Crystal structure of the first eukaryotic bilin reductase GtPEBB reveals a flipped binding mode of dihydrobiliverdin.

Phycobilins are light-harvesting pigments of cyanobacteria, red algae, and cryptophytes. The biosynthesis of phycoerythrobilin (PEB) is catalyzed by the subsequent action of two ferredoxin-dependent bilin reductases (FDBRs). Although 15,16-dihydrobiliverdin (DHBV):ferredoxin oxidoreductase (PebA) catalyzes the two-electron reduction of biliverdin IXα to 15,16-DHBV, PEB:ferredoxin oxidoreductase (PebB) reduces this intermediate further to PEB. Interestingly, marine viruses encode the FDBR PebS combining both activities within one enzyme. Although PebA and PebS share a canonical fold with similar substrate-binding pockets, the structural determinants for the stereo- and regiospecific modification of their tetrapyrrole substrates are incompletely understood, also because of the lack of a PebB structure. Here, we solved the X-ray crystal structures of both substrate-free and -bound PEBB from the cryptophyte Guillardia theta at 1.90 and 1.65 Å, respectively. The structures of PEBB exhibit the typical α/ß/α-sandwich fold. Interestingly, the open-chain tetrapyrrole substrate DHBV is bound in an unexpected flipped orientation within the canonical FDBR active site. Biochemical analyses of the WT enzyme and active site variants identified two central aspartate residues Asp-99 and Asp-219 as essential for catalytic activity. In addition, the conserved Arg-215 plays a critical role in substrate specificity, binding orientation, and active site integrity. Because these critical residues are conserved within certain FDBRs displaying A-ring reduction activity, we propose that they present a conserved mechanism for this reaction. The flipped substrate-binding mode indicates that two-electron reducing FDBRs utilize the same primary site within the binding pocket and that substrate orientation is the determinant for A- or D-ring regiospecificity.

Evolution and molecular mechanism of four-electron reducing ferredoxin-dependent bilin reductases from oceanic phages.

Ferredoxin-dependent bilin reductases (FDBRs) are a class of enzymes reducing the heme metabolite biliverdin IXα (BV) to form open-chain tetrapyrroles used for light-perception and light-harvesting in photosynthetic organisms. Thus far, seven FDBR families have been identified, each catalysing a distinct reaction and either transferring two or four electrons from ferredoxin onto the substrate. The newest addition to the family is PcyX, originally identified from metagenomics data derived from phage. Phylogenetically, PcyA is the closest relative catalysing the reduction of BV to phycocyanobilin. PcyX, however, converts the same substrate to phycoerythrobilin, resembling the reaction catalysed by cyanophage PebS. Within this study, we aimed at understanding the evolution of catalytic activities within FDBRs using PcyX as an example. Additional members of the PcyX clade and a remote member of the PcyA family were investigated to gain insights into catalysis. Biochemical data in combination with the PcyX crystal structure revealed that a conserved aspartate-histidine pair is critical for activity. Interestingly, the same residues are part of a catalytic Asp-His-Glu triad in PcyA, including an additional Glu. While this Glu residue is replaced by Asp in PcyX, it is not involved in catalysis. Substitution back to a Glu failed to convert PcyX to a PcyA. Therefore, the change in regiospecificity is not only caused by individual catalytic amino acid residues. Rather the combination of the architecture of the active site with the positioning of the substrate triggers specific proton transfer yielding the individual phycobilin products. ENZYMES: Suggested EC number for PcyX: 1.3.7.6 DATABASES: The PcyX X-ray structure was deposited in the PDB with the accession code 5OWG.