Dr Mervi T Hyvönen
email: mervi.hyvonen@uku.fi

Michael T Howard

Christine B Anderson

Nikolay Grigorenko

Alex R Khomutov

Jouko Vepsäläinen

Leena Alhonen

Juhani Jänne

Tuomo A Keinänen

A I Virtanen

Divergent regulation of the key enzymes of polyamine metabolism by chiral α-methylated polyamine analogs

Keywords: polyamines, optical isomers, frameshifting, upstream open reading frame, alternative splicing, difluoromethylornithine

Polyamines are achiral organic polycationic compounds that play a critical role as regulators of important cellular processes such as proliferation, differentiation and modulation of ion channels [START_REF] Tabor | Polyamines[END_REF]. As expected for a molecule with multiple cellular roles, polyamine levels are tightly controlled through a series of feedback circuits to maintain appropriate intracellular concentrations. The key regulatory enzymes in the polyamine biosynthesis are ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (AdoMetDC). During catabolism of polyamines, spermidine/spermine N 1 -acetyltransferase (SSAT) is the rate-controlling enzyme of polyamine oxidase (PAO)-mediated back-conversion of spermine and spermidine to spermidine and putrescine, respectively. Based on the observations that increased polyamine levels are correlated with, and can promote neoplastic growth, it has been proposed that reducing or depleting intracellular polyamines is an appropriate therapy for cancer and other diseases involving undesired cellular proliferation. Towards this end, inhibitors of the ODC and AdoMetDC [START_REF] Metcalf | Catalytic irreversible inhibition of mammalian ornithine decarboxylase (E.C. 4.1.1.17) by substrate and product analogues[END_REF][START_REF] Williams-Ashman | Methyl glyoxal bis(guanylhydrazone) as a potent inhibitor of mammalian and yeast S-adenosylmethionine decarboxylases[END_REF][START_REF] Casara | 5'-{[(Z)-4-amino-2butenyl]melthylamino}-5'-deoxyadenosine: a potent enzyme-activated irreversible inhibitor of Sadenosyl-L-methionione decarboxylase from Escherichia coli[END_REF] have been identified and polyamine analogues have been designed with the intent of affecting multiple targets in the polyamine pathway. Several of these compounds alone or in combination are currently in clinical trials [START_REF] Wolff | A phase II study of the polyamine analog N1,N11diethylnorspermine (DENSpm) daily for five days every 21 days in patients with previously treated metastatic breast cancer[END_REF][START_REF] Simoneau | The effect of difluoromethylornithine on decreasing prostate size and polyamines in men: Results of a year-long phase IIb randomized placebo-controlled chemoprevention trial[END_REF][START_REF] Hahm | Phase I study of N(1),N(11)-diethylnorspermine in patients with non-small cell lung cancer[END_REF][START_REF] Meyskens | Development of difluoromethylornithine (DFMO) as a chemoprevention agent[END_REF] as potential anti-cancer agents.

Due to their positive charge at physiological pH, a major fraction of polyamines is electrostatically bound to anionic cellular sites such as RNA, DNA and phospholipids. Thus, polyamines are involved in regulation of many levels of gene expression, by influencing chromatin condensation, DNA stability and structure, RNA processing and translation. One very specific target of polyamine-mediated regulation is the programmed frameshifting of eukaryotic ODC antizyme [START_REF] Matsufuji | Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme[END_REF]10] and TY1 transposon [START_REF] Balasundaram | Spermidine deficiency increases +1 ribosomal frameshifting efficiency and inhibits Ty1 retrotransposition in saccharomyces cerevisiae[END_REF]. Antizyme is a small regulatory protein which binds to and inhibits ODC and targets it to the proteasome for degradation. In addition, antizyme negatively regulates the polyamine uptake system [START_REF] Mitchell | Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells[END_REF][START_REF] Suzuki | Antizyme protects against abnormal accumulation and toxicity of polyamines in ornithine decarboxylaseoverproducing cells[END_REF]. The synthesis of antizyme is controlled by cellular polyamine concentration at the level of translation, whereby polyamines greatly increase the efficiency of +1 ribosomal frameshifting, allowing the production of functional, full-length protein, thus completing an autoregulatory circuit controlling polyamine synthesis and transport.

The feedback regulation of AdoMetDC by spermidine and spermine involves control of translational initiation. The AdoMetDC 5' leader sequence harbours an upstream open reading frame (uORF), which codes for a small regulatory peptide with the sequence MAGDIS [START_REF] Ruan | The upstream open reading frame of the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive translational control element[END_REF]. When polyamine levels are low ribosomes pause after uORF termination but are able to continue to AdoMetDC reading frame. By contrast, elevated polyamine levels stabilize the ribosomal pause in the vicinity of uORF termination codon, and inhibit the completion of uORF peptide synthesis reducing translation of the AdoMetDC ORF.

We recently discovered a novel regulation mechanism for the expression of SSAT, where polyamines modulate the alternative splicing of SSAT pre-mRNA [START_REF] Hyvönen | Polyamine-regulated unproductive splicing and translation of spermidine/spermine N1-acetyltransferase[END_REF]. Low polyamine levels result in accumulation of an unproductive splice variant (SSAT-X), which is rapidly degraded by the protein synthesisdependent mRNA surveillance pathway known as nonsense-mediated mRNA decay (NMD). By contrast, high polyamine levels favour generation of the productive mRNA variant (SSAT), resulting in synthesis of active SSAT enzyme protein, leading to decreased polyamine levels.

Although complex feedback mechanisms have been found for all key metabolic enzymes controlling cellular polyamine levels, the underlying regulatory mechanisms of these specific processes are not yet understood. Thus, new specific tools are needed to manipulate the polyamine metabolism. α-Methylated polyamine analogs are functional mimetics for natural polyamines both in vivo and in vitro [START_REF] Lakanen | Alpha-methyl polyamines: Metabolically stable spermidine and spermine mimics capable of supporting growth in cells depleted of polyamines[END_REF][START_REF] Järvinen | Metabolic stability of alpha-methylated polyamine derivatives and their use as substitutes for the natural polyamines[END_REF][START_REF] Keinänen | Alpha-methylated polyamines as potential drugs and experimental tools in enzymology[END_REF]. αmethylated spermidine (MeSpd) and spermine (Me 2 Spm) are not cytotoxic and fulfil many cellular functions of the natural polyamines and support proliferation. Because they are metabolically much more stable than their natural counterparts and replace the natural polyamines by enhancing polyamine efflux and inhibiting uptake, the α-methylated analogs are convenient tools for polyamine research. Unlike the natural polyamines, the α-methylated analogs are chiral. Since chirality plays an important role in nature by controlling the binding and metabolic transformation of the compounds in living systems, we rationalized that the stereoisomers of α-methylated polyamine analogs may possess divergent biological properties. Indeed, the investigation of these novel chiral analogs led to the discovery of the hidden A c c e p t e d M a n u s c r i p t stereospecificity of several enzymes of polyamine metabolism, such as polyamine oxidase (PAO) [START_REF] Järvinen | Alpha-methyl polyamines: Efficient synthesis and tolerance studies in vivo and in vitro. first evidence for dormant stereospecificity of polyamine oxidase[END_REF], spermine oxidase (SMO) and deoxyhypusine synthase [START_REF] Hyvönen | Role of hypusinated eukaryotic translation initiation factor 5A in polyamine depletion-induced cytostasis[END_REF].

Here we investigated the differences between the stereoisomers of MeSpd and Me 2 Spm in their cellular uptake and their ability to regulate the key enzymes of polyamine metabolism. Our results revealed significant differences between the stereoisomers on the major known polyamine feedback regulatory mechanisms which control intracellular polyamine levels.

EXPERIMENTAL Materials

Human epithelial kidney cell line (HEK293) and human prostate carcinoma cell line (DU145) were obtained from American Type Culture Collection. Nontransgenic mouse primary foetal fibroblasts were prepared as previously published [START_REF] Alhonen | Correlation of polyamine and growth responses to N1,N11-diethylnorspermine in primary fetal fibroblasts derived from transgenic mice overexpressing spermidine/spermine N1-acetyltransferase[END_REF]. The optical isomers of α-methylspermidine (MeSpd) and α,ωbismethylspermine (Me 2 Spm) were synthesized as described in [START_REF] Grigorenko | Synthesis of novel optical isomers of alpha-methylpolyamines[END_REF]. Difluoromethylornithine (DFMO) was obtained from ILEX oncology Inc. [ 14 C]-labelled spermine tetrahydrochloride (specific activity 112 mCi/mmol), spermidine trihydrochloride (specific activity 113 mCi/mmol) and carboxyl-S-adenosyl-Lmethionine (specific activity 54 mCi/mmol) were from GE Healthcare.

Cell culture HEK293 cells were cultured in DMEM, 10 % FBS at +37°C and 5 % CO 2 . Primary fetal fibroblasts and DU145 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Sigma) supplemented with 10 % heat-inactivated FBS (Gibco) and 50 µg/ml gentamycin (Gibco), and incubated in humidified atmosphere +37 °C, 10 % CO 2 . When natural polyamines were used, 1 mM aminoguanidine was included to prevent the oxidation of Spd and Spm by serum amine oxidases. Analog concentration of 100 µM was used to effectively replace the natural polyamines with the analogs. The cells were harvested by trypsinization, washed with PBS, pelleted and stored in -70 °C. The cell number was measured electronically with Coulter Counter model Z1. Cell pellets were lysed in buffer (20 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.1 % Triton X-100, 1 mM dithiothreitol, Complete EDTA-free (Roche)) and incubated for 20 min on ice. For AdoMetDC activity assay, the cell samples were lysed in buffer containing 50 mM potassium phosphate buffer pH 7.2, 0.1 mM EDTA, 0.1 % Triton X-100 and 1 mM dithiothreitol. Samples for polyamine measurement were taken and the rest of the lysate was centrifuged for 12 000 x g 20 min +4 °C. The supernatant fraction was used for enzymatic assays of SSAT, ODC and AdoMetDC. Total protein concentration was measured using Coomassie Brilliant Blue (Bio Rad) with dilutions of bovine serum albumin (Sigma) as standards.

Enzyme activities and polyamines

Intracellular polyamines, polyamine analogs and nucleosides were measured with HPLC according to the published method [START_REF] Hyvönen | Monitoring of the uptake and metabolism of aminooxy analogues of polyamines in cultured cells by high-performance liquid chromatography[END_REF]. The amount of DNA was measured from pellets of polyamine samples according to the method of Giles and Myers [START_REF] Giles | An improved diphenylamine method for the estimation of deoxyribonucleic acid[END_REF]. ODC and SSAT activities were measured as described earlier [START_REF] Jänne | On the purification of L-ornithine decarboxylase from rat prostate and effects of thiol compounds on the enzyme[END_REF][START_REF] Bernacki | Antitumor activity of N,N'bis(ethyl)spermine homologues against human MALME-3 melanoma xenografts[END_REF]. AdoMetDC activity was measured by a similar method as ODC activity, using reaction buffer consisting of 50 mM potassium phosphate buffer pH 7.4, 1.25 mM dithiothreitol, 3 mM putrescine, 0.2 mM S-adenosyl-L-methionine and 25 µCi/ml [ 14 C-carboxyl]-S-adenosyl-L-methionine.

Alternative splicing of SSAT

Primary mouse foetal fibroblasts were plated on 6-well plates and grown overnight. Fresh medium containing 10 µg/ml cycloheximide was changed. After an hour, polyamine analogs (100 µM) were added, and the cells were further incubated for 7 h. RNA extraction, DNase-treatment, cDNA synthesis and quantitative RT-PCR were carried out as previously published [START_REF] Hyvönen | Polyamine-regulated unproductive splicing and translation of spermidine/spermine N1-acetyltransferase[END_REF]. A c c e p t e d M a n u s c r i p t DU145 cells were plated onto 6-well plates at density of 0.3 × 10 6 cells/well. After overnight incubation, pre-warmed serum-free medium supplemented with 10 µM [ 14 C]-Spd or [ 14 C]-Spm (specific activity 25 mCi/mmol) and competing polyamine analogs (1-100 µM) was changed. After 10-min incubation, the medium was aspirated, the plates washed twice with ice-cold PBS and the cells lysed in 500 µl of 0.5 M NaOH. Four hundred µl of the lysate was mixed with 3 ml of Optiphase HiSafe scintillation cocktail (Perkin Elmer) and counted by liquid scintillation counter (1450 Microbeta PLUS, Wallac). Total protein concentration was measured from the lysate using Coomassie Brilliant Blue (Bio-Rad).

Antizyme frameshifting in HEK293 cells and in vitro

Frameshifting assay was essentially carried out as described in [START_REF] Petros | Polyamine analogs with xylene rings induce antizyme frameshifting, reduce ODC activity, and deplete cellular polyamines[END_REF][START_REF] Grentzmann | A dualluciferase reporter system for studying recoding signals[END_REF]. In brief, HEK293 cells were plated at 7x10 3 cells/well of ½-area 96-well plate. After 48 hours culture in 2.5 mM DFMO the cells were transfected with 50 ng/well p2luc reporter plasmid [START_REF] Howard | Cell culture analysis of the regulatory frameshift event required for the expression of mammalian antizymes[END_REF] and 0.2 µl of Lipofectamine 2000. Reporter plasmid contains antizyme frameshifting sequences needed for polyamine induction between the Renilla and firefly luciferase genes. The firefly luciferase ORF lacks an initiation codon and can only be expressed as a fusion protein with Renilla luciferase if translational frameshifting occurs. After 12 hours, polyamine analogs, 1 mM aminoguanidine and 2.5 mM DFMO were added. The cells were lysed 24 hours later and Relative Light Units were measured following an autoinjection of the Dual Luciferase Reporter Assay reagents using a Veritas Luminometer (Turner Biosystems). In vitro frameshifting assays were carried out according to manufacturer's specifications using the TNT Quick Coupled Transcription/Translation system (Promega) in a final volume of 10 µl supplemented with varying concentrations of polyamine analogs. After incubating for 1 hour at 30 °C, reactions were diluted 10-fold in Passive Lysis Buffer supplied with the Dual Luciferase Reporter Assay (Promega) reagents. Frameshifting efficiency was calculated as a ratio of the firefly to Renilla luciferase activities standardized to an in-frame control.

AdoMetDC uORF translation in vitro

Plasmids containing AdoMetDC uORF-luciferase fusion construct or a similar construct where uORF initiation codon AUG was mutated to AUA, were a kind gift from Dr. David Morris (USA) [START_REF] Law | Polyamine regulation of ribosome pausing at the upstream open reading frame of S-adenosylmethionine decarboxylase[END_REF]. The plasmids were produced in E. coli, purified with QIAFilter Maxi Kit (Qiagen) and linearized with EcoRI. Capped mRNAs were synthesized using RiboMAX Large Scale RNA production system (T7) in vitro transcription kit (Promega) and ribo m 7 G cap analog (Promega) according to the manufacturer's instructions. After purification, in vitro translation reactions (25 µl) were carried out using Flexi rabbit reticulocyte lysate (Promega) and added polyamine (100 µM Spm and Me 2 Spm, 250 µM Spd and MeSpd). The endogenous spermidine and spermine levels in the lysate were 440 and 40 µM, respectively. Final Mg 2+ and K + concentrations in the reactions were 3.2 and 75 mM, respectively. Luminescence was measured as quadruplicates with Victor 2 Multilabel Counter (Wallac) from 5 µl of the lysate mixed with 100 µl of Luciferase assay reagent (Promega).

Statistical analysis

Values are means ± S.D. One-way analysis of variance (ANOVA) with Tuckey's post-hoc test was used for multiple comparisons with the aid of a software package, GraphPad Prism 4.03 (GraphPad Software Inc.). *, ** and *** refer to p values of < 0.05, < 0.01 and < 0.001, respectively.

RESULTS

Polyamine concentrations, ODC, AdoMetDC and SSAT activities

To examine the effect of the stereoisomers of MeSpd and Me 2 Spm on intracellular polyamine concentrations and on the enzyme activities of ODC, SSAT and AdoMetDC, DU145 prostate cancer cells were treated with 100 µM of the analogs for 72 hours, with or without 5 mM difluoromethylornithine (DFMO), an irreversible ODC inhibitor. As shown in Tables 1 and2 A c c e p t e d M a n u s c r i p t even better than DFMO. By contrast, the analogs only slightly induced SSAT activity, Sand S,S-isomers being the most potent ones. All the stereoisomers accumulated in the cells to concentrations near that of the natural polyamines, and decreased the intracellular levels of natural polyamines, especially when used in combination with DFMO. S-MeSpd and S,S-Me 2 Spm accumulated in somewhat higher intracellular concentration than the other stereoisomers, and most effectively depleted the natural polyamines. When different analog concentrations (1, 10 or 100 µM) were tested, it was found that the higher the analog concentration, the more efficient reduction of the natural polyamines (data not shown). As we reported previously [START_REF] Hyvönen | Role of hypusinated eukaryotic translation initiation factor 5A in polyamine depletion-induced cytostasis[END_REF], all analogs supported cell growth during DFMO-induced polyamine depletion for 3 days (data not shown). Of Me 2 Spm stereoisomers, only S,S was metabolized to MeSpd (Table 2), and both MeSpd enantiomers were metabolized to α-MeSpm by spermine synthase (Table 1). These α-MeSpm enantiomers can be then further converted to α-MeSpd and Spd by SMO, with S-MeSpm being preferred to R-MeSpm (data not shown).

Table 3 depicts AdoMetDC activity and the levels of S-adenosylmethionine (SAM) and decarboxylated S-adenosylmethionine (deSAM) in DU145 cells treated with DFMO and the stereoisomers or natural polyamines for 24, 48 and 72 hours. As expected, DFMO caused an induction of the enzyme activity, and led to accumulation of deSAM, and the treatment with natural polyamines or their analogs suppressed the enzyme activity and reduced the amount of deSAM. Based on both activity and nucleoside data, S-MeSpd suppressed the enzyme activity more than R-MeSpm and R,R was the best suppressor of AdoMetDC activity among the Me 2 Spm stereoisomers. Table 3 also shows that the methylated analogs were not as efficient AdoMetDC suppressors as their corresponding natural polyamines.

Uptake of the stereoisomers

The ability of the stereoisomers to compete for uptake with the natural polyamines was investigated in DU145 cells by 10-min competition assays with [ 14 C]-labelled spermidine or spermine (Figure 1). In such a short time, the antizyme does not downregulate the polyamine uptake. S-MeSpd and S,S-Me 2 Spm were clearly least effective competitors. Similar results were obtained with mouse primary fetal fibroblasts (data not shown). Since we found differences in the ability of the stereoisomers to inhibit ODC activity, we hypothesized that the stereoisomers might differently affect the synthesis of the functional antizyme by inducing ribosomal frameshifting on antizyme mRNA. Therefore, antizyme-dependent uptake of the stereoisomers was studied by blocking antizyme expression by pre-incubating DU145 cells with a translation inhibitor, cycloheximide (CHX). After 1 hour, the analogs were added for further 2 h and the intracellular concentrations of the analogs were measured with HPLC. As shown in Figure 2, without CHX treatment there were no differences in the accumulation of the distinct stereoisomers of MeSpd or Me 2 Spm. By contrast, when CHX was added, Sand S,S-isomers accumulated in higher concentrations than the others, suggesting that these stereoisomers more effectively induce antizyme expression.

ODC antizyme frameshifting

To investigate in more detail whether Sand S,S-isomers induce antizyme frameshifting more efficiently than the other isomers, HEK293 cells were transfected with a dual luciferase reporter plasmid containing the antizyme sequences required for polyamine induced frameshifting between the Renilla and firefly luciferase genes [START_REF] Howard | Cell culture analysis of the regulatory frameshift event required for the expression of mammalian antizymes[END_REF]. The firefly luciferase ORF lacks an initiation codon and can only be expressed as a fusion protein with Renilla luciferase if translational frameshifting occurs. Frameshifting efficiency was calculated as a ratio of the firefly to Renilla luciferase activities standardized to an in-frame control. Figure 3a indicates that S-MeSpd and S,S-Me 2 Spm were more potent than the other stereoisomers at inducing antizyme frameshifting. To rule out the possibility that the better frameshifting efficiency was caused by higher intracellular analog concentrations, frameshifting was also tested in rabbit reticulocyte lysate. As depicted in Figure 3b, differences existed between the stereoisomers, S-MeSpd and S,S-Me 2 Spm again being more efficient than the others. Since the stereoisomers differently affected SSAT activity, we next tested whether there were differences between the stereoisomers in their ability to regulate the alternative splicing of SSAT pre-mRNA. Mouse primary foetal fibroblasts were first exposed to the protein synthesis inhibitor CHX in order to block the NMD pathway and rapid degradation of the unproductive splice variant. After 1 hour, polyamine analogs were added, and incubation was continued for 7 hours. Figure 4 shows the amount of the unproductive splice variant (SSAT-X) relative to total SSAT mRNA (SSAT + SSAT-X) as measured by quantitative RT-PCR. The treatment with CHX led to marked accumulation of the unproductive variant due to inhibition of NMD, and S,S-Me 2 Spm and S-MeSpd were the most effective isomers in decreasing the generation of the unproductive variant of SSAT.

Alternative

Translational regulation of AdoMetDC in vitro

The expression of AdoMetDC is regulated mainly through translation of its uORF, where polyamines suppress the translation of downstream AdoMetDC protein [START_REF] Law | Polyamine regulation of ribosome pausing at the upstream open reading frame of S-adenosylmethionine decarboxylase[END_REF]. Since our cell culture data showed differences in the AdoMetDC activity and deSAM level between the stereoisomers, we further investigated whether they differently affected the translation of AdoMetDC uORF. We translated uORFluciferase fusion construct in rabbit reticulocyte lysate with or without added natural polyamines or the analogs. As expected, all polyamines suppressed the translation of the fusion construct (Figure 5). However, marked differences existed between the tested compounds, the natural polyamines being better suppressors than the analogs. In line with our cell culture data we found that S-MeSpd and R,R-Me 2 Spm were significantly more efficient suppressors of AdoMetDC uORF translation than the other stereoisomers.

DISCUSSION

Polyamines participate in several important cellular processes such as cell cycle regulation, differentiation, apoptosis, protein synthesis and modulation of ion channels. Some of those processes require covalent binding of polyamines to proteins, of which a well-known example is the hypusination of eukaryotic translation initiation factor 5A (eIF5A) protein [START_REF] Park | Identification of hypusine, an unusual amino acid, in a protein from human lymphocytes and of spermidine as its biosynthetic precursor[END_REF]. Other polyamine effects are mediated through their ionic interactions with cellular anionic sites. The difference between polyamines and inorganic cations such as Mg 2+ is that the charges are distributed along a conformationally flexible carbon backbone, allowing them to interact with various targets, especially the polynucleotides RNA and DNA. Through these interactions, the polyamines influence the expression of many important genes, among which are the genes involved in their own metabolism.

Our present data revealed significant differences between the different stereoisomers of MeSpd and Me 2 Spm in their ability to regulate several components of polyamine homeostasis, such as uptake and the expression of the rate-limiting biosynthetic and catabolic enzymes, ODC, AdoMetDC and SSAT. Earlier studies have indicated that polyamines and their close structural analogs induce +1 ribosomal frameshifting in ODC antizyme mRNA [START_REF] Matsufuji | Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme[END_REF][START_REF] Balasundaram | Spermidine deficiency increases +1 ribosomal frameshifting efficiency and inhibits Ty1 retrotransposition in saccharomyces cerevisiae[END_REF][START_REF] Petros | Polyamine analogs with xylene rings induce antizyme frameshifting, reduce ODC activity, and deplete cellular polyamines[END_REF][START_REF] Mitchell | Antizyme induction by polyamine analogues as a factor of cell growth inhibition[END_REF]. Our results from uptake and frameshifting studies show that Sand S,S-isomers were more effective than the other stereoisomers of MeSpd and Me 2 Spm at inducing antizyme frameshifting, and the results from in vitro frameshifting are in line with the cell culture studies. Furthermore, Sand S,S-stereoisomers were the most efficient in downregulating ODC activity. In fact, S,S-Me 2 Spm alone was even more potent in decreasing ODC activity than the inhibitor DFMO.

Since antizyme is known not only to promote ODC degradation but also to inhibit polyamine uptake [START_REF] Mitchell | Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells[END_REF][START_REF] Suzuki | Antizyme protects against abnormal accumulation and toxicity of polyamines in ornithine decarboxylaseoverproducing cells[END_REF][START_REF] Sakata | Properties of a polyamine transporter regulated by antizyme[END_REF], we examined the uptake and intracellular accumulation of these analogs. In 10-min uptake experiments, where the analogs apparently did not affect the amount of antizyme, S-MeSpd and S,S-Me 2 Spm were found to be the least efficient inhibitors of the uptake of Spd and Spm, respectively. However, unexpectedly, after longer time of culture Sand S,S-stereoisomers accumulated at slightly higher intracellular concentrations than Rand R,R-isomers. When the concentrations of the added analog as well as its metabolic products were added together (Tables 1 and2), the differences between the stereoisomers were smaller, but still present. The reason why Sand S,S-isomers induced antizyme frameshifting, but still accumulated at higher concentration than the other isomers, is not understood. The analogs may divergently modulate some yet unidentified cellular regulatory systems.

The mechanisms of polyamine-mediated regulation of the key enzymes of polyamine metabolism are currently not fully understood. It is possible that polyamines directly interact with specific site(s) of target mRNA(s). This view is supported by the work of Higashi and colleagues, who recently found that spermidine binds to and induces a structural change in a bulged-out region of double-stranded RNA, and that the effect is different than that induced by Mg 2+ [START_REF] Higashi | Selective structural change of bulged-out region of double-stranded RNA containing bulged nucleotides by spermidine[END_REF]. However, it has been also suggested that polyamines may modulate frameshifting through interactions with the translational machinery, such as tRNAs or the ribosome, and further detailed mechanistic studies are needed to clarify this point.

The interpretation of our cell culture results is complicated by the fact that the intracellular concentrations of S-MeSpd and S,S-Me 2 Spm were somewhat higher than those of the other isomers. However, this observation must be interpreted cautiously as the intracellular localization and the level of free analog can not readily be determined. To overcome these technical problems, we used in vitro systems to verify the results from cell culture experiments, which indicated that differences existed between the stereoisomers. Our view that the regulation is due to the stereospecificity is also supported by our recent findings, that the stereoisomers of α-methylpolyamines significantly differ in their abilities to condense DNA and protect it from hydrogen peroxide-induced oxidative stress in in vitro systems where no endogenous polyamines are present (Dr. TJ Thomas and Dr. MT Hyvönen, unpublished).

Earlier publications indicate that some polyamine metabolic enzymes are stereospecific for their substrates, such as spermidine synthase [START_REF] Pankaskie | Inhibitors of polyamine biosynthesis. 9. Effects of S-adenosyl-L-methionine analogues on mammalian aminopropyltransferases in vitro and polyamine biosynthesis in transformed lymphocytes[END_REF] and ODC [START_REF] Qu | Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine[END_REF]. The use of novel chiral α-methylated polyamines led to the discovery of a hidden stereospecificity for polyamine oxidase [START_REF] Järvinen | Alpha-methyl polyamines: Efficient synthesis and tolerance studies in vivo and in vitro. first evidence for dormant stereospecificity of polyamine oxidase[END_REF], spermine oxidase and deoxyhypusine synthase [START_REF] Hyvönen | Role of hypusinated eukaryotic translation initiation factor 5A in polyamine depletion-induced cytostasis[END_REF]. In addition, others have reported that deoxyhypusine hydroxylase [START_REF] Byers | Cytostasis induced in L1210 murine leukaemia cells by the S-adenosyl-L-methionine decarboxylase inhibitor 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine may be due to hypusine depletion[END_REF][START_REF] Byers | Effects of chronic 5'-([(Z)-4-amino-2butenyl]methylamino)-5'-deoxy-adenosine (AbeAdo) treatment on polyamine and eIF-5A metabolism in AbeAdo-sensitive and -resistant L1210 murine leukaemia cells[END_REF] and similarly spermine synthase [START_REF] Pegg | Role of unsaturated derivatives of spermidine as substrates for spermine synthase and in supporting growth of SV-3T3 cells[END_REF] show stereocontrol for unsaturated spermidine analogs.

A vast number of polyamine analogs, of which most are achiral, have been synthesized for studying the physiological roles of polyamines and developing drugs for the treatment of cancer and parasitic diseases. Among these compounds are N-alkylated, C-alkylated, unsaturated, fluorinated and aminooxy analogs [START_REF] Pegg | Use of aminopropyltransferase inhibitors and of non-metabolizable analogs to study polyamine regulation and function[END_REF]. The increasing knowledge about polyamine metabolism and their physiological roles has opened up possibilities to develop potent antimetabolites such as the cytotoxic N-alkylated analog N 1 N 11diethylnorspermine (DENSPM), which is currently undergoing trials for the chemotherapy of colon carcinoma. One key issue in the drug development is acceptable host toxicity, since total polyamine depletion is difficult to achieve and also may be highly toxic to host. An important goal is to find differences in polyamine metabolism and cellular targets between the host and parasite, or normal and malignant tissue, which can be used to selectively target the drugs. Functional polyamine mimetics with diverse effects on polyamine metabolism may offer the means to selectively regulate key metabolic pathways. Here we show that the optical isomers of α-methylated polyamine analogs are promising examples of this approach, by providing evidence that their metabolism and biological effects can be modulated by relatively minor changes such as the configuration of the chiral center. For example, S-MeSpd supports hypusine synthesis in DU145 cells during prolonged polyamine depletion with DFMO whereas R-MeSpd does not [START_REF] Hyvönen | Role of hypusinated eukaryotic translation initiation factor 5A in polyamine depletion-induced cytostasis[END_REF]. Thus, the combined use of DFMO and R-MeSpd may offer practical means to achieve eIF5A-dependent cytostatic response in vivo. In further analog development studies the size, position and chemical nature of substituents can be altered to achieve additional novel biological or metabolic properties for research and/or therapeutic use.

Uptake competition experiments 3 Biochemical

 3 Journal Immediate Publication. Published on 12 Jun 2009 as manuscript BJ20090737 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090737

  , S-MeSpd and S,S-Me 2 Spm were the most effective stereoisomers to inhibit ODC activity. In fact, S,S-Me 2 Spm downregulated ODC activity 4 Biochemical Journal Immediate Publication. Published on 12 Jun 2009 as manuscript BJ20090737 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090737

splicing of SSAT 5 Biochemical

 5 Journal Immediate Publication. Published on 12 Jun 2009 as manuscript BJ20090737 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090737 A c c e p t e d M a n u s c r i p t

6

  Biochemical Journal Immediate Publication. Published on 12 Jun 2009 as manuscript BJ20090737 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090737 A c c e p t e d M a n u s c r i p t

8Figure 1

 1 FIGURE LEGENDS

Figure 2

 2 Figure 2 Effect of antizyme on the uptake of the MeSpd and Me 2 Spm stereoisomers. DU145 cells were incubated with or without cycloheximide (CHX, 10 µg/ml) for 1h, after which the analogs (100 µM) were added, and incubation continued for 2h. No metabolism of the analogs were detected by HPLC in 2 h. Data are means ± S.D., n=3.

Figure 3

 3 Figure 3 Induction of ODC antizyme frameshifting by the stereoisomers of MeSpd and Me 2 Spm.(a) HEK293 cells were pretreated for 48h with 2.5 mM DFMO and transfected with dual luciferase reporter plasmid containing antizyme frameshift site. Polyamines and analogs, 2.5 mM DFMO and 1 mM aminoguanidine were added 12h after transfection and the cells collected 24 hours later. The analogs were tested at 5 different concentrations; the results from experiments performed with optimal concentrations for frameshifting by MeSpd and Me 2 Spm are shown (1.25 µM and 2.5 µM, respectively). Data are means ± S.D., n=10-13. (b) Dual luciferase reporter plasmid was translated in rabbit reticulocyte lysate using optimal concentrations of MeSpd (400 µM) and Me 2 Spm (100 µM). Results are means ± S.D., n=12 from three independent experiments, statistical analysis was done using Student's t-test.

Figure 4

 4 Figure 4 Effect of the stereoisomers of MeSpd and Me 2 Spm on the alternative splicing of SSAT pre-mRNA.Primary mouse foetal fibroblasts were treated with protein synthesis inhibitor cycloheximide (CHX, 10 µg/ml) for 8 h, of which the last 7 h with the stereoisomers (100 µM). The relative expression level of SSAT-X to total SSAT mRNA was analyzed by qRT-PCR. Data are means ± S.D., n=3, statistical comparisons are to CHX-treated cells.

Figure 5

 5 Figure 5 Ability of the natural polyamines and the stereoisomers of MeSpd and Me 2 Spm to suppress AdoMetDC uORF translation in vitro.A fusion construct containing AdoMetDC uORF and luciferase reporter was translated in rabbit reticulocyte lysate in the absence or presence of added polyamines and analogs (Spd and MeSpd 250 µM, Spm and Me 2 Spm 100 µM). The expression levels were compared to a control plasmid (=100 %) where the initiation codon of uORF was mutated to AUA (direct translation of luciferase). Data are expressed as means ± S.D., n=6.

Figure 1 . 15 Biochemical

 115 Figure 1.

Figure 2 . 16 Biochemical

 216 Figure 2.

Figure 3 17

 3 Figure 3

Figure 4 18

 4 Figure 4

Figure 5 19

 5 Figure 5

  Biochemical Journal Immediate Publication. Published on 12 Jun 2009 as manuscript BJ20090737 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090737

Table 1 Effect of MeSpd stereoisomers on ODC and SSAT activities and intracellular polyamine pools in DU145 cells after 72 h treatment DU145

 1 cells were treated with 100 µM analogs with and without 5 mM DFMO for 72 hours. N 1 -acetylspermidine and N 1 -acetylspermine levels were below detection limit. Results are means ± S.D., n=3. n.d., not detectable; rac, racemic.

	Treatment	ODC	SSAT	Pu	Spd	Spm	MeSpd	MeSpm	MeSpd+
									MeSpm
		(pmol/h/µg DNA)	(pmol/10 min/ µg DNA)			(pmol/µg DNA)
	Control	14.56 ± 0.14	11.2 ± 0.3	123 ± 4	464 ± 8	201 ± 2
	Rac	0.40 ± 0.06	12.8 ± 0.3	n.d.	40 ± 1	167 ± 15	413 ± 23	100 ± 14	513
	R	0.74 ± 0.05	11.2 ± 0.4	n.d.	45 ± 1	177 ± 5	375 ± 9	126 ± 17	501
	S	0.46 ± 0.03	14.7 ± 0.4	n.d.	32 ± 0	154 ± 3	477 ± 10	80 ± 9	557
	DFMO	0.14 ± 0.01	11.4 ± 0.0	n.d.	21 ± 3	195 ± 10
	DFMO+Rac	0.06 ± 0.01	13.1 ± 1.3	n.d.	< 5	50 ± 1	599 ± 10	180 ± 5	779
	DFMO+R	0.08 ± 0.01	17.0 ± 9.4	n.d.	n.d.	45 ± 2	554 ± 18	191 ± 15	745
	DFMO+S	0.04 ± 0.01	22.6 ± 7.0	n.d.	< 5	57 ± 3	650 ± 11	112 ± 9	762

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Table 2 Effect of Me 2 Spm stereoisomers on ODC and SSAT activities and intracellular polyamine pools in DU145 cells after 72 h treatment DU145

 2 cells were treated with 100 µM analogs with and without 5 mM DFMO for 72 hours. N 1 -acetylspermidine and N 1 -acetylspermine levels were below detection limit. Results are means ± S.D., n=3. n.d., not detectable; rac, racemic.

		ODC	SSAT	Pu	Spd	Spm	MeSpd	Me 2 Spm	MeSpd + Me 2 Spm
	Treatment	(pmol/h/µg DNA)	(pmol/10 min/ µg DNA)				(pmol/µg DNA)
	Control	5.99 ± 0.46	11.5 ± 1.5	139 ± 7 402 ± 18	251 ± 20
	Rac	1.06 ± 0.12	12.6 ± 1.7	23 ± 2	78 ± 7	65 ± 3	17 ± 3	480 ± 21	497
	R,R	1.58 ± 0.03	12.4 ± 0.1	34 ± 2	99 ± 7	62 ± 4	n.d.	474 ± 29	474
	S,S	0.05 ± 0.00	24.0 ± 2.3	n.d.	11 ± 0	26 ± 2	74 ± 6	551 ± 51	625
	R,S	0.92 ± 0.09	14.6 ± 0.7	19 ± 2	79 ± 2	55 ± 3	n.d.	545 ± 24	545
	DFMO	0.17 ± 0.04	10.7 ± 1.0	n.d.	15 ± 4	205 ± 7
	DFMO+Rac	0.12 ± 0.02	14.1 ± 0.3	n.d.	6 ± 1	10 ±1	15 ± 2	551 ± 37	566
	DFMO+R,R	0.15 ± 0.01	12.3 ± 0.0	n.d.	8 ± 1	10 ± 1	n.d.	573 ± 13	573
	DFMO+S,S	0.03 ± 0.02	22.2 ± 2.5	n.d.	< 5	12 ± 0	75 ± 2	557 ± 4	632
	DFMO+R,S	0.14 ± 0.01	14.6 ± 0.4	n.d.	< 5	10 ± 1	n.d.	594 ± 14	594

Biochemical Journal Immediate Publication. Published on 12 Jun 2009 as manuscript BJ20090737 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090737 A c c e p t e d M a n u s c r i p t

Table 3 Ability of the natural polyamines and the stereoisomers of MeSpd and Me 2 Spm to suppress AdoMetDC activity and to affect intracellular nucleoside (deSAM, SAM) levels in DU145 cells DU145

 3 cells were cultured with 5 mM DFMO and 100 µM analogs for 24, 48 and 72 h. All plates contained also 1 mM aminoguanidine to prevent the oxidation of Spd and Spm by serum amine oxidases. Data are expressed as means ± S.D., n=3. n.d., not detectable. Biochemical Journal Immediate Publication. Published on 12 Jun 2009 as manuscript BJ20090737 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090737

			AdoMetDC			deSAM			SAM
	Treatment	(pmol/h/10 6 cells)		(pmol/10 6 cells)			(pmol/10 6 cells)
		24h	48 h	72 h	24h	48h	72h	24h	48h	72h
	Control	879 ± 11	518 ± 39	569 ± 59	n.d.	n.d.	n.d.	108 ± 9 157 ± 12 189 ± 9
	DFMO	1589 ± 165 2149 ± 114 7244 ± 44 11.5 ± 0.4 160.3 ± 13.7 96.1 ± 14.9 103 ± 5 128 ± 4 128 ± 16
	DFMO+Spd	1262 ± 63	843 ± 27	1600 ± 35	n.d.	10.2 ± 0.4	n.d.	145 ± 12 170 ± 20 219 ± 26
	DFMO+R-MeSpd	1818 ± 52 1103 ± 36 2163 ± 82 11.3 ± 1.0 43.9 ± 5.3	5.0 ± 0.9 154 ± 23 195 ± 25 299 ± 32
	DFMO+ S-MeSpd	1495 ± 65 1003 ± 42 2072 ± 151	n.d.	28.5 ± 2.3	2.4 ± 0.3 168 ± 34 205 ± 22 251 ± 55
	DFMO+Spm	602 ± 3	346 ± 17	184 ± 21	n.d.	n.d.	n.d.	146 ± 19 168 ± 20 209 ± 34
	DFMO+R,R-Me 2 Spm 639 ± 54	336 ± 25	251 ± 15	n.d.	n.d.	0.4 ± 0.2 155 ± 19 192 ± 16 232 ± 15
	DFMO+S,S-Me 2 Spm	769 ± 34	468 ± 40	406 ± 35	n.d.	5.9 ± 0.2	1.5 ± 0.1 136 ± 13 209 ± 36 227 ± 14
	DFMO+R,S-Me 2 Spm	742 ± 39	449 ± 23	394 ± 61	n.d.	9.1 ± 0.9	0.7 ± 0.3 180 ± 23 254 ± 4 260 ± 12

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

This manuscript is dedicated to the memory of Professor Juhani Jänne. The authors warmly thank Dr. David Morris (USA) for providing AdoMetDC uORF-luciferase fusion constructs, and Mrs Anne Karppinen, Mrs Arja Korhonen and Mrs Tuula Reponen (University of Kuopio, Finland) for skilful technical assistance.

Foundation for Basic Research [grant number 08-04-91777].

FUNDING 7 Biochemical Journal Immediate Publication. Published on 12 Jun 2009 as manuscript BJ20090737 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090737


Stefano Donini

Manuela Ferrari

Chiara Fedeli

Marco Faini

Ilaria Lamberto

Ada Serena Marletta

Lara Mellini

Michela Panini

Riccardo Percudani

Loredano Pollegioni

Laura Caldinelli

Stefania Petrucco

Alessio Peracchi
email: peracchi@unipr.it

Recombinant production of eight human cytosolic aminotransferases and assessment of

Keywords: pyridoxal-phosphate, glyoxylate, transaminase, hyperoxaluria

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

INTRODUCTION

Primary hyperoxaluria type 1 (PH1) is an autosomal-recessive inborn disease occurring in 1 per 120,000 live births [START_REF] Cochat | Primary hyperoxaluria type 1[END_REF]. It is caused by a deficiency of the enzyme alanine:glyoxylate aminotransferase (AGXT), which normally localizes to the peroxisomes in hepatocytes [START_REF] Cochat | Primary hyperoxaluria type 1[END_REF][START_REF] Danpure | Molecular aetiology of primary hyperoxaluria type 1[END_REF][START_REF] Coulter-Mackie | Genetic heterogeneity in primary hyperoxaluria type 1: impact on diagnosis[END_REF]. AGXT uses pyridoxal phosphate (PLP) as a cofactor [START_REF] Zhang | Crystal structure of alanine:glyoxylate aminotransferase and the relationship between genotype and enzymatic phenotype in primary hyperoxaluria type 1[END_REF] and catalyzes the nearly-irreversible transfer of the amino group from alanine to glyoxylate, to yield glycine and pyruvate [START_REF] Thompson | Isolation and characterization of an L-alanine: glyoxylate aminotransferase from human liver[END_REF][START_REF] Cellini | Human wild-type alanine:glyoxylate aminotransferase and its naturally occurring G82E variant: functional properties and physiological implications[END_REF] (Figure 1).

In PH1 patients, glyoxylate cannot be efficiently converted to glycine and is instead oxidized to oxalate, leading to hyperoxalemia and hyperoxaluria. In turn, this causes the deposition of insoluble calcium oxalate in the kidney and in other tissues, leading to nephrolithiasis, nephrocalcinosis, systemic tissue damage and eventually kidney failure [START_REF] Cochat | Primary hyperoxaluria type 1[END_REF][START_REF] Coulter-Mackie | Genetic heterogeneity in primary hyperoxaluria type 1: impact on diagnosis[END_REF][START_REF] Danpure | Molecular etiology of primary hyperoxaluria type 1: new directions for treatment[END_REF].

Since the human organism produces a variety of aminotransferases, and since these enzymes often show a broad substrate specificity [START_REF] Mehta | The molecular evolution of pyridoxal-5'phosphate-dependent enzymes[END_REF], it is somewhat surprising that other transaminases may not functionally compensate for the deficiency of AGXT. As a matter of fact, another alanine:glyoxylate aminotransferase (known as AGXT2; [START_REF] Lee | Molecular cloning and sequencing of a cDNA encoding alanine-glyoxylate aminotransferase 2 from rat kidney[END_REF]) is found in mammalian liver, but this enzyme localizes solely into the mitochondria [10] and from this subcellular compartment it cannot surrogate the function of AGXT. Furthermore, several pre-genomic studies suggest that other enzymes occurring in the liver of mammals can convert glyoxylate to glycine employing amino acids other than alanine as cosubstrates. For example, an aminotransferase that apparently prefers glutamate as amino-group donor has been purified from human liver [START_REF] Thompson | Isolation and characterization of a glutamate-glycine transaminase from human liver[END_REF]. Another glyoxylate transaminase that uses preferably aromatic amino acids as cosubstrates has been obtained from the liver of rodents and monkeys [START_REF] Harada | Purification and characterization of aromatic-amino-acid-glyoxylate aminotransferase from monkey and rat liver[END_REF] while an enzyme with glutamine-glyoxylate transaminase activity was isolated from rat liver [START_REF] Cooper | Isolation and properties of highly purified glutamine transaminase[END_REF]. All these activities are attributable to PLP-dependent enzymes, but none of these catalysts has ever been characterized at a molecular level, and their subcellular localization is not known.

Today, the availability of the complete human genome sequence offers an opportunity to clarify the role of different aminotransferases in the metabolism of glyoxylate. The genes coding for aminotransferases can be identified based on homology criteria and their overall number is relatively modest [START_REF] Percudani | A genomic overview of pyridoxalphosphate-dependent enzymes[END_REF]. Understanding which of these gene products can use glyoxylate as an amino-group acceptor would integrate the earlier biochemical literature and perhaps suggest new routes to the treatment of primary hyperoxaluria type 1. In fact, should some aminotransferase prove capable of efficiently converting glyoxylate to glycine, then stimulation of this enzyme (by means of drugs, or simply by providing a dietary supplement of the preferred amino-group donor) could represent a viable therapy to AGXT deficiency.

Herein, we report the cloning and recombinant expression of eight cytosolic aminotransferases and a preliminary assessment of their efficiencies and cosubstrate preferences in the transamination of glyoxylate.

Enzymes and chemicals

Recombinant glycine oxidase (GO, EC 1.4.2.19) from Bacillus subtilis was expressed in Escherichia coli and purified as described [15]. The final enzyme preparation showed a specific activity of ~0.7 U/mg protein on glycine as substrate. Rabbit muscle lactate dehydrogenase (LDH) was purchased from Fluka; malate dehydrogenase from pig heart, glutamate dehydrogenase from bovine liver and horseradish peroxidase were from Sigma. All the chemicals were of the highest commercial purity available.

Molecular cloning

A plasmid carrying the AGXT coding sequence (NCBI accession n. X53414) was kindly provided by Prof. C.J. Danpure (University College London). Clones of the complete cDNAs for the other aminotransferases were obtained from ImaGenes (Berlin, Germany). The full-length coding sequences were PCR-amplified from these clones, using the PfuUltra High Fidelity Taq polymerase (Stratagene) or the Deep Vent DNA polymerase (New England Biolabs). The ID of the clones and the genespecific PCR primers (purchased from MWG Biotech, Ebersberg, Germany) are listed in Table 1. All the primers bore 5'-tails such that the amplification products contained CpoI target sequences (uppercase letters) near to both ends.

A schematic description of the molecular cloning strategy is outlined in Figure 2. Amplicons were blunt-end cloned into an intermediate vector (usually pBlueScript II KS+ [Stratagene] treated with SmaI) using published protocols [START_REF] Bolchi | A general one-step method for the cloning of PCR products[END_REF]. Ligated plasmids were then transformed into XL1B E. coli cells (Stratagene) and the inserts were sequence-verified. The plasmids were subsequently treated with CpoI, to extract the fragments corresponding to the amplified full-length cDNAs, ready for subcloning into the expression vector pET28-CpoI.

This plasmid is a derivative of pET28 (Novagen) modified to present a single CpoI restriction site in the cloning region, downstream to a sequence encoding a hexahistidine tag (Angelo Bolchi, unpublished; Supplementary Figure 1). CpoI cleaves its target sequence (CGGTCCG/CGGACCG) after the first CG, generating two nonidentical 3' overhangs (GTC on one strand and GAC on the other). Exploiting these features, the CpoI-cleaved fragments were cloned in-frame and directionally into pET28-CpoI.

Heterologous gene expression and protein purification

The expression vectors carrying the cloned inserts were transformed into BL21-CodonPlus®-RIL (Stratagene) or BL21 Star® (Invitrogen) E. coli cells.

The general expression protocol was as follows. A subculture was used to inoculate a flask of broth containing 50 µg/ml kanamycin (for BL21-CodonPlus®-RIL cells, also 170 µg/ml chloramphenicol). Bacteria were grown at 37 °C in vigorous aeration and agitation, until the cell density reached the appropriate value (usually, OD 600 nm ∼0.6), at which point isopropyl β-D-1-thiogalactopyranoside was added to a final concentration of 1 mM. After further growth of the induced culture, cells were harvested by centrifugation and sonicated. Subsequently, the recombinant proteins present in the soluble lysate fraction were purified by immobilized metal affinity chromatography. The protein fractions were analyzed by gel electrophoresis and A c c e p t e d M a n u s c r i p t 5 those fractions with a purity > 90% were pooled, dialyzed against a buffer containing 4 µM PLP and 1 mM dithiothreitol, and stored at -80 °C.

Details on (i) the culture conditions adopted for the overexpression of individual proteins, (ii) the specific purification procedures used for each protein and (iii) the overall protein yields are provided in the Supplementary Data.

The concentration of each purified recombinant protein was estimated based on the extinction coefficient at 280 nm calculated by ProtParam (www.expasy.ch/tools/protparam.html). Activity of the recombinant enzymes (when their specific reactions were known) was assessed through established methods (See Supplementary Table 1).

Glycine oxidase (GO) assay

The kinetics of glycine production were determined by a discontinuous assay based on the use of GO. The principle of the assay is illustrated in Supplementary Figure 2. Briefly, glyoxylate (1-10 mM final concentration) and one or more potential amino-group donors were equilibrated at the desired reaction temperature in 25 mM potassium phosphate, pH 7.4 before starting the reaction with addition of the aminotransferase (0.05-3 µM final concentration).

Aliquots (20 µl each) were removed at appropriate intervals, and further reaction was stopped by mixing with 2 µl of 1 M phosphoric acid to reach pH ~2. These samples were then heated at 95 °C for 5 min to completely inactivate the aminotransferase. Each aliquot was subsequently mixed with 178 µl of a solution containing Tris-HCl pH 8.0 (100 mM final concentration), GO (4 µM final, corresponding to about 0.03 units), 3 units of horseradish peroxidase and 1 mM odianisidine, and incubated at 37 °C for 30 min (the time required to reach an endpoint). Finally, the mixtures were supplemented with 50 µl of sulfuric acid (7 N final concentration) to dissolve the occasional precipitates of oxidized o-dianisidine and augment the sensitivity of the assay [START_REF] Washko | Determination of glucose by an improved enzymatic procedure[END_REF], before measuring absorption spectra in the 530 nm region to quantitate the extent of o-dianisidine oxidation.

In parallel, a calibration line was prepared. 2-40 nmoles of glycine (from a 10 mM standard solution) were added to a reaction mixture containing 1 mM odianisidine, 4 µM GO, 15 units/ml horseradish peroxidase, 10 mM phosphoric acid and 100 mM Tris-HCl pH 8.0 in a final volume of 200 µl. The reaction mixtures were incubated and treated as above, and the absorbance at 530 nm vs. the initial amount of glycine was used to generate a calibration line. Control reactions were performed in which glyoxylate and the potential amino-group donors were incubated together, in the absence of transaminases; under these conditions, the amount of spontaneous glyoxylate transamination was negligible.

GO-based assay in a 96-well format

To screen for the ability of recombinant aminotransferases to produce glycine, using a variety of potential amino-group donors, the GO-based assay was also adapted to a 96-well microtiter format.

In each well, a single aminotransferase (1 µM) was incubated with glyoxylate (4 mM) and one or more potential amino-group donors (5-10 mM each) in a total volume of 40 µl. After 2.5 hours of incubation at 25 °C, the enzymes were inactivated by adding phosphoric acid to a final concentration of 90 mM (pH~2). Then, the reaction mixtures were supplemented with 180 µl of Tris-HCl 200 mM (pH 8) A c c e p t e d M a n u s c r i p t containing glycine oxidase (1 µM final), peroxidase (8 units/ml) and dianisidine (1 mM) and stored at 37°C for one hour. Finally 50 µl of sulfuric acid were added to each well and the plate was scanned with an automated plate reader.

Continuous spectrophotometric assays for alanine-glyoxylate, serine-glyoxylate and glutamate-glyoxylate transaminase activities

Alanine-glyoxylate, serine-glyoxylate and glutamate-glyoxylate transamination reactions were measured using a continuous spectrophotometric assay at 220 nm [18,[START_REF] Cooper | L-alanineglyoxylate aminotransferase II of rat kidney and liver mitochondria possesses cysteine S-conjugate β-lyase activity: a contributing factor to the nephrotoxicity/hepatotoxicity of halogenated alkenes?[END_REF]. At this wavelength, most α-keto acids possess an extinction coefficient about tenfold higher than glyoxylate (Supplementary Figure 3) so that their formation can be easily quantified [18].

Typically the reaction mixture (0.9 ml, final volume) contained 1-150 mM amino acid (L-Glu, L-Ala or L-Ser) and 1-10 mM glyoxylate in 25 mM potassium phosphate at pH 7.5. Reactions were started by adding the recombinant enzyme, and the increase in absorbance was monitored continuously using a Cary 400 double-beam spectrophotometer (Varian). Initial velocities were calculated graphically and expressed as µmoles of keto acid product (e.g., pyruvate) formed per minute, per mg of protein. The kinetic data were analyzed using Sigma Plot (Systat Software Inc.).

The kinetics of the glutamate-glyoxylate transamination (catalyzed by, e.g., the enzyme PSAT1) could also be monitored spectrophotometrically by a coupled assay, in which the recombinant enzyme (0.1-0.5 µM), glutamate and glyoxylate were incubated in the presence of glutamate dehydrogenase, NH 4 + and NADH. The reaction mixture (200 µl final volume) contained 25 mM potassium phosphate (pH 7.5), 32 mM ammonium acetate, 0.25 mM NADH and 15 units/ml glutamate dehydrogenase. The disappearance of NADH was monitored at 340 nm.

RESULTS

Identification, selection and cloning of the aminotransferase genes

An inventory of PLP-dependent enzymes encoded in the human genome [14] reveals 18 genes coding for aminotransferases -eight of which are predicted to localize in mitochondria. However, two arguments suggest that mitochondrial enzymes cannot compensate for the deficiency of peroxisomal AGXT. First, the existence of a mitochondrial AGXT isoenzyme (AGXT2) does not prevent the development of primary hyperoxaluria. Second, it is well known that PH1 is often caused by mutations that mistarget AGXT from peroxisomes to mitochondria [20,[START_REF] Danpure | Alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting in human hereditary kidney stone disease[END_REF]. Even though AGXT retains its catalytic function in the mitochondria, it is metabolically ineffective because it is prevented from acting on the peroxisomal glyoxylate pool [START_REF] Danpure | Alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting in human hereditary kidney stone disease[END_REF]. Instead, the glyoxylate produced in the peroxisomes can presumably be exported to the cytosol [22]. Thus, we focused in this study on the nine genes predicted to encode cytosolic transaminases, plus AGXT that was used as a positive control. The list of genes is shown in Table 1.

For all these genes, complete cDNAs were available from the IMAGE consortium; when multiple splice variants of a gene product were documented, we chose to clone the variant encoding the longest polypeptide. The coding sequences for the enzymes were then PCR-amplified from the cDNAs, subcloned, sequenceverified and finally cloned into E. coli expression vectors (Figure 2). The enzymes were overexpressed as His6-tagged proteins in E. coli cells, and purified by immobilized metal affinity chromatography (see Experimental). In most cases it was possible to obtain a good yield of soluble protein. However, production of the protein corresponding to the gene GOT1L1 encountered several problems: upon induction, multiple overexpressed protein species were detected in the cell lysate, and none of them could be purified by metal affinity chromatography. We thus decided not to pursue further the purification and characterization of this gene product.

The recombinant transaminases were soluble, could bind the PLP cofactor (as shown by their characteristic spectra; Supplementary Figure 4), reacted with potential substrates and could catalyze their specific reactions (when known) at rates comparable to those reported by other researchers (Supplementary Table 1).

Development and validation of an AGXT assay using glycine oxidase and peroxidase as coupling enzymes

To assess the formation of glycine by the various transaminases, we used a GO/peroxidase coupling system. GO catalyzes the conversion of glycine to glyoxylate and H 2 O 2 [15,23]. The latter is then used by peroxidase to oxidize odianisidine, yielding an intensely colored product [START_REF] Washko | Determination of glucose by an improved enzymatic procedure[END_REF] (Supplementary Figure 2).

In principle, the use of GO and peroxidase as indicator enzymes could allow monitoring the on-line formation of Gly by means of a continuous assay. However, in continuous coupled assays, the reaction catalyzed by the indicator enzyme must not be rate-limiting the overall kinetics. This requisite is difficult to meet by GO, which is a rather slow enzyme (

3 M cat 10 K k ≈ M -1 s -1 , under optimal conditions [24]
). Hence we resorted to a discontinuous method, which, despite being more cumbersome, is not limited by the rate of the coupled reaction, affords an improved reliability of the assay and provides a much greater flexibility in terms of kinetic conditions that can be explored.

The details of the GO-based assay are given in the Experimental section. For comparison, the rates for glycine formation by recombinant AGXT were determined through this assay and through a continuous assay based on the absorbance change at 220 nm (α-keto acids production) [18,[START_REF] Cooper | L-alanineglyoxylate aminotransferase II of rat kidney and liver mitochondria possesses cysteine S-conjugate β-lyase activity: a contributing factor to the nephrotoxicity/hepatotoxicity of halogenated alkenes?[END_REF], using the same buffer and substrate concentrations (Table 2). The rates obtained through the two methods were virtually identical, validating the use of the GO assay for measuring the kinetics of enzymatic glycine production.

Screening of the recombinant aminotransferases for their ability to use glyoxylate as an amino group acceptor

The GO-based assay was adapted for use in a 96-well microtiter format (see Experimental). In fact, we used this plate assay to screen for the ability of the recombinant enzymes to transaminate glyoxylate, using the standard amino acids as amino-group donors (Figure 3).

Besides AGXT (that was included as the positive control) only two aminotransferases appeared able to produce glycine from glyoxylate to an appreciable extent -namely alanine aminotransferase (GPT) and phosphoserine transaminase (PSAT1). The activity of GPT towards glyoxylate was consistent with the results of pre-genomic studies on the rat liver enzyme [START_REF] Noguchi | Glutamate-glyoxylate aminotransferase in rat liver cytosol. Purification, properties and identity with alanine-2-oxoglutarate aminotransferase[END_REF][START_REF] Kobayashi | Identity of alanine:glyoxylate aminotransferase with alanine:2-oxoglutarate aminotransferase in rat liver cytosol[END_REF] and, not unexpectedly, L-Ala and L-Glu were by far the best amino-group donors (Figure 3). On the other hand PSAT1 could use several different amino acid cosubstrates. These included acidic amino acids (L-Glu and L-Asp, but also Lhomocysteate) and small neutral amino acids such as L-Ala and L-Ser (Figure 3 and data not shown). A series of control kinetic experiments showed that L-Glu was the preferred amino-group donor both at 25°C (Table 3 and data not shown) and at 37°C (Supplementary Table 2), consistent with the notion that phosphoserine transaminase uses L-Glu as its standard cosubstrate [START_REF] Hirsch | Studies on phosphoserine aminotransferase of sheep brain[END_REF]. In spite of the enzyme's name, phosphoserine was not an efficient amino-group donor (not shown).

A surprising result of the plate assay was the apparent inability of CCBL1 (also known as glutamine transaminase K) to act on glyoxylate. This was in contrast with a previous study by Han et al. [START_REF] Han | pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I[END_REF], where the same enzyme was reported to employ quite efficiently glyoxylate as an amino-group acceptor. Nevertheless, the reaction conditions in that earlier study were significantly different from those of our plate assay (45°C, higher ionic strength and the nonstandard amino acid L-kynurenine used as the amino-group donor). More importantly, the reaction rates estimated in that earlier study were based on a single time-point, collected 10 min after starting the reaction [START_REF] Han | pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I[END_REF]. When we performed specific kinetic assays on the reaction between phenylalanine, glutamine or methionine and glyoxylate, catalyzed by CCBL1, we observed modest initial transamination rates that declined after a few minutes, coming to a near complete stop well before completion of the reaction (Table 3 and Supplementary Figure 5A). Control experiments showed that the enzyme was not undergoing irreversible inactivation, but rather that it was being strongly inhibited by the keto acid products of the reaction (Supplementary Figure 5B and data not shown).

Reactions of GPT and PSAT1 with glyoxylate

We investigated in more detail the activity of GPT and PSAT1 towards glyoxylate. To this end, we measured the reaction rates of the two enzymes as a function of glyoxylate concentration, either in the presence of L-Ala (for GPT only; Figure 4A) or L-Glu (for both enzymes; Figure 4B-C). In these experiments the reaction conditions were chosen to approximate the intracellular environment (potassium phosphate buffer, pH 7.4, 37° C) and even the concentrations of aminogroup donors employed (1 to 5-10 mM) represented rough lower and upper limits for the cytosolic concentration of L-Ala and L-Glu [START_REF] Fafournoux | Control of alanine metabolism in rat liver by transport processes or cellular metabolism[END_REF][START_REF] Geerts | In situ measurement of glutamate concentrations in the periportal, intermediate, and pericentral zones of rat liver[END_REF].

GPT transaminated glyoxylate with higher efficiency. Reaction rates changed little when the concentration of L-Ala or L-Glu was raised from 1 to 5 mM, implying that GPT was essentially saturated by the amino-group donors at 1 mM concentrations. The apparent specificity constant,

Glyoxylate M cat K k
, fell in all cases in the range 6000-10000 M -1 s -1 (as it can be calculated from the data in Figure 4A-B, based on a molecular mass of 57 kDa for recombinant GPT).

PSAT1 was comparatively less efficient, even though its maximum activity (3.5 µmoles min -1 mg -1 , corresponding to a turnover number of about 2.5 s -1 ) was just fivefold less than the activity of PSAT1 toward its 'physiological' substrate couple, glutamate-phosphohydroxypyruvate. The apparent specificity constant doubled (from 900 to 1800 M -1 s -1 ) when L-Glu was raised from 1 to 10 mM, suggesting that PSAT1 was not fully saturated at the lowest L-Glu concentration tested. However, a further increase in glutamate concentration (to 20 mM) slightly depressed the apparent We have expressed in E. coli eight cytosolic aminotransferases, plus the peroxisomal alanine:glyoxylate aminotransferase. For some of these human enzymes, overproduction by recombinant means had already been described [START_REF] Han | pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I[END_REF][START_REF] Davoodi | Overexpression and characterization of the human mitochondrial and cytosolic branched-chain aminotransferases[END_REF][START_REF] Sivaraman | The narrow substrate specificity of human tyrosine aminotransferase-the enzyme deficient in tyrosinemia type II[END_REF][START_REF] Baek | Characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of L-serine biosynthesis[END_REF][START_REF] Liu | Expression, purification, and initial characterization of human alanine aminotransferase (ALT) isoenzyme 1 and 2 in High-five insect cells[END_REF]. For others (GOT1, AGXT2L1 and AGXT2L2) this is the first report of cloning and recombinant expression in bacteria. In the long term, the recombinant expression of all human transaminases will allow the production of enzyme arrays useful for a synoptic analysis of the respective contributions to metabolic pathways, for quick comparisons of substrate specificities and for the screening of selective inhibitors. Furthermore, aminotransferases are also important tools in biocatalysis, e.g. for the deracemization of natural and non-proteinogenic α-amino acids [START_REF] Tessaro | Chemo-enzymatic deracemization methods[END_REF].

An assay for glycine-forming transaminases

The specific goal of this study was the identification of aminotransferases that use glyoxylate as a substrate. To this end, we have devised a method for detecting the formation of glycine -a method whereby multiple potential amino-group donors can be tested simultaneously and where separation of the products from the substrates is not required. In fact, while GO is active on glycine and on a few Damino acids, as well as on some primary and secondary amines (such as sarcosine), L-amino acids are neither good substrates nor inhibitors of this flavoenzyme [15].

In the past, several assays have been used to monitor the reactions catalyzed by transaminases that use glyoxylate [37]. Early methods were based (like the GO assay we developed) on the quantitation of the glycine produced [START_REF] Alexander | A specific micromethod for the colorimetric determination of glycine in blood and urine[END_REF]. However, these techniques were laborious and often unreliable, so that most researchers turned instead to assays for the quantitation of the keto acid co-product (e.g., pyruvate for the AGXT reaction) [37].

The most accurate and sensitive of these methods entail the derivatization of the keto acid product with some phenylhydrazine dye, followed by HPLC separation and quantitation (e.g., see [START_REF] Cellini | Human wild-type alanine:glyoxylate aminotransferase and its naturally occurring G82E variant: functional properties and physiological implications[END_REF]). These assays however require specific equipment and instrumental setup, and are difficult to apply to large-scale screenings.

The continuous assays based on the differential absorbance of glyoxylate versus other keto acids at ~220 nm are simple and reasonably accurate, provided that the extinction coefficient of the specific keto acid product is known [18]. A major limit of the method is the need to work at a wavelength where most substances absorb, thus affecting the sensitivity of the assay.

Finally, continuous coupled assays based on dehydrogenases (e.g., malate dehydrogenase to monitor the formation of oxaloacetate) are very convenient to study the kinetics of specific transamination reactions and can be easily employed in large-scale screenings. However, these assays are not general, since detection of different keto acid products requires the use of different coupling enzymes. Additionally, some dehydrogenases can use glyoxylate as a substrate, complicating the kinetic analysis [39].

Cytosolic transaminases that act on glyoxylate

The glycine oxidase assay could be adapted to a 96-well format, and was used to screen our set of recombinant aminotransferases (as well as libraries of potential amino-group donors). Notably, only PSAT1 and GPT, out of the eight enzymes tested, could transaminate glyoxylate to glycine with substantial efficiency. The two homologs of the mitochondrial AGXT (AGXT2L1 and AGXT2L2) would not produce glycine using any standard amino acid as a co-substrate. Preliminary results suggest that these proteins are indeed transaminases, but they appear to strongly prefer pyruvate as the amino group acceptor. Studies are underway to elucidate the reaction preferences of these enzymes.

To our knowledge, the activity of PSAT1 on glyoxylate has never been described before. The observation that the most efficient amino-group donor for this enzyme was L-Glu, led us to consider the identification of PSAT1 with a glutamateglyoxylate transaminase partially purified from human liver by Thompson and Richardson, over 40 years ago [START_REF] Thompson | Isolation and characterization of a glutamate-glycine transaminase from human liver[END_REF]. The enzyme studied by those authors reportedly showed a 2 K Glyoxylate M = mM [START_REF] Thompson | Isolation and characterization of a glutamate-glycine transaminase from human liver[END_REF], similar to the one observed by us with PSAT1 (Figure 4C). However, the enzyme from human liver was unable to use L-Ser as an amino-group donor, while it was very active with L-Ala [START_REF] Thompson | Isolation and characterization of a glutamate-glycine transaminase from human liver[END_REF]. Based on these considerations, it is most likely that the enzyme partially purified by Thompson and Richardson was GPT.

Implications for glyoxylate metabolism

The results of this paper help integrate the current view of the cytosolic metabolism of glyoxylate. It is well appreciated that, in the hepatocyte cytosol, glyoxylate can be oxidized to oxalate by lactic dehydrogenase (LDH) or reduced to glycolate by glyoxylate reductase/hydroxypyruvate reductase (GRHPR) [40,[START_REF] Behnam | Reconstruction of human hepatocyte glyoxylate metabolic pathways in stably transformed Chinese-hamster ovary cells[END_REF]. Oxalate is a dead-end product of the metabolism, as it can only be excreted and eliminated, mainly through the kidney. Glycolate can also be excreted; otherwise, it may enter the peroxisomes and be reconverted to glyoxylate [22], which would establish a sort of futile cycle.

We have confirmed a third fate for cytosolic glyoxylate, namely its conversion to glycine, carried out by GPT and PSAT1 (and, to a much lesser extent, by CCBL1) (Figure 5). This transamination reaction is virtually irreversible [START_REF] Cellini | Human wild-type alanine:glyoxylate aminotransferase and its naturally occurring G82E variant: functional properties and physiological implications[END_REF][START_REF] Thompson | Isolation and characterization of a glutamate-glycine transaminase from human liver[END_REF], and has the advantage of producing a substance that the cell can readily incorporate into proteins or utilize in many biosynthetic pathways.

The actual importance of glyoxylate transamination, relative to the first two metabolic routes, remains to be established. The apparent Glyoxylate M cat K k values (mirroring catalytic performance at low glyoxylate concentrations) for GPT and PSAT1 fall in the 10 3 -10 4 M -1 s -1 range (Figure 4), which is lower than the values reported for LDH (3 × 10 4 to 10 5 M -1 s -1 ) and GRHPR (6×10 4 to 1.1 × 10 5 M -1 s -1 ) [40]. In vivo, however, the partitioning of cytosolic glyoxylate between oxidation, reduction and transamination will depend on several factors that may outweigh the intrinsic efficiencies of the individual catalysts. Such factors include the availability of specific coenzymes and cosubstrates (e.g., NADPH, PLP, L-Glu), the presence of inhibitors (e.g., keto acids that compete with glyoxylate) and above all the cytosolic concentration of the different enzymes, which in turn will depend on their relative expression levels.

Potential implications for the treatment of hyperoxaluria

It is believed that the excess oxalate produced in PH1 is formed mostly in the hepatocyte cytosol, via the LDH-catalyzed reaction [START_REF] Danpure | Molecular aetiology of primary hyperoxaluria type 1[END_REF]42]. This implies that cytosolic transaminases are inefficient at competing with LDH for the common substrate, glyoxylate, released by the peroxisomes. However if expression of the genes GPT A c c e p t e d M a n u s c r i p t and PSAT1 were increased substantially above normal, this should steer more and more glyoxylate toward transamination -perhaps enough to significantly diminish oxalate production and the symptoms that ensue.

Are there practical ways to stimulate the expression of GPT and PSAT1, so as to test the above hypothesis? It is known that GPT expression is induced by corticosteroids -indeed, the gene promoter includes a glucocorticoid response element -and by prescription drugs such as the hypolipidemic compound fenofibrate [43]. As for PSAT1, its expression in hepatocytes seems highly regulated and may be strongly influenced by nutritional factors. For example, a survey of the data available at GEO profiles (http://www.ncbi.nlm.nih.gov) suggests that PSAT1 in the mouse liver is highly induced by a ketogenic regimen (i.e., a diet based on fats and proteins, but low in carbohydrates) [START_REF] Kennedy | A high-fat, ketogenic diet induces a unique metabolic state in mice[END_REF].

The possibility that off-the-shelf drugs and dietary changes may help manage PH1, while admittedly speculative, seems worth being explored. Since transgenic mice exist which recapitulate the metabolic defect of PH1 [45], it could be relatively straightforward to test whether treatments that augment the expression of GPT and PSAT1 can decrease oxalate excretion in these animal models. Such studies would provide a better understanding of oxalate production in vivo and, in the best scenario, offer concrete suggestions for the therapy of genetic hyperoxaluria. Fig. 2. Overview of the general procedure adopted for the molecular cloning and recombinant expression of human aminotransferase genes. Fig. 3: A plate assay for the ability of recombinant aminotransferases to produce glycine from glyoxylate. Each well contained the indicated amino acids (10 mM each, except for aromatic amino acids that were 5 mM), glyoxylate (4 mM) and the specified transaminase (1 µM). The transamination reactions were allowed to proceed for 2.5 hours before testing for the formation of glycine (see Experimental). values may mirror the fact that the carbonyl group of glyoxylate is >95% hydrated to the gem diol form in neutral aqueous solution [START_REF] Meany | The dehydration of glyoxalate hydrate: general-acid, general-base, metal ion and enzymic catalysis[END_REF]. GPT encodes the cytosolic alanine transaminase. PSAT1 encodes phosphoserine aminotransferase. TAT encodes the cytosolic tyrosine aminotransferase. † Forward primers were designed to introduce a CpoI restriction site (uppercase letters) just before the start codon of the coding sequence (underlined). Reverse primers were usually designed to pair with the 5'-UTR of the cDNAs; however, when such sequence was unknown or too short, the primers were designed to pair with the 5'-end of the coding sequence, including the stop codon (doubly-underlined). ‡ The length reported here refers to the recombinant protein, which includes a 23-aa Nterminal tail encompassing a hexahistidine tag. § A vector containing the complete coding sequence of AGXT was kindly provided by Dr. C. J. Danpure (University College London). ║ Before subcloning in the expression vector, the cDNA contained in this EST clone was manipulated to remove a small intervening sequence and an internal CpoI restriction site. The concentration of glyoxylate was 4 mM in all cases. Activities were measured in 25 mM potassium phosphate buffer (pH 7.4) at 25°C, using either the continuous assay at 220 nm or the GO-based assay. Upper limits are given for several enzymes that, at a 1 µM concentration, failed to yield a detectable amount of glycine in six hours (i.e., they formed less than 100 µM glycine, the minimum amount measurable in our GO assay setup). * β-alanine was tested as a potential substrate for AGXT2L1 and AGXT2L2 since the homologous enzyme AGXT2 is known to transaminate this amino acid.

Biochemical

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A c c e p t e d M a n u s c r i p t 9 DISCUSSION

 9 to substrate inhibition (not shown). Biochemical Journal Immediate Publication. Published on 23 Jun 2009 as manuscript BJ20090748 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090748

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A c c e

  p t e d M a n u s c r i p t Biochemical Journal Immediate Publication. Published on 23 Jun 2009 as manuscript BJ20090748 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090748 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited 13 A c c e p t e d M a n u s c r i p t

Fig. 1 .

 1 Fig. 1. The reaction catalyzed by aminotransferases that use glyoxylate as an amino-group acceptor, producing glycine. For AGXT, the second substrate (amino-group donor) is L-alanine and the second product is pyruvate.

Fig. 4 :

 4 Fig. 4: Rates of the glyoxylate transaminations catalyzed by GPT and PSAT1, as a function of substrate concentration. Reactions were carried out at 37°C, in 25 mM potassium phosphate, and monitored by following the change in absorbance at 220 nm. Data were fitted to Michaelis-Menten hyperbolas. (A) Reaction of GPT with L-alanine as the amino-group donor. The titrations collected at 1 mM and 5 mM L-Ala yielded similar V max values (5.7 and 7.2 µmoles min -1 mg -1 , respectively) and similar apparent Glyoxylate M K values (1 and 0.8 mM, respectively). (B) Reaction of GPT with L-glutamate as the donor. The titrations collected at 1 mM and 5 mM L-Glu were practically undistinguishable. V max = 6.7 µmoles min -1 mg -1 and Glyoxylate M K = 0.75 mM. (C) Reaction of PSAT1 with L-glutamate as the amino-group donor. The catalytic parameters were: V max =3.5 µmoles min -1 mg -1 and Glyoxylate M K = 5.5

Fig. 5 :

 5 Fig. 5: Metabolic fates available to glyoxylate in the cytosol of hepatocytes. GRHPR indicates glyoxylate reductase/hydroxypyruvate reductase.LDH is lactate dehydrogenase. Examination of the NCBI EST profile database (http:// www.ncbi.nlm.nih.gov/unigene) suggests that LDH isozymes are normally expressed in the liver at higher levels than GRHPR, GPT or PSAT1.

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Biochemical

  Figure 1

Figure 2 Protein

 2 Figure 2

Figure 3 Biochemical

 3 Figure 3

  Figure 4

  Figure 5

Table 1 : cDNAs corresponding to the aminotransferases cloned in this study Gene* EST Clone ID PCR primers † Protein size (aa) ‡

 1

	AGXT §	-	FW: atCGGTCCGatggcctctcacaagctgctg	415
			RE: taCGGACCGtcacagcttcttcttggggc
	AGXT2L1 IMAGE 4797767	FW: atCGGTCCGatgtgcgagctgtacagtaagc	522
			RE: atCGGACCGatcttgctttaaaatgcaaatcag
	AGXT2L2 IMAGE 5220434	FW: atCGGTCCGatggccgcagaccagcgc	473
			RE: atCGGACCGtaggcagagcagggctggc
	BCAT1	DKFZp686E12175Q FW: atCGGTCCGatggattgcagtaacggatgc	408
	CCBL1	IMAGE 4137942 ║	RE: taCGGACCGttccattgtatcctctattttcc FW: atCGGTCCGatggccaaacagctgcaggc	445
			RE: taCGGACCGccaaggcgtgacttcaggg
	GOT1	IMAGE 2820849	FW: atCGGTCCGatggcacctccgtcagtctttg	436
			RE: taCGGACCGtactggacgggtggtgtttc
	GOT1L1 IMAGE 5273324	FW: atCGGTCCGatgcccaccctttcagtgttc	444
			RE: atCGGACCGttgcaaagactaaagttttattcca
	GPT	IMAGE 4179699	FW: atCGGTCCGatggcctcgagcacaggtg	519
			RE: taCGGACCGccagcctggccccagctg
	PSAT1	IMAGE 3883843	FW: atCGGTCCGatggacgcccccaggcag	393
			RE: taCGGACCGagtatatcctggttaggatgtg
	TAT	IMAGE 40146565	FW: atCGGTCCGatggacccatacatgattcag	477
			RE: taCGGACCGtcaggagaatggatgcaggc

* AGXT encodes the peroxisomal alanine-glyoxylate aminotransferase. AGXT2L1 and AGXT2L2 encode two homologs (presumably cytosolic) of AGXT2 (the mitochondrial alanine-glyoxylate aminotransferase). BCAT1 encodes the cytosolic branched-chain amino acid transaminase. CCBL1 (official HUGO Gene Nomenclature Committee symbol) encodes a cytosolic, promiscuous enzyme known by different names: glutamine transaminase K, kynurenine aminotransferase I and cysteine-conjugate β-lyase I. GOT1 encodes the cytosolic aspartate aminotransferase. GOT1L1 encodes a GOT1 homolog.

Table 2 : Comparing the reaction rates of AGXT measured through the GO assay or through a continuous spectrophotometric assay (α-keto acids formation)

 2 Reactions were measured in 25 mM potassium phosphate buffer (pH 7.4) at 25°C. The reported activity values represent the average of at least two independent determinations.

	Biochemical Journal Immediate Publication. Published on 23 Jun 2009 as manuscript BJ20090748
					Activity
	Glyoxylate	L-Ala	L-Ser	(µ µ µ µmol min -1 mg -1 )
	(mM)	(mM)	(mM)	GO assay	Abs 220 nm assay
	10	2		1.3± 0.2	1.5± 0.3
	10	10		7.9 ± 2.3	6.9 ± 1.6
	10	150		15.9 ± 1.5	17.8 ± 0.5
	2	10		6.4 ± 0.2	5.5 ± 0.4
	10		10	0.7 ± 0.2	0.6 ± 0.3

Table 3 : Glycine-forming activities of the recombinant aminotransferases, using selected amino-group donors Enzyme Amino-group donor

 3

	Activity
	(µ µ µ µmol min -1 mg -1 )
	1 mM donor	10 mM donor

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

We are indebted with Andrea Mozzarelli, Simone Ottonello and Angelo Bolchi for support, as well as with Filippo Monica, Francesca Passeri and Francesca Donati for technical assistance.

FUNDING

The financial support of Telethon-Italy (grant GGP06183) is gratefully acknowledged. Parts of this work were also supported by a FAR (Fondo di Ateneo per la Ricerca) grant to L. Pollegioni and by a grant from the Italian Ministry of Research (COFIN 2007).