group of CoA to produce one molecule of 2-nitro-5-thiobenzoic acid (TNB), which can be followed colorimetrically.

To validate the in vitro reconstituted ASCT/SCS cycle assay, the dependence on each assay component was investigated (Fig. S4A). When TbASCT, acetyl-CoA, or succinate was removed, no activity was detected. However, when either SCS, ADP, phosphate or MgCl 2 was removed, a minimal amount of activity remained, ranging from 3.76 to 14.7 µmol/min/mg protein, which was consistent with the non-enzymatic degradation of succinyl-CoA produced by TbASCT in the assay system (equivalent to 15.5 µmol/min/mg protein with 0.1 mM succinyl-CoA).

Biochemical characterization of TbASCT

Purified recombinant TbASCT was biochemically characterized using the in vitro reconstituted ASCT/SCS cycle assay. TbASCT activity exhibited a linear dose response over the range of 6.25 to 200 ng/ml in the assay mixture, with an R 2 value of 0.981 (Fig. S4B). As such, subsequent assays were conducted using 100 ng/ml TbASCT. The optimum temperature and pH for the reaction were determined to be 30°C and 7.0, respectively (Fig. S4C,D). The K m values for acetyl-CoA and succinate were 0.046 mM and 13.8 mM, respectively (Fig. 3A,B and Table 1). At a high concentration of succinate, substrate inhibition was clearly observed, with a K i of 75.7 mM (Fig. 3B). Considering substrate inhibition, the V max of succinate was calculated as 221 µmol/min/mg (k cat = 191 s -1 ) according to the following equation:

𝑉 = ! !"# × ! ! ! ! ! × ! ! ! ! ! .
Next, the TbASCT reaction mechanism was examined. The reaction of TbASCT requires two substrates (acetyl-CoA and succinate). When the enzyme activity was determined by varying the concentration of one substrate under a fixed concentration of the second substrate and vice versa, double reciprocal plots of both cases exhibited parallel lines (Fig. 4A,B), indicating that TbASCT follows a ping-pong bi-bi catalytic mechanism (Fig. 4C) [START_REF] Segel | Enzyme kinetics[END_REF]. Table 1. Biochemical parameters of WT and E319A mutant TbASCT.

Acetyl-CoA Succinate K m (mM) k cat (s -1 ) K m (mM) k cat (s -

Blue Native (BN)-PAGE and in-gel ASCT-activity staining

To investigate the oligomeric state of TbASCT in solution, BN-PAGE and in-gel ASCT activity staining were performed (Fig. 4D). Two bands corresponding to 225 and 414 kDa were detected by CBB staining (100 ng TbASCT/lane), while a third band at 564 kDa could be observed by ASCT activity staining (10 ng TbASCT/lane), which are consistent with calculated molecular weight of one, two and three tetramers of TbASCT, respectively (Fig. 4D, red arrows). At lower concentration of TbASCT, the 225 kDa band become predominant indicating that in solution TbASCT is a tetramer. At high concentration, smearing above 225 kDa band was observed for both staining methods, highlighting the sticky nature of TbASCT.

Surprisingly, in-gel ASCT activity staining showed 1,000 times more sensitivity compared with CBB staining (Fig. 4D) and can be used to detect TbASCT as low as 0.1 ng/lane.

Specificity of CoA donors to the TbASCT reaction

Although acetyl-CoA is known to be a substrate for TbASCT, the specificity of CoA donors has not been demonstrated. Hence, we examined the ability of TbASCT to utilize different CoA donors (Fig. 5A) and found that in the presence of n-propionyl-CoA, acetoacetyl-CoA, or malonyl-CoA, the activity remaining relative to acetyl-CoA was 60%, 6%, and 13%, respectively. Other CoA donors tested in this study exhibited negligible

CoA-transferase activity (Fig. 5A). As TbASCT exhibited significantly high CoA-transferase activity with n-propionyl-CoA, we determined the kinetic parameters and found K m , V max , k cat values of 0.018 mM, 49.7 µmol/min/mg, and 42.9 s -1 , respectively. At n-propionyl-CoA concentrations above 0.1 mM, CoA-transferase activity was inhibited, with a K i value of 0.633 mM (Fig. 5B). and K i values for propionyl-CoA were 0.018 ± 0.002 mM, 49.7 ± 2.75 µmol/min/mg, 42.9 ± 2.38 s -1 , and 0.633 ± 0.175 mM, respectively. Kinetic parameters described above represent the average ± SE (n = 3) calculated using GraphPad Prism 7.

Biochemical characterization of HsSCOT, ASCT/SCOT reaction specificity and site-directed mutagenesis of TbASCT

Because TbASCT shares 52% amino acid sequence identity with HsSCOT, and TbASCT had SCOT activity (Table S5), we also examined whether HsSCOT exhibits ASCT activity.

Recombinant HsSCOT was purified, and after removal of the His 10 -SUMO tag, a single 52-kDa band corresponding to HsSCOT was detected by SDS-PAGE (Fig. S3B). HsSCOT activity exhibited a linear dose response over the concentration range of 58 to 1,860 ng/ml in the assay mixture, with an R 2 value of 0.998 (Fig. S5A); therefore, subsequent assays were conducted using 232 ng/ml. The optimum temperature and pH for this reaction were determined to be 37°C and 8.6, respectively (Fig. S5B,C). Under optimal conditions, purified recombinant HsSCOT followed classical Michaelis-Menten kinetics, with K m , V max , and k cat values for acetoacetate and succinyl-CoA of 0.765 mM, 121 µmol/min/mg, and 105 s -1 and 0.971 mM, 219 µmol/min/mg, and 190 s -1 , respectively (Table S3). Acetoacetate exhibited substrate inhibition at high concentrations, with a K i value of 285 mM (Fig. S5D).

The ASCT and SCOT activities of TbASCT and HsSCOT were also analyzed and compared, with the activity assayed under optimal conditions. The SCOT activity of WT TbASCT was 0.038 µmol/min/mg, whereas HsSCOT did not exhibit detectable ASCT activity. Thus, TbASCT exhibited 3,000-fold greater ASCT than SCOT activities.

Crystal structure of TbASCT

To compare detailed structural difference between ASCT and SCOT, the crystal structure of TbASCT was investigated. The ligand-free TbASCT crystal (Fig. S6D andS7A) was solved by molecular replacement using the structure of HsSCOT (Fig. S6C between the two structures for 456 Cα atoms (Fig. S8D). Succinyl-CoA is a product and a substrate of ASCT and SCOT, respectively, which is likely to bind to common substrate/product tunnel and reacts with the active site glutamate residue. In the crystal structure of SsSCOT complexed with CoA [START_REF] Fraser | Catalytic role of the conformational change in succinyl-CoA:3-oxoacid CoA transferase on binding CoA[END_REF] (Fig. S6B), CoA was found forming thioester bond with the active site glutamate residue (E344) in the SENG motif conserved in family IA ASCTs and SCOTs. In the crystal structure of TbASCT, E319 corresponding to active site glutamate residue in the SENG motif could be clearly identified (Fig. S6D) as predicted previously [START_REF] Tielens | Acetate formation in the energy metabolism of parasitic helminths and protists[END_REF]. Consistently, ASCT and SCOT activities of TbASCT were abolished in the E319A mutant (Table S5), even with 10-fold more protein than used in the WT assay (Table 1).

Discussion

The results of this study demonstrate an essential role of mitochondrial acetate production in the T. brucei BSF energy metabolism and provide the first biochemical characterization of an ASCT family enzyme. The gene encoding T. brucei ASCT was identified in 2004 [START_REF] Riviere | Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism[END_REF] followed by ACH [START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF], it has been shown to play an essential role in mitochondrial acetate production. The acetate production catalyzed by ASCT is coupled to the generation of ATP via the ASCT/SCS cycle [START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF]. Although the RNAi ASCT.i mutant BSF lacks a growth phenotype (Fig. 2), it has been previously demonstrated in PCF that the knockdown of F 1 β subunit of F o /F 1 -ATP synthase is lethal in Δasct background, indicating that TbASCT and ATP synthase are both responsible for ATP production in mitochondria.

Since in BSF, the ATP synthase functions to maintain the electrochemical gradient by pumping protons using ATP (reverse reaction) [START_REF] Schnaufer | The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function[END_REF][START_REF] Brown | ATP synthase is responsible for maintaining mitochondrial membrane potential in bloodstream form Trypanosoma brucei[END_REF], the TbASCT/SCS cycle can be the main or the only source of mitochondrial ATP.

Nevertheless, the growth inhibition observed in the Δach/ RNAi ASCT.i double mutant generated in this study demonstrated that mitochondrial acetate production is indeed essential not only in PCF [START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF], but also in BSF. It is noteworthy that the Δach/ RNAi ASCT cell line obtained in this study exhibited an important leakage (i.e., reduced TbASCT expression even in the absence of tetracycline) (Day 0 post-induction in Fig. 2D). TbASCT was rapidly re-expressed (Day 7 in Fig. 2D) with restoration of normal growth at 7 days post-induction.

Re-expression of RNAi-targeted genes upon induction is commonly observed in the BSF as well as PCF when the targeted gene is essential for their growth [START_REF] Tyc | Mitochondrial heat shock protein machinery hsp70/hsp40 is indispensable for proper mitochondrial DNA maintenance and replication[END_REF][START_REF] Klein | The role of the zinc finger protein ZC3H32 in bloodstream-form Trypanosoma brucei[END_REF]. The quite early reversion of growth, correlated with TbASCT re-expression supports an essential role for ASCT-ACH genes in the BSF. Thus, collectively, these data demonstrate that although TbASCT and TbACH catalyze distinct reactions, they share a common role in terms of acetate production to feed the de novo fatty acid biosynthesis [START_REF] Mazet | Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for parasite viability[END_REF][START_REF] Riviere | Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes[END_REF], act redundantly, and are indispensable for growth of BSF trypanosomes as previously observed for the PCF [START_REF] Millerioux | ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma[END_REF].

Biochemical and structural biology studies of ASCT have thus far been hampered due to a difficulty to express and purify the recombinant enzyme because of its low expression level and solubility and high degree of instability [START_REF] Riviere | Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism[END_REF][START_REF] Van Grinsven | Acetate:succinate CoA-transferase in the anaerobic mitochondria of Fasciola hepatica[END_REF][START_REF] Van Grinsven | Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization[END_REF]. Expression of recombinant TbASCT fused with a His 6 -SUMO tag at the N-terminus in this study is therefore critical for increasing the expression level as well as solubility of the enzyme [START_REF] Malakhov | SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins[END_REF][START_REF] Zuo | Expression and purification of SARS coronavirus proteins using SUMO-fusions[END_REF][START_REF] Marblestone | Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO[END_REF][START_REF] Wang | Expression, purification, and crystallization of type 1 isocitrate dehydrogenase from Trypanosoma brucei brucei[END_REF], and the addition of glycerol [START_REF] Shiba | Structure of the trypanosome cyanide-insensitive alternative oxidase[END_REF][START_REF] Wang | Expression, purification, and crystallization of type 1 isocitrate dehydrogenase from Trypanosoma brucei brucei[END_REF] in all purification steps is necessary to stabilize the enzymatic activity.

Previously, a method to assay ASCT using [1-14 C]acetyl-CoA was reported [START_REF] Van Hellemond | Trypanosomatidae produce acetate via a mitochondrial acetate:succinate CoA transferase[END_REF][START_REF] Van Grinsven | Acetate:succinate CoA-transferase in the anaerobic mitochondria of Fasciola hepatica[END_REF][START_REF] Van Grinsven | Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization[END_REF].

However, such radioisotope (RI) methods require specialized facilities and the purification of released [1-14 C]acetate using anion-exchange chromatography, which is not suitable for steady-state kinetic studies and high-throughput screening of inhibitors. Hence, a new assay method was developed based on real-time quantification of free CoA released by the in vitro reconstituted ASCT/SCS cycle. This assay is totally dependent on TbASCT substrates, which enables monitoring of the initial velocity, thus allowing us to perform the first detailed biochemical characterization of TbASCT. The affinity (K m ) values determined for acetyl-CoA (0.046 mM) and succinate (13.8 mM) were similar to reported values determined using a RI method with partially purified ASCT from L. mexicana promastigotes (0.26 mM for acetyl-CoA and 6.9 mM for succinate) [START_REF] Van Hellemond | Trypanosomatidae produce acetate via a mitochondrial acetate:succinate CoA transferase[END_REF]. This result suggests that the trypanosomatid ASCT/SCS cycle functions under high concentrations of succinate, which is a major end-product of metabolism in the T. brucei PCF [START_REF] Millerioux | The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control[END_REF][START_REF] Van Weelden | Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation[END_REF][START_REF] Coustou | Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei[END_REF][START_REF] Smith | Metabolic reprogramming during the Trypanosoma brucei life cycle[END_REF] and BSF [START_REF] Van Grinsven | Adaptations in the glucose metabolism of procyclic Trypanosoma brucei isolates from tsetse flies and during differentiation of bloodstream forms[END_REF]. In T. brucei PCF, succinate is produced by glycosomal and mitochondrial NADH-fumarate reductases (NADH-FRDg and NADH-FRDm1, respectively), from which mitochondrial NADH-FRDm1 is responsible for up to 44% of total succinate excreted [START_REF] Coustou | A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei[END_REF]. Although succinate production from glucose metabolism is reduced by 92% in the PCF RNAi FRDg/m1

double mutant [START_REF] Coustou | A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei[END_REF], the cytosolic dihydroorotate dehydrogenase, which used fumarate as the electron acceptor, may also significantly contributes to succinate production [START_REF] Takashima | Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi[END_REF][START_REF] Arakaki | Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies[END_REF]. Thus, succinate is expected to be present at high concentrations in T. brucei mitochondrion. Under optimal conditions, the ASCT/SCS cycle exhibits a turnover (k cat ) of 191 s -1 . Considering a stoichiometry of 1 mole of ATP produced per mole of CoA detected, the k cat of the TbASCT/SCS cycle is higher than the reported values for purified F o /F 1 -ATP synthase from several organisms, including Bacillus PS3 (16 s -1 ) [START_REF] Soga | Efficient ATP synthesis by thermophilic Bacillus F o F 1 -ATP synthase[END_REF], E. coli (27 s -1 ) [START_REF] Fischer | ATP synthesis catalyzed by the ATP synthase of Escherichia coli reconstituted into liposomes[END_REF], and

Saccharomyces cerevisiae (120 s -1 ) [START_REF] Forster | Proton transport coupled ATP synthesis by the purified yeast H + -ATP synthase in proteoliposomes[END_REF], clearly demonstrating that the ASCT/SCS cycle produces ATP with high efficiency. The F o /F 1 -ATP synthase is a multi-subunit mega-complex enzyme, with the simplest form of E. coli composed of 8 subunits, α 3 β 3 γδ (F o ) and ab 2 c 10 (F 1 ) [START_REF] Okuno | Rotation and structure of F o F 1 -ATP synthase[END_REF]. Compared to the reported ATP synthases, the ASCT/SCS cycle is encoded by a total of three genes (for ASCT α 4 and SCS αβ subunits) and does not interact each other. The most prominent advantage of the ASCT/SCS cycle is the ability to synthesize ATP and acetate independently of the presence of oxygen, an electrochemical gradient, and OXPHOS, hence, this cycle fuels and boosts ATP and acetate production in the parasite's mitochondria. Such an observation is consistent with the fact that the ASCT/SCS cycle is conserved in the hydrogenosome and mitochondria of many anaerobic protists and helminths [START_REF] Tielens | Acetate formation in the energy metabolism of parasitic helminths and protists[END_REF], in which ATP synthase is not conserved (hydrogenosome) or exhibits limited activity due to low environmental oxygen concentrations.

Despite the high amino acid identity between TbASCT and HsSCOT, our comprehensive biochemical analysis clearly demonstrates that TbASCT have low SCOT activity and that catalysis. As a crystal structure of ligand free TbASCT was obtained (PDB: 6LP1), study on the binding site of the CoA acceptor by solving the crystal structure of TbASCT in complex with acetate or succinate is on going.

Conclusion

In conclusion, we demonstrated that mitochondrial acetate production is essential for growth of the BSF of trypanosomes. Previously, we have reported the remarkable effect of combination of ascofuranone and glycerol targeting TAO and glycerol kinase, respectively, for trypanocidal activity against BSF [START_REF] Balogun | Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei[END_REF]. Considering the growth phenotype of Δach/ RNAi ASCT.i double mutant described in this study, a dual inhibitor targeting TbASCT and TbACH can be a suitable drug candidate to combat African trypanosomes due to their common function in terms of acetate production, although they catalyze distinct reaction.

Furthermore, the absence of the ACH gene in the T. cruzi (American trypanosome) genome suggests that ASCT is the sole source of mitochondrial acetate and may be a potential drug target against Chagas disease. Because of the high degree of amino acid identity, the kinetic characterization of TbASCT reported here might facilitate the development of high-throughput screening systems and development of useful tools for structure-based design of ASCT-specific inhibitors targeting African and American trypanosomiasis.

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Fig. 1 .

 1 Fig. 1. Schematic representation of the reactions catalyzed by (A) the ASCT/SCS cycle, (B) ACH and SCOT reaction. Abbreviations: AcCoA, acetyl-CoA; Ace, acetate; Suc, succinate; SucCoA, succinyl-CoA; AcAce, acetoacetate; AcAcCoA, acetoacetyl-CoA.

  15 hours. The pre-culture was inoculated (to a final OD 600 of 1.0) into 6 Ultra Yield TM flasks (Thomson), each containing 600 ml of TB medium supplemented with 50 µg/ml kanamycin and 20 µM isopropyl β-D-thiogalactopyranoside (IPTG), and cultured for 12 hours at 20°C on a rotary shaker (200 rpm; BioShaker GBR-300; Taitec). The E. coli cells were harvested by centrifugation at 7,000 × g, 10 min, 4°C and resuspended in 210 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 5 mM imidazole, 20% [v/v] glycerol, 300 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA, 250 µM phenylmethylsolfonyl fluoride [PMSF]). Cells were broken by a single passage through a French Press (Ohtake) at 180 MPa and 4°C. Unbroken cells and debris were removed by centrifugation at 30,000 × g, 30 min, and 4°C (R20A2 rotor, Hitachi), and the cytosolic fraction was obtained as the supernatant following ultracentrifugation at 200,000 × g, 90 min, and 4°C (45TI rotor, Hitachi). A pre-equilibrated 2-ml volume of Ni-NTA agarose (QIAGEN) was mixed with the cytosolic fraction overnight, loaded onto Econo-Pac ® chromatography columns (Bio-Rad), and washed with 2 ml of lysis buffer. The column was consecutively washed with 5 ml of wash buffer A (50 mM Tris-HCl pH 8.0, 20 mM imidazole, 20% [v/v] glycerol, 10 mM ATP, 300 mM NaCl, 2.5 mM MgCl 2 , 250 µM PMSF) and 30 ml of wash buffer B (50 mM Tris-HCl pH 8.0, 20 mM imidazole, 20% [v/v] glycerol, 300 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA, 250 µM PMSF), and recombinant TbASCT was eluted with 10 volumes of elution buffer (50 mM Tris-HCl pH 8.0, 250 mM imidazole, 20% [v/v] glycerol, 300 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA, 250 µM PMSF),

  with 4 × SDS-PAGE loading buffer (240 mM Tris-HCl pH 6.8, 8% [w/v] SDS, 40% [v/v] glycerol, 0.04% [w/v] bromophenol blue, and 5% [v/v] β-mercaptoethanol) and heated for 5 min at 95°C. Protein preparations were loaded onto the gel and run alongside broad-ranged Precision Plus Protein TM Standards (Bio-Rad). The electrophoresis was carried out at 25 mA for 120 min at room temperature, and the gel was stained with GelCode TM Blue Safe Protein Stain (Thermo Fisher Scientific).

  Screen 2, and PEG-ION 1, 2, and Grid Screen PEG6000 from Hampton Research; and Wizard Classic 1, 2, and Wizard Cryo 1, 2, from Molecular Dimensions). A droplet made by mixing 0.5 µl of purified TbASCT (5, 10 mg/ml) with an equal volume of reservoir solution was equilibrated against 100 µl of the reservoir solution at 293 K. After the initial crystallization, the crystals of TbASCT were generated under two conditions (PEG-ION Screen No. 5 and PEG-ION 2 Screen No. 48); crystallization conditions were optimized by fine-tuning the pH and precipitant concentration using the hanging drop vapor diffusion method at 293 K. The best crystals were obtained using a reservoir solution containing 0.05 M HEPES-NaOH buffer pH 7.4, 18% (w/v) PEG3350, 0.35 M CaCl 2 with 10 mg/ml TbASCT dissolved in 20 mM Tris-HCl pH 8.5, 100 mM NaCl, and 10% (v/v) glycerol.

Fig. 2 . 1 ~○, 5 )

 215 Fig. 2. Analysis of TbASCT and TbACH mutant cell lines. Growth curves of the (A) ∆ach and (B) RNAi ASCT mutants incubated in the presence (.i, ○) or absence (.ni, •) of 10 µg/ml tetracycline. Bottom panel show the Western blot analyses of the PCF, parental BSF trypanosomes, and mutant cell lines using immune serum indicated in the left margin. Integration of targeting vector into the ACH locus in the ∆ach cell line. (C) The top panel shows results of PCR analysis of genomic DNA isolated from the parental 427 MITat1.2 13-90 BSF strain (WT) and ∆ach cell lines. The lower panel shows PCR fragments (○, 1 ~○, 5 ) amplified using primers based on sequences that flank the 5'UTR used to target the ACH gene for disruption (black boxes), internal sequences from ACH, and puromycin resistance (PAC) or phleomycin (BLE) resistance genes. As expected, using primers derived from the ACH gene and drug resistance genes, PCR amplification was only observed for the parental 427 (WT) and ∆ach cell lines, respectively. (D) Growth curves of the ∆ach/ RNAi ASCT mutants incubated in the presence (.i, ○) or absence (.ni, •) of 10 µg/ml tetracycline. Bottom panel show the Western blot analyses of the parental BSF trypanosomes, and ∆ach/ RNAi ASCT mutant cell line using immune serum indicated in the left margin.

Fig. 2 .

 2 Fig. 2. Analysis of ASCT and ACH mutant cell lines.

Fig. 3 .

 3 Fig. 3. Biochemical characterization of TbASCT. Kinetic parameters of TbASCT were determined by varying the concentration of (A) acetyl-CoA under a fixed succinate concentration of 20 mM or (B) succinate under a fixed acetyl-CoA concentration of 0.5 mM, and the Lineweaver-Burk plot is shown in the inset. Error bars are ± SD (n = 3). At high succinate concentrations, substrate inhibition was observed, with a K i of 75.7 mM.

  Values represent the average ± SE (n = 3) calculated using GraphPad Prism 7. N.D. indicates non-detectable level.

Fig. 4 .

 4 Fig. 4. Reaction mechanism of TbASCT. The double reciprocal plot of varying the concentration of (A) acetyl-CoA under a set of fixed succinate concentrations of 5, 10, and 20 mM and varying the concentration of (B) succinate under a set of fixed acetyl-CoA concentrations of 0.02, 0.05, and 0.1 mM. (C) Schematic representation of ping-pong bi-bi mechanism catalyzed by TbASCT. (D) BN-PAGE and In-gel ASCT activity staining of TbASCT, M: Protein marker. Major bands identified from both staining are shown in red arrows with calculated molecular weight of 225, 414 and 564 kDa, which corresponds to one, two or three tetramers of TbASCT, respectively.

Fig. 5 .

 5 Fig. 5. CoA donor specificity of TbASCT. (A) TbASCT shows activity in the presence of n-propionyl-CoA, acetoacetyl-CoA, and malonyl-CoA of 60%, 6%, and 13% relative to acetyl-CoA (100%), respectively. Error bars are ± SD (n = 4). (B) Biochemical characterization of propionyl-CoA:succinate CoA transferase activity of TbASCT. Lineweaver-Burk plot is shown in the inset. Error bars are ± SD (n = 3). The K m , V max , k cat ,

  and S7B, PDB entry 3DLX) and Sus scrofa SCOT (SsSCOT, Fig. S6A, PDB entry 3OXO) as search models and refined to 2.01-Å resolution. Four TbASCT protomers were identified in the crystallographic asymmetric unit (chains A-D; Fig S8A). Each TbASCT protomer associated to form a tight homotetramer, consistent with the oligomerization state revealed by BN-PAGE and in-gel ASCT-activity staining (Fig. 4D). Each protomer of TbASCT was shown to have a similar structure, as indicated by root-mean-square (RMS) deviations of 0.73-0.97 Å as calculated for 463 superimposed Cα positions between the chains. The RMS deviation of monomers within TbASCT (Chain A) and HsSCOT (Chain A) was 1.06 Å

Acknowledgements

We thank all of the staff members of beamline BL44XU (SPring-8) and BL-17A (KEK-PF) for assistance with X-ray diffraction data collection. The synchrotron radiation experiments were performed at BL-17A of the Photon Factory (proposal nos. 2013G055, 2013G107, 2015G510, and 2017G022) and at BL44XU of SPring-8 (proposal nos.

Funding source This work was supported in part by Infectious Disease Control from the Science and Technology Research Partnership for Sustainable Development (SATREPS, nos. 10000284 to K.K. and 14425718 to D.K.I.); a Grant-in-aid for the Bilateral Joint Research Project (no. 16035611-000722 to K.K.); a Grant-in-aid for Scientific Research on Priority Areas 18073004 (to K.K.); a Creative Scientific Research Grant 18GS0314 (to K.K.) from the Japan Society for the Promotion of Science; a Grant-in-aid from the Program for the Promotion of Basic and Applied Research for Innovations in Bio-Oriented Industry (BRAIN) (no. 26020A to S.H. and K.K.); Grants-in-aid for Scientific Research (B) 16K19114 to D.K.I and (C) 23570131 and 26234567 to T.S.; and The Leading Initiative for Excellent Young Researchers (LEADER) 16811362 to D.K.I. and (B) 19H03436 to K.K. and D.K.I. and (C) 19K07523 to D.K.I. from the Japanese Ministry of Education, Science, Culture, Sports and Technology (MEXT). This work was also supported by the Japanese Initiative for Progress of Research on Infectious Diseases for Global Epidemics (JP18fm0208027 to D.K.I. and T.S.) from the Agency for Medical Research and Development (AMED); a Grant-in-aid for research on emerging and re-emerging infectious diseases from the Japanese Ministry of Health and Welfare (to K.K.); and the Strategic Japanese-French Cooperative Program 11102218 (to K.K.). This work was also supported by the Centre National de la Recherche Scientifique (CNRS), the Université de Bordeaux, the Agence Nationale de la Recherche (ANR) through grants ACETOTRYP of the ANR-BLANC-2010 call and GLYCONOV of the "Générique" call and the Laboratoire d'Excellence (LabEx) ParaFrap ANR-11-LABX-0024 to F.B.

Data availability

The Table S4 and S5 include mutant analyses data. The ligand-free structure of TbASCT with PDB accession code 6LP1 is described in METHODS. Source data for Fig. S6 and7 can be found in Table S6.

HsSCOT does not have ASCT activity, in accordance with the lack of experimental evidence of β-oxidation in T. brucei [START_REF] Allmann | Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei[END_REF]. Moreover, we investigated the reaction mechanism of TbASCT and found that TbASCT employs a ping-pong bi-bi mechanism of catalysis, similar to other family IA CoA transferases, including SCOTs [START_REF] Tielens | Acetate formation in the energy metabolism of parasitic helminths and protists[END_REF]. We also analyzed the substrate specificity in terms of CoA donor and found that TbASCT can utilize n-propionyl-CoA with an affinity higher than that for acetyl-CoA, with similar catalytic efficiency (k cat /K m ): 2,375 and 2,267 s -1 mM -1 , respectively. Interestingly, F. hepatica excretes acetate and propionate as end products of glucose catabolism [START_REF] Mansour | Studies on the carbohydrate metabolism of the liver fluke Fasciola hepatica[END_REF], and it was demonstrated that F. hepatica ASCT utilizes both propionyl-CoA and acetyl-CoA as CoA donors [START_REF] Van Grinsven | Acetate:succinate CoA-transferase in the anaerobic mitochondria of Fasciola hepatica[END_REF]. It is important to note that T. brucei does not accumulate propionate [START_REF] Millerioux | The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control[END_REF][START_REF] Van Weelden | Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation[END_REF][START_REF] Coustou | Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei[END_REF][START_REF] Smith | Metabolic reprogramming during the Trypanosoma brucei life cycle[END_REF][START_REF] Van Grinsven | Adaptations in the glucose metabolism of procyclic Trypanosoma brucei isolates from tsetse flies and during differentiation of bloodstream forms[END_REF]; consistent with these reports, n-propionyl-CoA was not detected in a previous metabolome analysis [START_REF] Creek | Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose[END_REF]. Taken together, our results indicate that acetyl-CoA is the physiological and the only CoA donor for TbASCT in vivo.

The catalytic glutamate residue located within the SENG motif was identified in SsSCOT by site-directed mutagenesis [START_REF] Rochet | Identification of glutamate 344 as the catalytic residue in the active site of pig heart CoA transferase[END_REF] and analysis of the crystal structure in complex with CoA (PDB: 3OXO) [START_REF] Fraser | Catalytic role of the conformational change in succinyl-CoA:3-oxoacid CoA transferase on binding CoA[END_REF]. Although TbASCT exhibited extremely low SCOT activity (0.038 µmol/min/mg), the enzyme has 52% identity with SsSCOT and the SENG motif is conserved. In contrast to homodimeric mammalian SCOTs, the TbASCT forms a tight homotetramer as revealed by the crystal structure obtained in this study (Fig. S8) and by BN-PAGE followed by CBB and activity staining (Fig. 4D). The RMS deviation of monomers within TbASCT (Chain A) and SsSCOT (Chain E, PDB: 3OXO) was 1.50 Å between the two structures for 454 Cα atoms. In the crystal structure of SsSCOT, CoA was found covalently bound to E344, which corresponds to E319 of TbASCT. In addition, the carbonyl group of M423 located within the X-CoA tunnel from SsSCOT, which is not conserved in TbASCT and is replaced by P398, is found making hydrogen bond with the amine group of β-mercaptoethylamine of bound CoA. As the ASCT activity of the E319A and P398A mutants were completely abolished (Table . S4 and S5), we confirmed that E319 is the catalytic glutamate residue and P398 is located in the predicted X-CoA tunnel from TbASCT.

In the structure of SsSCOT bound to CoA, a second tunnel, adjacent to CoA binding site, is found with potential to bind the CoA acceptor (Fig. S6). In the second tunnel, several residues conserved in mammalian SCOTs (L327, L350, Y353), which are not conserved in trypanosomatid ASCTs (T302, M325, F328) are identified (Fig. S7). However, TbASCT mutants targeting T302, M325 and F328 showed no drastic changes in ASCT or SCOT activities (Table S4 andS5) and thus, we conclude that the second tunnel is not involved in

Conflict of interest

The authors declare no conflict of interests.

Appendix A. Supplementary data Supplementary data to this article can be found online.


Synthesis of N-Glycosyl-2-oxindoles by Pd-Catalyzed N-Arylation of 1-Amidosugars

Boris Letribot, a Wafa Redjdal, a,b Belkacem Benmerad, b Franck Le Bideau, a Mouâd Alami, a and Samir Messaoudi a * a Université Paris-Saclay, CNRS, BioCIS, 92290, Châtenay-Malabry, France b Laboratoire de Physico-Chimie des Matériaux et Catalyse, Faculté des Sciences Exactes, Université de Bejaia, 06000 Bejaia, Algeria Supporting Information Placeholder ABSTRACT: An efficient intramolecular Pd-catalyzed N-arylation of o-iodo-amidosugars to the synthesis of N-glycosylated oxindoles has been reported. The coupling reaction takes place in toluene and involves Pd(OAc)2/RuPhos catalytic systems in the presence of K2CO3. This versatile approach was extended successfully to the synthesis of N-glycosylated other heterocycles.

The 2-oxindole scaffold has attracted considerable attention due to its widespread application in organic synthesis, and in the design of pharmaceuticals. [START_REF] Millemaggi | 3-Alkenyl-oxindoles: Natural Products, Pharmaceuticals, and Recent Synthetic Advances in Tandem/Telescoped Approaches[END_REF] This common motif was found in many biological active compounds with applications in various therapeutic areas. [START_REF] Kaur | A chemical prism carrying plethora of therapeutic benefits[END_REF] Of the many drugs developed so far containing a 2-oxindole core, four marketed molecules have emerged (Figure 1-A). Sunitinib is used to treat renal cell carcinoma, gastrointestinal stromal and pancreatic neuroendocrine tumours. Ropinirol is recommended in Parkinson's disease, nintedanib is a medication delivered against idiopathic pulmonary fibrosis and ziprasidone is used to treat schizophrenia and bipolar disorder. Other 2-oxindole derivatives are also under clinical trials against various diseases. [START_REF] Wishart | Drugbank: a comprehensive resource for in silico drug discovery and exploration[END_REF] The incorporation of a sugar moiety in bioactive molecules may have several benefits such has enhancing their bioavailability, [START_REF] Moradi | Glycosylation, an Effective Synthetic Strategy to Improve the Bioavailability of Therapeutic Peptides[END_REF] their solubility [START_REF] Cheng | Enzyme-Catalyzed Glycosylation of Curcumin and Its Analogues by Glycosyltransferases from Bacillus Subtilis ATCC 6633[END_REF] or their biological activity by targeting for example, selectively cancer tissues through the Warburg effect. 6 Such a transformation was achieved in the 2-oxindole series, by chemical synthesis, 7 and recently by biotransformation using Streptomyces sp. SANK 60895, [START_REF] Ohnuki | Practical One-Step Glucuronidation via Biotransformation[END_REF] delivering N-glycosylated oxindoles which displayed interesting biological activities. Interestingly, these latter structures were also found in the living systems (Figure 1-B) as illustrated by compound A, extracted from the seeds of Ziziphus jujuba var. spinosa 9 and compound B, found in the stems of Brucea mollis. [START_REF] Chen | Indole Alkaloids and Quassinoids from the Stems of Brucea Mollis[END_REF] Despite the high importance of N-glycosyl-2-oxindoles, direct methods for their synthesis are scarce. To our knowledge, only two methods based on a multi-steps sequence synthesis are reported. The most known one 7d relies on a four-steps synthesis (Scheme 1, a) starting from aniline and glucose, to lead to Nglycosyl isatines. The extra C3-carbonyl group was used to Scheme 1. Strategies to the synthesis of N-glycosyl oxindoles further functionalization. This method however, suffers from drawbacks such as low overall yields and difficulties of purification linked to the  anomeric composition of the starting materials (up to 2:1). Another approach to N-glycosyl-2-oxindoles involves a six-steps procedure including N-glycosylation of indoline, oxidation, protection, functionalization and deprotection. Only one unsubstituted oxindole was reported through this procedure in 26% overall yield (Scheme 1, b). [START_REF] Messaoudi | Synthesis and Biological Evaluation of Oxindoles and Benzimidazolinones Derivatives[END_REF] As part of our continuing effort in functionalizing sugars under transition-metal catalysis, 12 we became interested to develop an efficient and convergent approach to the synthesis of substituted N-glycosyl oxindoles. We postulated that these glycosides maybe obtained simply through an intramolecular N-arylation of 1-amidosugar using a catalytic amount of palladium (Scheme 1, c). Herein, we report our success in the development of such protocol. In order to determine optimal conditions for the selective intramolecular arylation of 1-amidosugars, (2-iodophenyl)acetamidoglycopyranose (3a, Table 1) was initially selected as a model substrate. This compound was prepared by reacting 2-iodophenylacetic acid (1a) with tetraacetylated 1-amino-β-glycopyranose (2a) in the presence of hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl). Under these coupling conditions, compound 3a was obtained in 67% yield as a β:α mixture in a ratio of 20:1 (determined by 1 H NMR, comparing integrations of the two anomeric protons, see supporting information). With a sub-sequent amount of 3a in hand, we turned our attention to examine the intramolecular N-arylation of this substrate. The first tentative was accomplished by using our previously reported Pd-catalyzed conditions [START_REF] Luong | Intramolecular Pd-Catalyzed Arylation of 1-Amidosugars: A New Route to N-Glycosyl Quinolin-2-ones[END_REF] for the synthesis of N-glycosyl quinolin-2-ones [Pd(OAc)2 (5 mol%), n-Bu4NOAc (1.5 equiv), dioxane, 100°C, 1 h]. However, under these conditions, even traces of 4a were not identified by LCMS analysis. Therefore, we turned our attention toward a systematic screening of literature reaction conditions 14 which have proven their efficiency to accomplish the intramolecular N-arylation of secondary amines or amides (Table 1 and SI.). Among this series, only conditions using Pd(dba)2/DavePhos 14e (entry 1) provided traces (by LCMS) of the desired cyclized product thus establishing the starting point of our optimizing process. We next evaluated monophosphine SPhos (entry 2) and bidentate phosphine ligand (XantPhos, see SI) usually involved in such pallado-catalyzed coupling reaction type to find that only the SPhos (entry 2) was able, in toluene and acetonitrile solvents, to afford the desired glycoside oxindole 4a in 15% yield as a β:α mixture (20:1 ratio). Doubling the catalyst ratio to 10 mol% (entry 3) allowed a small improvement of the yield (20%) and gratefully, removing the polar aprotic acetonitrile solvent, the yield of the reaction raised to 55 % in only 3 hours (entry 4). An increase of the Pd/L ratio from 1:1 to 1:2 leads to an improved yield (entry 4, 55% yield vs entry 5, 72% yield). Shifting from SPhos to a more hindered ligand RuPhos allowed a slight improvement of the yield (entry 6, 78%). In addition, performing the reaction with Pd(OAc)2 pre-catalyst instead of Pd(dba)2 lead to 4a in 82% yield (entry 7). We next succeeded in reaching an excellent yield (90%) by combining 10 mol% of Pd(OAc)2 and 40 mol% of RuPhos (entry 8). It can be noted that 4a was isolated in all cases as a mixture of β:α anomers in 20:1 ratio, indicating that no anomerization occurred during the process. This was confirmed by conducting the intramolecular coupling reaction from a pure β-anomer 3a which led to the β-glycosylated oxindole 4a without traces of the α-anomer. With optimal conditions in hand, the scalability and robustness of this methodology were demonstrated on 1 mmol scale. As shown in Scheme 2, 4a was obtained in 83% yield when the Narylation reaction of 3a was conducted with 1 mmol scale. Scope and limitations of the reaction were then examined with a diversity of substituted 1-amido-sugars 3 (Scheme 2). These later were prepared from a readily available carboxylic acids 1 and the appropriate 1-amino-β-sugars. In all cases, the expected β-amidosugars 3 were obtained as the major glycosides contaminated in some cases, by a small amount of the α-isomer (see Supporting Information for the β/α ratios), and this mixture was used for the next step.

Scheme 2 Scope of the intramolecular N-arylation of 1-amidosugars 3

Reaction conditions: 1-amidosugar 3 (0.036 to 0.1 mmol), Pd(OAc)2 (10 mol %), RuPhos (40 mol%), K2CO3 (2 equiv) in toluene (0.015 to 0.021 M) at 130 °C for 3 h. a Yield of isolated product. b reaction was conducted at 1 mmol scale. c the β:α ratio was determined by 1 H NMR after purification on flash chromatography. d 4k and his precursor 3k were obtained as a racemic mixture regarding to the stereogenic center.

We were pleased to note that the coupling conditions were efficient either starting from iodo-or bromo-and chloro-aryl substrates, delivering product 4a with the same yield (90%). Interestingly, in contrast to the iodide substrate 3a (β:α = 20:1) used, the bromide 3a' (β:α = 20:1) or chloride 3a'' (β:α = 9:1) analogues delivered a pure β-N-glycosyl oxindole 4a although they were used as a mixture of β/α anomers. This result indicates clearly that anomerization occurs during the process when bromide or chloride substrates (3a' and 3a'', respectively) were used. Substrates with electron donating (3b-c, 3e-f) and electron withdrawing (3d, 3g-i) substituents on various positions of the aromatic ring were also successfully converted into the corresponding N-glycosyl oxindoles in good to excellent yields (up to 90%). Additionally, the chemoselectivity of this coupling was examined with the dihalogenated substrate 3h bearing two different C-X bonds (C-I and C-Cl) at the orth-ortho' positions of the benzene ring. Interestingly, under our optimized conditions, only the C-I reacted delivering the corresponding glycoside 4h in 84% yield and anomeric stereoselectivity β:α of 20:1. The presence of aryl fused ring was also well tolerated as shown with compound 4j. Moreover, the N-glycosyl congested tetracycle 4k was obtained in 84%. In addition, the spiro-oxindole N-glycosylated compound 4l with a cyclopropyl ring on the C3position was prepared in good 58% yield as a pure β-anomer.

The successful synthesis of N-glucosides 4a-l from 1-amidoglucose 3a-l encouraged us to investigate other sugar partners on this coupling. Pleasantly, the coupling reaction was extended with success to substrates with various peracetylated mono-, diand tri-glycosides (3m-p). The corresponding oxindoles bearing a mannose (4m), a galactose (4n), a cellobiose (4o) or a maltotriose (4p) were all prepared with excellent yields (up to 90%) and high stereoselectivity.

Encouraged by these results, the feasibility of this approach was investigated in the aim to synthesize N-glycosylated 6-membered heterocycles (Scheme 3). In this context, we were pleased to see that substrates 5a, 5b bearing an ether linkage or their thioether congener 5c all reacted successfully under our reaction conditions to furnish N-glycosylated dihydroquinolinone 6a, benzoxazinone 6b and benzothiazinone 6c in 70%, 68% and 24% yields, respectively. These results represent the first examples of diversification of this family of N-heterocycle glycosides through this methodology. With substantial amounts of 4a in hand (Scheme 4), we focused our attention on demonstrating whether our method could be employed for molecular diversity. At first, the unprotected glycoside-indolin-2-one 7a was synthesized by deacetylation of 4a under Zamplen conditions. This N-glucosyloxindole 7a was thus obtained in a 6-steps procedure, starting from 1a, in 22% overall yield. Finally, this methodology was applied to the synthesis of bioactive analogues. As shown in Scheme 4, β-N-glucosyl SUGEN derivative 8a which is a N-glycosylated analogue of semaxanib (SU5416, a tyrosine-kinase inhibitor used a cancer therapeutic), was obtained in 72 % yield by post-functionalization of the N-glycosyl oxindole 4a, via condensation with 3,5-dimethyl-1H-pyrrole-2-carbaldehyde. As it was previously described, [START_REF] Sun | Synthesis and Biological Evaluations of 3-Substituted Indolin-2-Ones: A Novel Class of Tyrosine Kinase Inhibitors That Exhibit Selectivity toward Particular Receptor Tyrosine Kinases[END_REF] only Z isomer of the alkene was observed presumably thanks to the hydrogen bond formed between the nitrogen of the pyrrole and the carbonyl of the oxindole. The deprotected analogue 9a was further prepared by removal of the acetate groups in presence of a catalytic amount of potassium carbonate in methanol in 84% yield.

Scheme 4 Further functionalisation of 4a and application to the synthesis of a glycosylated Sugen derivative 9a

a Yield of isolated product. b the β:α ratio was determined by 1 H NMR after purification on flash chromatography.

In summary, we succeeded the synthesis of N-glycosylated oxindoles by intramolecular Pd-catalyzed of N-arylation 1-amidosugars. This is the first methodology reporting an efficient way to prepare a large variety of substituted N-glycosyl oxindoles. This coupling reaction tolerates various functional groups and sugar moieties. Moreover, this approach was extended to the synthesis of N-glycosylated dihydroquinolinone, benzoxazinone and benzothiazinone. Finally, thanks to this methodology, we described for the first time, the synthesis of a N-glycosylated SUGEN derivatives and we expect to apply this general methodology to other therapeutic compounds.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures, spectroscopic data and NMR spectra of new compounds.

Figure 1 .

 1 Figure 1. Example of biologically active oxindoles

Scheme 3 :

 3 Scheme 3: Extending the methodology to other structures

Table 1

 1 Optimization of the coupling reaction of tetraacetylated β-amidoglucose 3a a

	en-try	Pd.cat	ligand	Solvent	t (h)	yield (%) b
	1	Pd(dba)2	DavePhos	toluene /MeCN	24	traces
		(10 mol%)	(10 mol%)	(3/1)
	2	Pd(dba)2 (5 mol%)	SPhos (5 mol%)	toluene /MeCN (3/1)	12	15%
	3	Pd(dba)2 (10 mol%)	SPhos (10 mol%)	toluene /MeCN (3/1)	12	20%
	4	Pd(dba)2 (10 mol%)	SPhos (10 mol%)	toluene	3	55%
	5	Pd(dba)2 (10 mol%)	SPhos (20 mol%)	toluene	3	72%
	6	Pd(dba)2 (10 mol%)	RuPhos (20 mol%)	toluene	3	78%
	7	Pd(OAc)2 (10 mol%)	RuPhos (20 mol%)	toluene	3	82%
	8	Pd(OAc)2 (10 mol%)	RuPhos (40 mol%)	toluene	3	90%

a A sealable tube was charged with 1-amidoglucose 3a (1 equiv, 0.2 mmol), [Pd pre-catalyst] (n mol%), ligand (x mol%), K2CO3, (2 equiv) in solvent (1.0 mL, 0.015M). b Yield of isolated product.

ACKNOWLEDGMENTS

Authors acknowledge support of this project by CNRS, University Paris-Sud, ANR (ANR-15-CE29-0002), and by la Ligue Contre le Cancer through an Equipe Labellisée 2014 grant. We also thank the Algerian Ministry of Education and Research for a fellowship (PNE) to Wafa Redjdal. Our laboratory is a member of the Laboratory of Excellence LERMIT supported by a grant (ANR-

AUTHOR INFORMATION

Corresponding Author * samir.messaoudi@u-psud.fr

Author Contributions

The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.