Indika Kumarapperuma

Lynn Kes

Cong Joseph

Linta Wang

Irin Biju

Kourtney Tom

Théophile Weaver

Frédéric Grébert

Wendy Partensky

Xiaojing Schluchter
email: xiaojing@uic.edu

Kes Lynn

Cong Wang

Linta M Biju

Irin P Tom

Kourtney D Weaver

Théophile Grébert

Frédéric Partensky

Wendy M Schluchter

Xiaojing Yang

Crystal structure and molecular mechanism of an E/F type bilin lyase-isomerase

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

1 Introduction A critical step in biogenesis of the light-harvesting complexes in photosynthetic cyanobacteria involves post-translational modifications that covalently attach bilin pigments to specific cysteine residues in phycobiliproteins [START_REF] Schluchter | Phycobiliprotein Biosynthesis in Cyanobacteria: Structure and Function of Enzymes Involved in Post-translational Modification[END_REF]. Such reactions are catalyzed by an important family of enzymes called bilin lyases. Phycobiliprotein a and b subunits incorporated with 1-3 bilins each are assembled as donut-shaped trimers or hexamers in megadalton phycobilisomes (PBS) where the rods project out from the core to allow light harvesting and directional energy transfer to the reaction centers of photosystems [START_REF] Glazer | Light guides. Directional energy transfer in a photosynthetic antenna[END_REF] (Fig. S1A). Bilin lyases are directly responsible for the extensive pigment diversity of the PBS and ubiquity of cyanobacteria on Earth by enabling them to adapt and thrive in different light niches [START_REF] Flombaum | Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus[END_REF][START_REF] Grébert | Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria[END_REF]Sanfilippo et al., 2019a).

Among three phylogenetically distinct types of bilin lyases [START_REF] Bretaudeau | CyanoLyase: a database of phycobilin lyase sequences, motifs and functions[END_REF][START_REF] Fairchild | Phycocyanin a-subunit phycocyanobilin Iyase[END_REF][START_REF] Scheer | Biliprotein maturation: the chromophore attachment: Biliprotein chromophore attachment[END_REF][START_REF] Schluchter | Phycobiliprotein Biosynthesis in Cyanobacteria: Structure and Function of Enzymes Involved in Post-translational Modification[END_REF][START_REF] Shen | Identification and Characterization of a New Class of Bilin Lyase THE cpcT GENE ENCODES A BILIN LYASE RESPONSIBLE FOR ATTACHMENT OF PHYCOCYANOBILIN TO CYS-153 ON THE β-SUBUNIT OF PHYCOCYANIN IN SYNECHOCOCCUS SP. PCC 7002[END_REF][START_REF] Shen | Biogenesis of Phycobiliproteins. I. cpcS-I and cpcU mutants of the cyanobacterium Synechococcus sp. PCC 7002 define a heterodimeric phycocyanobilin lyase specific for beta -phycocyanin and allophycocyanin subunits[END_REF][START_REF] Zhou | The cpcE and cpcF genes of Synechococcus sp. PCC 7002. Construction and phenotypic characterization of interposon mutants[END_REF], the S/U and T types have the b-barrel folds while the E/F type adopts an all-helical structure [START_REF] Kronfel | Structural and Biochemical Characterization of the Bilin Lyase CpcS from Thermosynechococcus elongatus[END_REF][START_REF] Overkamp | Insights into the Biosynthesis and Assembly of Cryptophycean Phycobiliproteins[END_REF][START_REF] Zhao | Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F[END_REF][START_REF] Zhou | The cpcE and cpcF genes of Synechococcus sp. PCC 7002. Construction and phenotypic characterization of interposon mutants[END_REF][START_REF] Zhou | Structure and Mechanism of the Phycobiliprotein Lyase CpcT[END_REF].

These enzymes differ in their protein scaffolds and confer different substrate specificities for pigments, acceptor proteins and target residues. In essence, they all catalyze the same chemical reaction resulting in formation of a thioether linkage between the sulfhydryl group of a cysteine residue in phycobiliprotein and the C3 1 atom in the A-ring of a bilin pigment [START_REF] Scheer | Biliprotein maturation: the chromophore attachment: Biliprotein chromophore attachment[END_REF][START_REF] Schirmer | Refined three-dimensional structures of two cyanobacterial C-phycocyanins at 2.1 and 2.5 Å resolution[END_REF] (Fig. S1B). It has been proposed that this reaction involves transient association between the bilin pigment and lyase mediated by a nucleophilic residue such as histidine or cysteine from the lyase via the C10 atom [START_REF] Stumpe | The addition of methyl-2mercaptoacetate to phycocyanobilin dimethyl ester: A model reaction for biliprotein biosynthesis?[END_REF][START_REF] Tu | Toward a Mechanism for Biliprotein Lyases: Revisiting Nucleophilic Addition to Phycocyanobilin[END_REF][START_REF] Zhao | Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon[END_REF]. However, the chemical identity of catalytic residue(s) and their roles in the enzymatic action remain elusive [START_REF] Gasper | Distinct Features of Cyanophage-encoded T-type Phycobiliprotein Lyase ΦCpeT: THE ROLE OF AUXILIARY METABOLIC GENES[END_REF][START_REF] Overkamp | Insights into the Biosynthesis and Assembly of Cryptophycean Phycobiliproteins[END_REF][START_REF] Scheer | Biliprotein maturation: the chromophore attachment: Biliprotein chromophore attachment[END_REF]Tu et al., 2009;[START_REF] Zhao | Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F[END_REF][START_REF] Zhao | Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon[END_REF][START_REF] Zhou | Structure and Mechanism of the Phycobiliprotein Lyase CpcT[END_REF]. For the E/F type bilin lyases, there has been only one structure solved for heterodimeric CpcE/F [START_REF] Zhao | Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F[END_REF].

To understand the molecular mechanism underlying the bilin ligation reaction, we investigate a single-chain E/F type bilin lyase MpeQ that incorporates PUB at the Cys83 site of the phycoerythrin-II a-subunit, a phycobiliprotein encoded by the mpeBA operon in marine Synechococcus [START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF]. The newly identified MpeQ is involved in Type IV chromatic acclimation (CA4) process, a widespread phenomenon where marine Synechococcus vary the molar ratio between the blue-light absorbing phycourobilin (PUB) and the green-light absorbing phycoerythrobilin (PEB) in phycobiliproteins depending on the ambient light color [START_REF] Everroad | Biochemical Bases of Type IV Chromatic Adaptation in Marine Synechococcus spp[END_REF][START_REF] Grébert | Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria[END_REF][START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF][START_REF] Palenik | Chromatic Adaptation in MarineSynechococcus Strains[END_REF]Sanfilippo et al., 2019a;[START_REF] Shukla | Phycoerythrin-specific bilin lyaseisomerase controls blue-green chromatic acclimation in marine Synechococcus[END_REF]. The CA4 phenomenon is attributed to light-regulated expression of a PEB lyase or lyase-isomerase encoded in a small CA4-conferring genomic island and competing with a constitutively expressed homolog (lyase-isomerase or lyase) encoded in the large genomic region involved in the biosynthesis of phycobilisome rods [START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF][START_REF] Mahmoud | Adaptation to Blue Light in Marine Synechococcus Requires MpeU, an Enzyme with Similarity to Phycoerythrobilin Lyase Isomerases[END_REF][START_REF] Sanfilippo | Self-regulating genomic island encoding tandem regulators confers chromatic acclimation to marine Synechococcus[END_REF]Sanfilippo et al., , 2019a;;[START_REF] Shukla | Phycoerythrin-specific bilin lyaseisomerase controls blue-green chromatic acclimation in marine Synechococcus[END_REF]. Compared to PEB lyases, bilin lyaseisomerases such as MpeQ have acquired an additional ability to isomerize PEB to PUB during the ligation reaction [START_REF] Blot | Phycourobilin in Trichromatic Phycocyanin from Oceanic Cyanobacteria Is Formed Post-translationally by a Phycoerythrobilin Lyase-Isomerase[END_REF][START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF][START_REF] Shukla | Phycoerythrin-specific bilin lyaseisomerase controls blue-green chromatic acclimation in marine Synechococcus[END_REF] (Fig. S1C).

However, the molecular basis for this isomerase action is not known. MpeQ thus offers a superb model system not only for dissecting the molecular mechanism of bilin lyases but also for addressing how these highly homologous enzymes attach different pigments to the same site of phycobiliproteins using the same substrate [START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF][START_REF] Humily | A Gene Island with Two Possible Configurations Is Involved in Chromatic Acclimation in Marine Synechococcus[END_REF]Sanfilippo et al., 2019b). This work reports the crystal structure of MpeQ, a representative bilin lyase-isomerase from the E/F family. This structure has revealed a hitherto unknown "question-mark" architecture and active site geometry that call for a major revision of the current model for the E/F bilin lyases [START_REF] Zhao | Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F[END_REF]. Based on structural analyses, site-directed mutagenesis and enzyme assays, we identify key residues responsible for the lyase and isomerase activities, respectively.

We propose a general tyrosine-mediated reaction scheme for bilin lyases in the E/F family and beyond. We further advance a model for the enzyme-substrate complex that elucidates how MpeQ accommodates both the bilin and protein substrates to catalyze the ligation reaction while employing stereoselectivity to confer the isomerase activity. These findings provide a structural framework and mechanistic insights into one of the most important enzymatic reactions in photosynthesis.

Results

MpeQ adopts a "question-mark" architecture

We have determined the crystal structure of MpeQ at 2.5 Å resolution by the single-wavelength anomalous diffraction (SAD) method using Se-methionine-derivatized crystals (Table 1). In addition to the N-terminal histidine tag, all 398 residues of MpeQ have been accounted for in the electron density map with two protein molecules in an asymmetric unit. The a-solenoid structure of MpeQ adopts a "question-mark" scaffold in which a total of 23 a-helices are supercoiled in a right-handed manner (Fig. 1A; Fig. 1D). The N-terminal (aa. 14-255) and C-terminal (aa. 274-398) domains connected by an extended linker are denoted the E and F domains, respectively, corresponding to the CpcE and CpcF subunits of a prototypical CpcE/F bilin lyase from Nostoc sp. PCC 7120 [START_REF] Zhao | Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F[END_REF]. However, the overall architecture of MpeQ starkly contrasts with the crescent-shaped structure proposed for CpcE/F [START_REF] Zhao | Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F[END_REF] (Fig. 2A,B).

Interestingly but not surprisingly, a revised CpcE/F model based on an alternative arrangement of the E and F subunits presents a heterodimeric structure highly comparable to that of MpeQ (Fig. 1B; Fig. 2). Remarkably, four out of five strictly conserved residues (Tyr76, Pro108, Arg112 and Trp151; numbering in CpcF) between heterodimeric CpcE/F and single-chain E/F lyases such as MpeQ line up in the tight turns between helices facing an enclosed cavity in the question-mark architecture in contrast to the published CpcE/F structure. This suggests that the MpeQ structure, but not the reported structure of CpcE/F [START_REF] Zhao | Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F[END_REF], represents the biological relevant protein framework for bilin lyases in the E/F family.

Active site and key residues responsible for lyase activity

The E and F domains of MpeQ are roughly perpendicular to each other in terms of the overall helical direction (Fig. 1C). The E domain constitute the "stroke" part of the question mark while the F domain corresponds to the "dot" part (Fig. 1A, Fig. 1D). Besides the linker, the E and F domains are bridged by a long N-terminal extension (aa. 1-13) enclosing a large interior chamber (Fig. 1A, Fig. 3). In the crystal lattice, two molecules of MpeQ dimerize via their N-terminal extensions tethered as two anti-parallel b-strands traversing each other's chamber (Fig. 1E). The enclosed chamber is open on both sides although one side is notably wider than the other (Fig. 1C). Arg/Lys residues pointing towards the chamber interior form large positively charged surface patches on the ceiling and wall areas (Fig. 3A; Fig. 4A). Along a shallow cleft between the E and F domains, some highly conserved residues as well as those hallmark residues that distinguish the lyases and lyase-isomerases are clustered at the chamber floor constituting a putative active site (Fig. 4B; Fig. S2). This site marked by Tyr318, Lys353 and Glu285 is exposed to the wide side of the chamber (Fig. 3B). In crystal lattice, Tyr318 and Lys353 engage close interactions with the E' domain of a neighboring molecule via an interface with buried surface area of 2426 Å 2 (PISA) (Fig. S3A). In solution, however, MpeQ is monomeric according to the elution profile of size exclusion chromatography (Fig. S3B), which makes this site fully accessible for substrate binding.

To confirm the active site, we made several single mutations in this cluster (Fig. 4A,B) and examined how they affect the lyase and/or isomerase activities in MpeQ. By co-expressing MpeQ in E. coli with two other plasmids that produce the MpeA and PEB substrates, we measured the normalized enzyme activities by detecting the chromophorylated products, MpeA-PUB or MpeA-PEB, using Zn-fluorescence [START_REF] Berkelman | Visualization of bilin-linked peptides and proteins in polyacrylamide gels[END_REF] and/or absorption spectra of the purified MpeA (Fig. 4C-E; Fig. 5). The lyase activity was completely abolished in the single mutants of Y318F, Y318A and K353A, with no detectable chromophorylation of MpeA (Fig. 4C; Table S1). The E285A mutant showed significantly reduced lyase activity while the S222A, S224P and W383F variants retained the lyase-isomerase activities with MpeA-PUB produced at varying levels lower than WT (Fig. 4C; Fig. 5). These mutational data corroborate our structural findings, supporting that Tyr318 and Lys353 play essential roles in catalysis and/or substrate binding (Fig. 3B).

We also made single mutants on several Arg residues lining the chamber interior (Fig. 4A).

When Arg71, Arg79, Arg135 or Arg143 was individually mutated to Asp, no MpeA chromophorylation was detected (Fig. 5C). Similar results were observed for the R228A and R198A variants (Fig. 4C; Fig. S2; Table S1). The R71A, R79A, R228D, and R198K variants showed low-level production of MpeA-PUB (Fig. 5; Table S1). These results suggest that although Arg71, Arg79, Arg198 and Arg228 are not directly involved in catalysis, they are likely to act as protein anchors that help stabilize the bilin pigment via interactions with the negatively charged propionates.

Steric factors underlying isomerase activity

To determine the molecular basis of the isomerase activity, we analyzed a collection of protein sequences of highly homologous E/F type lyases and their counterpart lyase-isomerases.

Specifically, sequence comparisons of the MpeW and MpeY lyases with the MpeQ and MpeZ lyase-isomerases retrieved from 106 marine Synechococcus strains or single-amplified genomes (Dataset S1) highlighted nine residues swapped between the lyases and lyase-isomerases (Fig. S2 and Dataset S2). To examine whether these "swap" residues contribute to the gain of the isomerase function, we individually substituted five such residues at the active site of MpeQ with their counterparts in the PEB lyase MpeW (Fig. S4). All five single mutants (A100T, S224P, V319G, T320A, and Y323Q) largely retained their lyase activities (Fig. 5; Fig. S4). Only the V319G variant produced an additional absorption peak near 550 nm indicating the formation of MpeA-PEB (Fig. S4B). In other words, the Val!Gly substitution led to partial loss of the isomerase activity in MpeQ (Fig. 4D, Fig. S4B).

Val319 is located right next to the catalytic Tyr318. We systematically explored possible steric effects on the lyase-isomerase activity by replacing Val319 with Gly, Ala, Leu and Phe, individually (Fig. 4C,4D). With increasingly bulkier side chains at the position of Val319, these mutants exhibited a striking color tuning effect on the MpeA product (Fig. 5A). The V319G variant with the smallest side chain showed a reddish color indicating a low PUB:PEB ratio in MpeA. The V319A variant with a slightly larger side chain has an orange color suggesting a higher PUB:PEB ratio. With valine or leucine present at the position 319, the yellow-colored sample suggests exclusive formation of MpeA-PUB. Larger side chains at this position appear to interfere with the bilin attachment. As such, the lyase activity of the V319L variant was reduced by about 50%, while the V319F variant barely showed any detectable chromophorylated MpeA (Fig. 4C,D; Fig. 5).

It is not surprising that the single swap mutations (A100T, S224P, T320A and Y323Q) did not directly affect the isomerase function as these sites are further away from the catalytic Tyr318 (Fig. 4B; Fig. S4B). However, when these substitutions were made in combination with V319G, they significantly altered the PUB:PEB ratio in the MpeA products (Fig. 4E). The single mutant V319G exhibited a PUB:PEB ratio of 10:1 while the double mutant V319G/T320A lowered the PUB:PEB ratio to 3:1. This ratio was further reduced to 1.5:1 in the triple mutant V319G/T320A/Y323Q. Two quintuple mutants (MpeQ5.1: A100T/V319G/T320A/Y323Q/T352A; MpeQ5.2: S224P/V319G/T320A/Y323Q/T352A) evidently switched to make more MpeA-PEB than MpeA-PUB resulting in the PUB:PEB ratios of 1:1.5 and 1:3, respectively (Fig. 4E). It is noteworthy that higher PUB:PEB ratios coincide with bulkier side chains at these swap sites (Fig. 4A), suggesting that the formation of MpeA-PEB requires a larger reaction volume at the active site. To this end, exclusive attachment of PEB to MpeA-Cys83 by MpeW arises from collective steric effects from multiple swap sites beyond Val319 as exemplified by the sequence differences between MpeQ and its counterpart lyase MpeW (Fig. S2 and Fig. S5).

Tripartite model and reaction mechanism

Identification of the active site allowed us to explore the modes of substrate binding to MpeQ.

We first examined structural flexibility of both MpeQ and MpeA using molecular dynamics (MD) simulations (Fig. S6). A 72-ns MD simulation on MpeQ revealed two distinct dispositions of the long N-terminal extension (~aa. 1-15) (Fig. S6A). In a "latch-on" position similar to that in the crystal structure, the N-terminal extension encloses the active site chamber while the "latchoff" position allows better accessibility to the active site. The RMSD distance matrix derived from 100 simulated structures on the trajectory reveals three rigid bodies in the a-solenoid structure (aa. 30-97 and 117-237 in the E domain; aa. 250-398 in the F domain), which roughly correspond to the ceiling, wall and floor sections of the chamber, respectively (Fig. S6B; Fig. 3B).

The MD trajectory shows close coupling between the wall and floor sections while the ceiling section exhibits higher mobility likely due to its proximity to the flexible N-terminal extension.

We also performed the MD simulations on an apo-MpeA model (Swiss-model) [START_REF] Biasini | SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information[END_REF], which also reveals two distinct clusters of conformations in the cap-like loop (aa. 63-81) shielding MpeA-Cys83 from the molecular surface (Fig. S6C; Fig. S1B). The "cap-on" conformation resembles a well-folded a-subunit while the "cap-off" structure exposes MpeA-Cys83 for docking at the active site of MpeQ.

Based on surface complementarity, protein flexibility and active site geometry, we present a tripartite model in which MpeQ is complexed with the PEB and MpeA substrates (Fig. 6). First of all, the active site in monomeric MpeQ is fully accessible for substrate binding (Fig. 6A). Apo-MpeA is expected to approach the active site chamber from the wide side. This docking is likely facilitated by induced fit involving both MpeA and MpeQ (Fig. 6A; Fig. S6). Specifically, the Nterminal extension of MpeQ would adopt a "latch-off" position allowing proper positioning of MpeA-Cys83 at the active site (Fig. 6B). With the bilin binding pocket of MpeA open to the chamber interior, the active site of MpeQ is accessible to the PEB chromophore from the narrow side (Fig. 6C). This model features PEB in its final binding cleft of MpeA (Fig. 6B,C) with its Aring sandwiched between MpeQ-Tyr318 and MpeA-Cys83.

At the active site, the A-ring assumes an extended anti-conformation as in the final product. This disposition presents the C3=C3 1 double bond, the target site for ligation, to the hydroxyl group of Tyr318 while its lactam group is positioned to engage direct interactions with Lys353/Glu285 (Fig. 6D; Fig. S7A). This active site geometry fully agrees with the mutagenesis data (Fig. 4C) supporting that Tyr318 is critical for catalyzing the ligation reaction while Lys353/Glu285 are important for conferring specific orientation of the A-ring. In addition, this model places Val319 right next to the R-configured C2 atom, which perfectly explains the remarkable steric effects of Val319 on the lyase and isomerase activities (Fig. 4D).

This tripartite model clearly points to distinct chemical origins for the lyase and isomerase actions of MpeQ. To address whether the PEB!PUB isomerization occurs before or after bilin ligation to MpeA-Cys83, we performed a simple experiment by co-expressing MpeQ with a PEB-producing plasmid and the MpeA-C83A variant. We reason that since MpeA-C83A is unable to covalently attach the bilin pigment, the ligation reaction cannot complete thereby allowing us to detect the isomerization product PUB if it is formed in the absence of ligation.

We measured absorption spectra from the load, flowthrough and eluted fractions of the affinity chromatography column that was used to separate bulk proteins and free pigments from the His-tagged MpeQ and MpeA (Fig. S8A). Both the load and flowthrough samples showed the absorption band around 560 nm supporting that the PEB substrate was sufficiently available in this co-expression system. While PUB was barely detectable in the bulk samples of load and flowthrough, the eluted fraction exhibits a small yet clear peak at ~495 nm (Fig. S8A), suggesting that PUB is formed in the absence of ligation to MpeA-Cys83 and remains associated with MpeQ and/or MpeA.

Taken together, we advance a reaction scheme to elucidate a tyrosine-mediated bilin ligation and isomerization catalyzed by MpeQ. In essence, the net reaction catalyzed by a bilin lyase is a nucleophilic addition of the sulfhydryl (-SH) group of cysteine in the phycobiliprotein substrate to the C3=C3 1 double bond of the bilin substrate (Fig. 7). In the final product of MpeQ, a thioether bond is formed between the C3 1 atom and the sulfur atom of MpeA-Cys83 while the C3=C3 1 double bond is reduced. Such a nucleophilic addition to alkene requires a catalytic group to polarize the C=C double bond. Our structural and mutagenesis data strongly suggests that the hydroxyl group of MpeQ-Tyr318 serves this role. We propose that in the first step of the reaction, Tyr318 activates the bilin substrate resulting in a polarized C3=C3 1 double bond (Fig. 7). This bilin activation step is critical as no lyase activities were detected in the Y318F or Y318A mutants when this hydroxyl group is absent, or in V319F where the catalytic hydroxyl group cannot get close enough to the C3 atom (Fig. 4). In MpeW where the active site poses no major steric hinderance, the bilin activation would proceed to the nucleophilic addition reaction giving rise to the MpeA-PEB product. In MpeQ, on the other hand, the MpeA-PEB formation is not allowed due to incompatibility between the active site residue (Val319) and the chiral centers at the C3 and/or C2 positions in PEB. The addition reaction cannot take place unless such a conflict is resolved in a rearrangement reaction where PEB!PUB isomerization effectively shifts the chiral centers from the C2/C3 to C4/C5 positions (Fig. 6; Fig. 7). Selected by evolution, the side chain of Val319 is small enough to permit the catalytic action of Tyr318, yet bulky enough to exclude the PEB attachment. Similar to retinoid isomerase RPE65 [START_REF] Kiser | Catalytic mechanism of a retinoid isomerase essential for vertebrate vision[END_REF], MpeQ exemplifies a general molecular strategy to confer the isomerase activity via stereoselectivity of the active site geometry (Fig. 7).

Discussion

Prior to this study, no crystal structure has been reported for any bilin lyase-isomerase; and CpcE/F (a PCB lyase from Nostoc sp. PCC 7120) was the only structure available for the E/F type bilin lyases [START_REF] Zhao | Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F[END_REF]. However, the interface between CpcE and CpcF proposed by Zhao et al. is not present in the structure of single-chain MpeQ (Fig. 1E; Fig. 2B) [START_REF] Zhao | Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F[END_REF]. In fact, no consistent pattern or interface other than the question-mark arrangement is found between the crystal structures of MpeQ and CpcE/F (Fig. 2D). Therefore, it is unlikely that the crescent-shaped architecture (PDB ID: 5N3U) represents the biological assembly or functional unit of CpcE/F despite its high resolution. Because the crystal lattice of CpcE/F presents two crystallography-equivalent yet distinct dimeric arrangements (Fig. 2), it is susceptible to misinterpretation of the biologically relevant heterodimer. The single-chain MpeQ, on the other hand, is not prone to such ambiguity especially when all residues have been accounted for in the electron density map. Given the similarities in protein sequence and secondary structure (Fig. 1, Fig. S5), we posit that the E/F family bilin lyases including the single-chain and heterodimeric enzymes share a common structural framework represented by the question-mark architecture MpeQ.

We also note remarkable similarities in the active site geometry between MpeQ and other types of bilin lyases despite their distinct protein scaffolds (Fig. S7A). The catalytic triad of MpeQ is not only found in the revised structure of CpcE/F but also in CpcT/CpeT (PDB ID: 4O4O, 4O4S, 5HI8) and CpcS/CpeS (PDBID: 3BDR and 4TQ2) [START_REF] Gasper | Distinct Features of Cyanophage-encoded T-type Phycobiliprotein Lyase ΦCpeT: THE ROLE OF AUXILIARY METABOLIC GENES[END_REF][START_REF] Kronfel | Structural and Biochemical Characterization of the Bilin Lyase CpcS from Thermosynechococcus elongatus[END_REF][START_REF] Overkamp | Insights into the Biosynthesis and Assembly of Cryptophycean Phycobiliproteins[END_REF][START_REF] Zhao | Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F[END_REF][START_REF] Zhou | Structure and Mechanism of the Phycobiliprotein Lyase CpcT[END_REF]. Specifically, they all feature a conserved tyrosine at the rim of a large protein cavity where the phycobiliprotein and bilin substrates are supposed to bind. And CpcF-Tyr76 is perfectly aligned with MpeQ-Tyr318 (Fig. S5; Fig. S7A). Beyond its locality, this conserved active-site tyrosine seems to play a similar role.

Just like MpeQ-Y318F, no bilin attachment was detected in the single mutants of CpcF-Y76A and CpcT-Y65F [START_REF] Zhao | Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F[END_REF][START_REF] Zhou | Structure and Mechanism of the Phycobiliprotein Lyase CpcT[END_REF]. We speculate that the proposed tyrosinemediated reaction mechanism also applies to bilin lyases in the T and S/U clans (Fig. 7).

Regarding the origin of the isomerase activity, we have considered two scenarios. First, the E/F lyase and isomerase activities are resulted from two distinct enzymatic reactions involving different catalytic residues, and these swap residues simply play a role in tipping off the balance between these two reactions. Second, the isomerase activity is an "add-on" function associated with the lyase reaction catalyzed by Tyr318, which arises from collective steric effects from the swap residues near the active site. The first scenario implies that the catalytic residues responsible for the isomerase activity are near the C4=C5 double bond of PEB, and they are distinct from Tyr318. However, our current docking model shows that the only residues within a 5-Å radius of the C4=C5 double bond of PEB are MpeQ-Tyr318 (3.5 Å) and MpeA-Cys83 (4.4 Å) (Fig. 6). In addition, it is unlikely that MpeA-Cys83 is directly involved in isomerization (Fig. S8A). On the other hand, a bioinformatic study on isomerases [START_REF] Martinez Cuesta | The evolution of enzyme function in the isomerases[END_REF][START_REF] Martínez Cuesta | Exploring the chemistry and evolution of the isomerases[END_REF] suggested that the isomerase and lyase activities are evolutionarily related due to their similarity in chemistry, which goes along the second scenario. We propose that highly homologous MpeQ and MpeW share the similar reaction mechanism for their lyase actions while the substrate transformation between the activation and ligation steps is directly influenced by the geometry and steric factors of the active site (Fig. 7).

Our mutagenesis data show that the steric factor at the position of Val319 directly influences the isomerase activities (Fig. 4C,D, Fig. S8B,C). Consistently, among the 106 sequences of the MpeQ/W/Y/Z family retrieved from marine Synechococcus strains or SAGs [START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF]Sanfilippo et al., 2019a;[START_REF] Shukla | Phycoerythrin-specific bilin lyaseisomerase controls blue-green chromatic acclimation in marine Synechococcus[END_REF], all enzymes with the isomerase function have a valine residue at the this position, whereas all lyases have a glycine (Fig. S2 andDataset S2).

This includes MpeZ, a lyase-isomerase shown to be involved in the CA4 strain RS9916 (Sanfilippo et al., 2019a;[START_REF] Shukla | Phycoerythrin-specific bilin lyaseisomerase controls blue-green chromatic acclimation in marine Synechococcus[END_REF], which features Val next to the catalytic Tyr321 while its counterpart PEB lyase MpeY has Gly instead. However, the Val!Gly substitution alone is not sufficient (even in combination with some swap sites) to confer exclusive PEB attachment in MpeQ (Fig. 4E). Evidently, a complete switch from MpeQ to a MpeW-like PEB lyase requires contributions from other swaps sites, which collectively confer an active site spacious enough to accommodate the C2/C3 chiral centers of a non-planar A-ring during the ligation reaction (Fig. 4E; Fig. S2).

It is worth noting that the heterodimeric lyase-isomerase PecE/F lacks tyrosine corresponding to Tyr318. Homology modeling of PecE/F places its signature HC motif right at the space occupied by MpeQ-Tyr318 (SwissModel) (Fig. S7B). In addition, the single mutations in the HC motif abolished the lyase activity of PecE/F [START_REF] Zhao | Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F[END_REF]. It is possible that PecE/F employs histidine instead of tyrosine as a catalytic residue for substrate activation (Tu et al., 2009) (Fig. S7B). This exception lends further support to the active site geometry proposed for the E/F bilin lyases.

The active site geometry as illustrated in our tripartite model not only supports the respective roles of Tyr318 and Val319 but also has other implications. It is plausible that Lys353/Glu285 from the catalytic triad contribute to stabilizing the A-ring in a 5,anticonformation via hydrogen bond interactions with the lactam group (Fig. S7A; Fig. 6D). This feature hints on the hitherto unknown structural basis for the 5,anti-conformation of bilins exclusively found in phycobiliproteins, in contrast to the 5,syn-conformation observed in phytochromes (30,31). In other words, the characteristic 5,anti-conformation arises from substrate selectivity of the bilin lyases. As such, altered protein-chromophore interactions in the K353A and E285A mutants resulted in poor bilin attachment (Fig. 4C). Furthermore, positively charged residues (e.g. Arg79/Arg228) are positioned close to the bilin propionates in the active site chamber (Fig. 6D); such ionic interactions are reminiscent of the bilin binding modes observed in phytochromes and other bilin-binding proteins [START_REF] Wagner | A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome[END_REF][START_REF] Yang | Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: Photoconversion and signal transduction[END_REF]. Not surprisingly, the R79D and R228D mutants adversely affected the lyase activities of MpeQ (Fig. 5).

Needless to say, further studies are needed to validate the atomic details of the proposed reaction scheme, which are beyond the scope of this work. To this end, our attempts to directly introduce substrates to the MpeQ crystals by soaking have been unsuccessful because the current crystal form blocks any substrate binding to the active site (Fig. 1E, Fig. S3A). MpeQ complexed with pigment alone is also deemed unfeasible for co-crystallization due to the large size difference between the active site chamber and PEB. We reason that both substrates (MpeA and PEB) are needed to assemble stable complexes for structural studies by crystallography or cryoEM. To accumulate homogeneous species for static studies, the ligation reaction can be arrested by using either an inactive enzyme such as MpeQ-Y318F or a disabled substrate such as MpeA-C83A. For example, MpeQ-Y318F can be used to capture the "pre-activation" state, while MpeA-C83A allows trapping of later "pre-ligation" intermediate(s). It will be intriguing to see whether bilin activation mediated by Tyr318 involves any formation of a tyrosine radical and/or transient covalent intermediates.

Why do some E/F lyases such as CpcE/F employ two polypeptide chains while other homologous enzymes such as MpeQ achieve the same function using a single protein chain? Biswas et al. suggested that the CpcE/F family evolved after the CpcS/U family of lyases because some E/F members have the ability to remove bilins [START_REF] Biswas | Characterization of the Activities of the CpeY, CpeZ, and CpeS Bilin Lyases in Phycoerythrin Biosynthesis in Fremyella diplosiphon Strain UTEX 481[END_REF]. We speculate that E/F heterodimers have advantages in allowing mix-and-match of different E-like and F-like subunits. It is noteworthy that the gene fusion events seem to occur more frequently in phycoerythrin-containing organisms. For example, RpcG (a PEB lyase-isomerase) is a fusion protein with its N-terminal and C-terminal domains closely related to PecE and PecF, respectively [START_REF] Blot | Phycourobilin in Trichromatic Phycocyanin from Oceanic Cyanobacteria Is Formed Post-translationally by a Phycoerythrobilin Lyase-Isomerase[END_REF] while CpeY (a PEB lyase for a-phycoerythrin I) appears to result from the fusion of CpcE-like and CpcF-like domains [START_REF] Biswas | Characterization of the Activities of the CpeY, CpeZ, and CpeS Bilin Lyases in Phycoerythrin Biosynthesis in Fremyella diplosiphon Strain UTEX 481[END_REF]. These examples provide clues about how the E/F clan shifted from the mainly heterodimers to single-chain enzymes, the latter conformation being the rule for all phycoerythrin-related lyases and lyasesisomerases.

Taken together, results from this study have revealed distinct chemical origins for the lyase and isomerase actions of bilin lyase-isomerase in the E/F family. Our structural and mutagenesis data strongly support that stereoselectivity of the active site geometry plays a critical role in conferring the isomerase activity of MpeQ. We further propose a tyrosinemediated reaction scheme shared among different types of bilin lyases. This work thus presents a structural and mechanistic framework that has advanced the fundamental understanding of critical biochemical reactions underlying the phycobilisome biogenesis.

Inclusion and Diversity

One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. In MpeW, Gly319 next to the catalytic Tyr318 is compatible with the non-planar A-ring conformation of PEB. The activated PEB is thus allowed to proceed to the nucleophilic addition reaction forming a thioether bond between MpeA-Cys83 (blue) and the A-ring C3 1 atom. In MpeQ, the side chain of Val319 renders a steric conflict with the A-ring of PEB. The ligation reaction only proceeds when this conflict is resolved via the PEB!PUB isomerization resulting in the MpeA-PUB product.

RESOURCE AVAILABILITY

Lead Contact:

• Xiaojing Yang (xiaojing@uic.edu).

Material availability

• Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Xiaojing Yang (xiaojing@uic.edu).

Data and code availability

• The coordinates and structure factor amplitudes of MpeQ in the space group C2 and C2221

have been deposited in the Protein Data Bank under the accession numbers 7MC4 and 7MCH, respectively.

• This paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

The mpeQ and mpeW genes from the CA-B strain Synechococcus A15-62 were cloned into the pCDF-Duet 1 and pET-Duet 1 vectors using the restriction sites SacI/NotI (or BglII/EcoRV) and NcoI/HindIII, respectively. MpeQ carrying a N-terminal 6xHis affinity tag was over-expressed in E. coli BL21(DE3) by isopropyl b-D-1-thiogalactopyranoside (IPTG, 1 mM) induction followed by overnight shaking at 18°C.

METHOD DETAILS

Protein purification and site-directed mutagenesis

After cell harvesting and cell lysis, His-tagged MpeQ protein was extracted by Co 2+-affinity chromatography and further purified by anion exchange chromatography (HiTrap Q-HP column). The selenomethionine (SeMet)-derivatized MpeQ protein was prepared according to the standard protocols (Doublié, 1997) and purified using the same protocols as for the native MpeQ.

Site-directed mutagenesis was carried out using combined overlapping polymerase Chain Reaction (COE-PCR) as previously described with modifications [START_REF] Hussain | Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis[END_REF].

Mutagenic primers were created using the Thermo Fisher Scientific GeneArt primer and construct design tool (https://www.thermofisher.com/order/oligoDesigner/mutagenesis) or NEB primer design tool (http://nebasechanger.neb.com) (Table S2, Table S3). Quintuple mutants were created using the GeneArt Seamless Cloning and Assembly Enzyme Mix (Invitrogen). All mutants of MpeQ were over-expressed and purified using the same protocols as for the wild type (Table S4).

Crystallization, data collection and structure determination

Purified MpeQ protein was crystallized using the hanging drop vapor diffusion method by mixing the protein sample (5 mg/mL) with the crystallization solution (2.1 M DL-Malic acid, pH 7.0) in 1:1 ratio. Single crystals obtained by macro-seeding were cryoprotected in the mother liquor containing 25% glycerol for X-ray diffraction experiments. All diffraction images were collected at the Life-Science Consortium Access Team (LS-CAT) beam stations at the Advanced Photon Source, Argonne National Laboratory. The diffraction datasets were indexed, integrated and scaled using HKL2000 (Otwinowski and Minor, 1997) and/or xia2 in CCP4 [START_REF] Winter | xia2: an expert system for macromolecular crystallography data reduction[END_REF].

The crystal structure of SeMet-MpeQ was determined using the single-wavelength anomalous dispersion method. The initial SAD phasing was carried out using the MpeQ dataset of 2.9 Å resolution in the space group of C2221 (Shelx97) [START_REF] Sheldrick | A short history of SHELX[END_REF]). An initial model was used as a search model to determine the MpeQ structure in the space group C2 by molecular replacement (Phaser) [START_REF] Mccoy | Phaser crystallographic software[END_REF]. The final model was refined at 2.5 Å resolution with the R-factor and free R-factor of 0.232 and 0.285, respectively (Phenix.refine) [START_REF] Adams | PHENIX: a comprehensive Python-based system for macromolecular structure solution[END_REF]. The final model in the space group C2221 was refined at 2.95 Å resolution with the Rfactor and free R-factor of 0.245 and 0.307, respectively. Model building and molecular graphics were done using Coot [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF]) and/or PyMOL [START_REF] Delano | Pymol: An open-source molecular graphics tool[END_REF].

Sequence analysis and identification of switch residues

Sequence alignment of 106 bilin lyases and lyase-isomerases in the MpeQWYZ family were aligned using MUSCLE [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF]) (Dataset S1). Each sequence was assigned a lyase or lyaseisomerase function based on the published biochemical data [START_REF] Grébert | Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria[END_REF]Sanfilippo et al., 2019b;[START_REF] Shukla | Phycoerythrin-specific bilin lyaseisomerase controls blue-green chromatic acclimation in marine Synechococcus[END_REF]. Positions identical or differing between lyases and isomerases were identified using Biopython 1.77 [START_REF] Cock | Biopython: freely available Python tools for computational molecular biology and bioinformatics[END_REF] (Dataset S2). Sequence logo was generated using WebLogo [START_REF] Crooks | WebLogo: A Sequence Logo Generator[END_REF].

Enzyme activity assays based on a co-expression system

A three-plasmid co-expression system was used to test the lyase activities of MpeQ and MpeW. Specifically, MpeQ or MpeW was co-expressed in E. coli BL21(DE3) cells with MpeA (pCOLA-Duet vector) along with a pACYC-Duet vector carrying heme oxygenase (HO1) and PEB synthase (PebS) genes [START_REF] Kronfel | The roles of the chaperone-like protein CpeZ and the phycoerythrobilin lyase CpeY in phycoerythrin biogenesis[END_REF]. His-tagged MpeQ or non-tagged MpeW was then co-purified with MpeA using Co2+-affinity column (Talen). Eluted fractions were then examined by SDS polyacrylamide electrophoresis (SDS-PAGE) and absorption spectroscopy, and/or fluorescence spectroscopy. The bilin lyase activity was measured by the chromophorylated MpeA detected either by Zn-fluorescence [START_REF] Berkelman | Visualization of bilin-linked peptides and proteins in polyacrylamide gels[END_REF], absorption spectroscopy (Shimadzu UV-2600 UV-Vis spectrophotometer), and/or fluorescence spectroscopy (Perkin Elmer LS55 fluorescence spectrophotometer). MpeA-PUB and MpeA-PEB have characteristic absorption peaks at 495 nm and 550 nm, respectively, while their corresponding emission peaks are around 500 nm and 565 nm. The lyase activities of the wild type MpeQ and mutants were quantified and compared by the absorbance readings at 495 nm normalized by the corresponding MpeA band intensities detected by SDS-PAGE (ImageJ). The relative activity of a specific mutant (in percentage) was calculated in comparison to the wild type for which the lyase activity was set to 100%.

Molecular dynamics (MD) simulations

72-ns MD simulations were performed on the structures of apo-MpeA and MpeQ (PDB ID: 7MC4). The starting structure for apo-MpeA was based on a homology model of MpeA obtained by SwissModel [START_REF] Biasini | SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information[END_REF] with all the pigments removed. All minimization and MD simulation steps were performed using NAMD [START_REF] Phillips | Scalable molecular dynamics on CPU and GPU architectures with NAMD[END_REF] with CHARMM36m force field [START_REF] Huang | CHARMM36m: an improved force field for folded and intrinsically disordered proteins[END_REF]. The simulation was performed on 32 processors of a Cray T3E parallel supercomputer at University of Illinois Chicago High-Performance

Computing. Frames were collected at 100-ps intervals for the simulation length of 72 ns, giving 720 frames for conformational analysis.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical data in Table 1 for the crystallography data and structural analysis were obtained from the outputs of HKL2000 (Otwinowski and Minor, 1997) and Phenix [START_REF] Adams | PHENIX: a comprehensive Python-based system for macromolecular structure solution[END_REF].

KEY RESOURCES

----MAERFDNLVE-GLTEERAMAVILADPDSLERPVDKYMAATRLGASNSEE--SLDVL MpeW -MVPSSQRFDNLFS-WMGEQQAIELLSQNVSTLDNPGIKYIAATRLGACSSRD--SLDAL MpeZ* ----MAERFDVLVE-GISEAEALSLLFAKTADVPRPSDRYFAATRLGLCSSGA--TLDAL MpeY ----MAERFDILFE-GLSEEKALELLRTNPEDLSNPVEKYTAATRLAASRSEQ--SLEGL CpcE MIEPSVEEFPAENGPQLTPELAIANL-----QSSDLSLRYYAAWWLGKYRVKESAAVDAL PecE* ----------MTAEPILSPETAIAAL-----SGEDNQIRYYAAWWLGKHNVQA--GFTAL MpeQ* IQA-A-ELDPEHLFNRITRRKAIDALGRRKSPKALPSLFKALKCSDEAAVINSVEAITKI MpeW VVA-A-TGDRDNIFDSITRRKSIEALGRRRDLSTLPTIYEAMHSSDEQTIVNSVDTLIHF MpeZ* IRATY-ELKIDELFDRITRRKAIEALGRRKDAKAVSALVDVLGCTDTEAVINAISALIRI MpeY IEA-I-QLDPENLFNRITRRKVLEALGRRRDARALPALFTALAFDDETSVINAVDSIAQI CpcE IAALEDEADRTELGGYPLRRNAARALGKLGNRKAVPGLINCLECPD-FYVREAAAQS--L PecE* CVALFDERYRIPSGGYPLRRQAARALGLLKNPQAVPALIAALECDEDLRLREAVICS--L MpeQ* DAPLTEADHEKLLEALKGED------------I-QKRAVIQAFCRLGVPGV--INSISPL MpeW GIPLDAQFKSSLLKIIQNGS------------DVLKRVAIQCFTRLEMHEF--NGIIREQ MpeZ* GWSPSQVEHDQLLRLLDGED------------VTQSRAVIQAFGRLSIKSAASKSRIREL MpeY GAALNSNQSQKLLCALRGSD------------N-QKRSVIQTHTRLGLAGG--EEEIAAL CpcE EMLKDKTAAPALIKLLDGGVAQAVQVTGRPHLVQPYEAVLEALGAIGATDA--IPLIQPF PecE* AAIGDKRAVTPLLNLLQS------------SQAQPYEALIEALATLQVWSA--RPQIEPF MpeQ* QDDSNPLVAGAARAYMSKVALQPDGLEVLIPQLVDPIAGRRRSAVIDLGDAGDVTRLEAL MpeW ESNPNILVNGASIAYSIRVEDDKRKLHILENHLNDLNVIHRRSSVIDIGDAGDPSLLSSI MpeZ* CDHESALVSGAARSCLSKLYGEKHLMQPLLSQLTDLVAGKRRSAVIDIGDSGDHSLLFDL MpeY EQEENPLVAGAARAYAARVHGRLDRLVPLIHQLTDSIAGRRRAAVIDLGDAGDVSMLPHL CpcE LEHPVSRVQCAAARAMYQLTQEPVYGELLVKVLAGNDLNLRRVALGDLGAIGYLAAAEAI PecE* LQHYSERVQCAAARYMYLLTQESEYIERIVKNLNHDNMYLRWAAVFDLGAVAHQQAVQAI MpeQ* VTAPVSMSLRARSAFQLVD-----PDKTCQVPEKYA---ELITQLLQ-----------DN MpeW SKAAVSMPLRAKSAFKMTS-----KIK----TKSQR---TLINQMLK-----------DD MpeZ* VRAPVSMSLRAKSFLQV-D-----EDGHLLKTEKYQ---KLFKRLLL-----------DD MpeY VTCPVSMPLRAKSAFQLVD-----PEKTGTVPTAHS---DLIIQLLQ-----------DD CpcE ANAKAENSFKLIALKGLLEHQMSAESNALSISDQAIRVMNLMDSLL---------------276 PecE* LTAQVPNSLKLLNLKRILEAMLNNNSVNHE---KAALLFGAIDDLLIRQL---------- MpeQ* PQQ-LKLRKEWICDI----EPTEIENNLQHRDEARQYGGASSLMAMPKAERMILINEIKE MpeW SRH-LYFTQKFDAPN----DLEKVCDFLKHRDEERQYFGAKALFSWPASTVTDAIKTIWD MpeZ* PLL-LDLDDEWKCTL----NPEDIERNLSHRDEARQYGAAASLMQIDRRECLELIDSMQE MpeY PAM-LSLQKDWICSS----NLTEIEKNLQHRDEGKQYGGALSLMNMPQELQIETIDQLRS CpcF -PEKLVKAVQELALAKDVAAIPTLIAVFGYNNPTAAAIASTALVQLGEVAVPQLLTQIDD 73 PecF* -------MNQ---ASLSVDAITNLIEAFHHHHPAVRSAAVDELIKLGSITVNLLIAAYDD 59 MpeQ* KLWSDYVTHYYLTAVVGLQG----LEERSDLIRLALAETIPQYTKSRIA------AAWGC MpeW NHGSDYGVHYQVNCLVSQLG----LKELSYIIKESLLEPAPQYAKSKIA------ATWGC MpeZ* RLWSDYVTHYYLTCVIGLRA----LHEKSYLVRSALAETTPQYTKSRIA------AAWAC MpeY RYWSDYGANYLLTAVVGLKN----VEERSDVVRTALAETLPQYAKSRVA------AAWAC CpcF --Y-NYGARAYSIRTLAAIADPRALDVLIDAAAT---DFAP-SVR-RAAAKGLGNLHWHK PecF* --SQDQGFQAQIIQVLAQIGDAKALKLLAEVVGTSVANHCQGNVR-RIAARGLGKIASTT MpeQ* LRLGLVD------QKPLLEELSVSAFWLPLKWTCQRVLKQLS------------------ MpeW LTLGLME------CRSDIENLLLKSTWEPLRWTCAEVLREL------------------- MpeZ* VELQLDD------QLELLYELSNSSPWFPLRWACQQAFAQLVDQQQTSDDLALR------ MpeY LRLGLTD------QVELLRDIQNTATWIPLKWSCEHALKGLS------------------ CpcF LEFPDNQTAPKKALETLLF-ISQDAEW-SIRYAAIVGLQGLVNIPDLQQPIHTRLKEMLA PecF* SNTEI----INNAQEKLIWALLTPEDW-GLRYAAAVSLQEIATPKA-----KAALQQAIA CpcF SDAEKAVRARILLAQSQ 200

Figure 1 .

 1 Figure 1. Protein architecture, topology and crystal packing of MpeQ. A) Ribbon diagram ofMpeQ shows an a-solenoid "question-mark"-shaped structure with distinct E (aa. 14-255) and F (aa. 274-398) domains. A long N-terminal extension loops back to the F domain as a gate to the large interior chamber. The structure is colored in rainbow from the N-terminus (blue) to C-

Figure 2 .

 2 Figure 2. Crystal packing and dimer assemblies. A) Crystal packing of the CpcE/F structure shows two possible dimer assemblies of the E (blue) and F (green) domains, namely, the E/F assembly or E/F' assembly. In crystallography, E and F are equivalent to E' and F', respectively. B) The CpcE/F assembly proposed by Zhao et al. (PNAS, 2017) (top) shows a crescent-shaped protein architecture (bottom). C) The revised CpcE/F' assembly reveals a question-markshaped protein architecture (bottom). D) Comparisons between single-chain MpeQ (in rainbow) and two CpcE/F assemblies (E in blue; F in green). Magenta spheres mark the conserved residues (Tyr318, Pro349, Lys353 and Trp383: numbering in MpeQ; Tyr76, Pro108, Arg112 and Trp151: numbering in CpcF). See also Fig. S5.

Figure 3 .

 3 Figure 3. The active site chamber of MpeQ. A) The electrostatic surface viewed from the narrow (left) and wide (right) sides. The chamber interior is largely positively charged where Arg/Lys residues are clustered in the ceiling, wall and floor regions of the chamber. B) The catalytic triad at the floor consists of Tyr318, Lys353 and Glu285 (green spheres) located in the loops between helices in the F domain.

Figure 4 .

 4 Figure 4. Site-directed mutagenesis in the active site. A) Sites of mutation are highlighted in stick models: arginines (cyan), swap residues (magenta) and catalytic residues (green spheres). B) Conserved (green) and swap (magenta) residues form a surface patch at the chamber floor at the E/F interface. C) Coomassie blue staining (top) and Zinc fluorescence (bottom) of the same SDS-PAGE gel of MpeA obtained via co-expression with MpeQ. D) Normalized absorption

Figure 5 .

 5 Figure 5. Lyase and lyase-isomerase activities of the MpeQ variants in an E. coli coexpression system. A) The distinct sample color of MpeA co-expressed with MpeQ serves as a good indicator of the lyase and lyase-isomerase activities of MpeQ. The yellow color of MpeA-PUB is attributed to the lyase-isomerase activity while the red/orange color of MpeA-PEB is a result of the lyase activity as shown for V319G. B) Normalized absorption spectra of MpeA coexpressed with various single mutants of MpeQ. C) Relative activities of the MpeQ variants.Absorbance at 495 nm normalized by relative band intensities of MpeA (Fig.4C) is used to quantify the lyase activity. All relative activities are obtained in reference to that of wild type (WT) MpeQ (set to 100%).

Figure 6 .

 6 Figure 6. A docking model for substrates at the active site. A) The active site of MpeQ (green) is accessible from both sides of the chamber. MpeA (gray) approaches the active site from the wide opening while the PEB substrate enters from the narrow side (blue arrow). B) Viewed from the narrow side, the bound bilin (PEB in magenta; PUB in yellow) is docked at the final binding cleft in the MpeA structure (rainbow ribbon diagram). C) Viewed from the wide side, the catalytic Tyr318 is poised to activate the A-ring, which causes a steric clash between PEB and Val319. MpeQ resolves this conflict via PEB!PUB isomerization, which presents the ring A to MpeA-C83 for ligation. D) In this front view, PEB is docked with its ring A/B side lying in the cleft between the E and F domains while the propionate groups are stabilized by Arg79 and Arg228.

Figure 7 .

 7 Figure 7. Proposed reaction scheme of bilin addition reaction catalyzed by bilin lyase MpeW and lyase-isomerase MpeQ. In the first step of the reaction, the bilin substrate PEB is activated by a catalytic tyrosine in both MpeW and MpeQ resulting in a polarized C3=C3 1 double bond.

  amplified genomes (SAGs) from the Joint Genome/Institute Integrated microbial genomes & microbiomes (IMG/M) databases.Dataset S2. Summary of the residues found in 106 sequences MpeQ/W/Y/Z from marine Synechococcus isolates and single-cell amplified genomes (related to Fig.4).

  Fig. 1

  Fig. 3

Figure S1 .Figure S2 .

 S1S2 Fig. 6

Figure S3 .Figure S4 .

 S3S4 Figure S3. MpeQ is monomeric in solution. (Related to Fig. 1, Fig. 3). A) A zoom-in view of interface I highlights close intermolecular interactions between the active site (green) and residues (magenta) from the E' domain of a neighboring molecule (surface rendering). B) Elution profiles of size exclusion chromatography of MpeQ (black) and four marker (gray) proteins show that MpeQ is monomeric in solution. The number above each peak marks the corresponding molecular weight in kilo-Dalton.

Figure S5 .

 S5 Figure S5. Sequence alignment of six representative E/F type blin lyases. (Related to Fig. 2-3, Fig. 7). MpeQ/W/Z/Y are single-chain lyases while CpcE/F and PecE/F are heterodimeric lyases. Lyase-isomerases are marked with *. MpeW/Q and MpeY/Z are equivalent pairs from the CA4 strains Synechococcus A15-62 and RS9916, respectively. The sequences of CpcE/F and PecE/F are from Nostoc sp. 7120 and M. laminosus, respectively. Residues highlighted in cyan mark the conserved positions between MpeQ and CpcE/F.

Figure S6 .Figure S7 .Figure S8 .

 S6S7S8 Figure S6. Conformational flexibility of MpeQ and MpeA. (Related to Fig. 6 and STAR methods). A) Molecular dynamics (MD) simulations of MpeQ reveal two clustered conformations in the N-terminal latch region. The "close" conformations (red) correspond to that of the crystal structure while the "open" conformations (green) expose the catalytic Tyr318 (spheres). B) RMSD distance matrix of the simulated MpeQ structures reveals the highly flexible N-terminus along with three rigid frameworks (green triangles). C) MD simulations of MpeA without pigments reveals a flexible segment (aa. 63-82) in two distinct conformations. In the "close" conformation, this "cap" helps shield Cys83 from the molecular surface while the "open" conformation makes Cys83 accessible for the bilin ligation. D) RMSD distance matrix calculated from the simulated MpeA structures shows the flexible region (shaded box) showing large RMSD values while green triangles mark those rigid regions of small RMSD values. High mobility observed in the N-terminus in MpeA is likely due to the absence of b subunit in MD simulations.

Table 1 . Crystallography Data Collection and Refinement Statistics.

 1

	Structure	MpeQ SeMet	MpeQ SeMet
	Space group	C2	C2221
	Cell parameters
	a, b, c (Å)	83.84, 173.25, 114.42	136.67, 173.05, 113.7
	a, b, g (°)	90, 126.08, 90	90, 90, 90
	Residues	1-398	1-398
	Chain	A, B	A, B
	Water
	Diffraction data
	X-ray source	21-ID-F, APS	21-ID-F, APS
	Methods	Monochromatic	Monochromatic
		100 K	100 K
	Wavelength (Å)	0.97872	0.97872
	Resolution (Å)	56-2.5 (2.54-2.50)	50-2.90 (2.95-2.90)
	Rmerge	0.101 (0.605)	0.082 (0.761)
	Completeness (%)
	All	99.5 (99.7)	99.8 (95.8)
	Anomalous	97.2 (84.7)	99.8 (95.5)
	Redundancy	3.7 (3.5)	3.9 (1.6)
	I/σ(I)	7.4 (1.3)	4.7 (1.0)
	Refinement
	Resolution (Å)	20-2.50 (2.79-2.50)	39.3-2.95 (3.01-2.95)
	R	0.239 (0.279)	0.242 (0.361)
	Rfree	0.284 (0.329)	0.298 (0.303)
	RMSD
	Bond length (Å)	0.009	0.008
	Bond angle (°)	1.013	1.248
	Average B (Å 2 )	28.63	98.9
	Ramachandran
	Favored (%)	96.6	96.1
	Allowed (%)	2.0	3.3
	Disallowed (%)	0.4	0.55
	PDB entry	#7MC4	7MCH

TABLE REAGENT

 REAGENT

	pCDF Duet-1 vector	Novagen	CAT#:71340-3
	pET Duet-1 vector	Novagen	CAT#:71146-3
	Plasmid: A1562 Nus-MpeQ/pET44b	(Grébert et al., 2021) N/A
	Plasmid: A1562 Nus-MpeW/pET44b	(Grébert et al., 2021) N/A
	Plasmid: A1562 HT-MpeA/pCOLA	(Grébert et al., 2021) N/A
	Plasmid: NT-PebS/NT-HOI/pACYC	(Kronfel et al., 2019)	N/A
	Plasmid: A1562 NT-MpeQ/pCDF	This study	N/A
	Plasmid: A1562 HT-MpeQ/pCDF	This study	N/A
	Plasmid: A1562 NT-MpeW/pET	This study	N/A
	See Table S4 for more recombinant DNA
	or RESOURCE	SOURCE	IDENTIFIER
	Bacterial and virus strains
	Escherichia coli: DH5a	Invitrogen	CAT#:18-258-012
	Escherichia coli: BL21(DE3)	Invitrogen	CAT#:C600003
	Chemicals, peptides, and recombinant proteins
	Selenomethionine	Acros Organics	CAT#: 3211-76-5
	TALON resin	Takara	CAT#: 635504
	DL-Malic acid	Sigma Aldrich	LOT#: MKBW2323V
	Synechococcus sp. A1562 MpeQ	This study	Genbank: AGW21721
	Synechococcus sp. A1562 MpeW	This study	Genbank: AGW21717
	Synechococcus sp. A1562 MpeA	This study	Genbank: AVH76704
	See Table S2 for more recombinant proteins	This study
	Critical Commercial Assays
	GeneArt Seamless Cloning and Assembly	Invitrogen	CAT#: A14606
	Enzyme Mix
	Deposited data
	MpeQ crystal structure in space group C2	This paper	PDB:7MC4
	MpeQ crystal structure in space group C2221 This paper	PDB:7MCH
	Recombinant DNA

Table S1 . Summary of MpeQ mutants

 S1

	A Wild Type (WT) Normalized Absorbance Y318A or Y318F		MpeA-PUB* High not detected	PUB(491nm) PEB(560nm) WT load WT FT WT elute MpeA-PEB PUB:PEB Lyase vs. isomerase not detected 10:0 lyase-isomerase not detected n/a abolished/no switch	Comments catalytic
		K353A		very low		not detected		n/a	abolished/no switch	A-ring interaction
		300 E285A	350	400 very low		450 not detected	500	550 0.5:0	600 reduced/no switch 650	700 A-ring interaction
		W383F		medium		Wavelength (nm) not detected 2:0	reduced/no switch	MpeA binding
	B	R71A or R71D R79A R79D R135D R143D Normalized Absorbance R198A		very low very low not detected not detected very low very low	PUB(493 nm) PEB(546 nm) MpeW G319V+V320T MpeW WT MpeW G319V MpeW V320T not detected n/a abolished/no switch not detected n/a reduced/no switch not detected n/a abolished/no switch not detected n/a abolished/no switch not detected 0.1:0 reduced/no switch not detected n/a abolished/no switch	MpeA binding Propionate interaction Propionate interaction MpeA binding MpeA binding Substrate binding
		R198K		low		not detected		3:0	reduced/no switch	Substrate binding
		400 R228A	450 not detected		500 not detected	550	n/a	600 abolished/no switch 650	700 propionate interaction
		R228D		low		Wavelength (nm) not detected 0.7:0	reduced/no switch	propionate interaction
	C	R287A		high		not detected			reduced/no switch	substrate binding
		V319G		high		low		10:1	high/partial switch	Swap/active site
		V319A		high		Very low		10:0.5	high/partial switch	Swap/active site
		V319L		medium		not detected		5:0	reduced/partial	Swap/active site
									switch
		V319F		very low		not detected		n/a	abolished/no switch	Swap/active site
		A100T		high		not detected		4:0	high/no switch	Swap/remote site
		S222A		high		not detected		3:0	reduced/no switch	Swap site
		S224P		medium		not detected		6:0	high/no switch	Swap site
		T320A		high		not detected		4:0	high/no switch	Swap site
		Y323Q		high		not detected		4:0	high/no switch	Swap site
		Y323L		medium		not detected		2:0	high/no switch	Swap site
		T352A		high		not detected			high/no switch	Swap site
		V319G/T320A		high		low		3:1	high/partial switch	Double swap sites
		V319G/Y323Q		medium		low		3:1	medium/partial switch	Double swap sites
		T320A/Y323Q		very low		not detected		n/a	abolished/no switch	Double swap sites
		A100T/T352A		high		not detected		3:0	high/no switch	Double swap sites
	V319G/T320A/Y323Q		medium		low		3:2	high/partial switch	Triple swap sites
	V319G/A100T/T320A/		medium		medium		2:3	medium/switch	Five swap sites
	Y323Q/T352A (Q5.1)
	V319G/S224P/T320A/		medium		high		1:3	medium/switch	Five swap sites
	Y323Q/T352A (Q5.2)

Note: *Compared to WT, High: >75%; medium: 25-50%; low: <25%; very low: <10%

Acknowledgement

We thank the staff of the Life Science Consortium Access Team (LS-CAT) at the Advanced Photo Source (APS) for support in X-ray diffraction data collection. Use of the LS-CAT Sector 21 is supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor under Grant 085P1000817. Use of the Advanced Photon Source is supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. This work is supported by the National Science Foundation grant MCB 2017171 to WS and MCB 2017274 to XY, the National Institutes of Health grant R01EY024363 to XY and the French national agency for research (ANR) program EFFICACY (ANR-19-CE02-0019) to FP.

Software and algorithms HKL2000 (Otwinowski & Minor, 1997) https://hkl-xray.com/hkl-2000 XIA2 (CCP4) (Winter, 2010) https://www.ccp4.ac.uk/ Shelx97 (CCP4) (Sheldrick, 2008) https://www.ccp4.ac.uk/ Phaser (Phenix) (McCoy et al., 2007) http://www.phenixonline.org/ Phenix.refine (Adams et al., 2010) http://www.phenixonline.org/ Coot (Emsley and Cowtan, 2004) https://www2.mrclmb.cam.ac.uk/personal /pemsley/coot/ PyMOL (DeLano, 2002) https://pymol.org/2/ MUSCLE (Edgar, 2004) https://www.ebi.ac.uk/T ools/msa/muscle/ BioPython (Cock et al., 2009) https://biopython.org/ WebLOGO (Crooks, 2004) http://weblogo.threeplus one.com/ SwissMODEL (Biasini et al., 2014) https://swissmodel.expa sy.org/ NAMD (Phillips et al., 2020) https://www.ks.uiuc.edu /Research/namd/

Author contributions

Conceptualization, XY and WMS; Methodology, IK, KLJ, WMS and XY; Investigation, IK, KLJ, LB, IT, KDW, CW, WMS and XY; Analysis, IK, KLJ, TG, FP, WMS and XY; Writing -Original Draft, XY; Writing -Review and Editing, XY, WMS, TG and FP; Funding Acquisition, XY, WMS and FP; Resources, IK and KLJ; Supervision, XY and WMS.

Declaration of interests

The authors declare no competing interests.

SUPPLEMENTAL TABLES AND DATASETS

Table S1. Summary of MpeQ mutants (related to Fig. 4 and Fig. 5).

Table S2. Supplemental STAR methods Oligonucleotides (related to the STAR Methods Method Details). Table S3. Supplemental STAR methods Recombinant DNAs (related to the STAT Methods Experimental Model and Subject Details).

Table S4. Supplemental STAR methods