Abstract
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/β/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.
Highlights
Phycobilins are light-harvesting pigments of cyanobacteria, red algae, and cryptophytes
The flipped substrate-binding mode indicates that two-electron reducing ferredoxin-dependent bilin reductases (FDBRs) utilize the same primary site within the binding pocket and that substrate orientation is the determinant for Aor D-ring regiospecificity
The biosynthesis is initiated through the binding of BV by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA); once bound, the substrate is protonated by a highly conserved aspartate residue located on the central -sheet of the enzyme
Summary
Version of BV to PCB via a bound intermediate 181,182-DHBV, and phycoerythrobilin synthases PebS/PcyX that both catalyze the reduction of BV via 15,16-DHBV to PEB [6, 9, 10, 16, 17]. We solved the crystal structure of the PebB homolog PEBB (nuclear encoded proteins in eukaryotes are denoted with capital letters by convention) from the eukaryotic cryptophyte alga Guillardia theta. This organism evolved by secondary endosymbiosis and employs soluble phycobiliproteins in the thylakoid lumen of the chloroplast [7, 18]. The new crystal structures of GtPEBB presented gave some unexpected insights into the A-ring reduction of phycobilins. They thereby contribute to a better understanding of the underlying catalytic mechanism
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.