Structural and Biochemical Characterization of the Bilin Lyase CpcS from Thermosynechococcus elongatus

Abstract

Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded beta barrel with two alpha helices and belongs to the lipocalin structural family. TeCpcS-III catalyzes both cognate as well as non-cognate bilin attachment to a variety of phycobiliprotein subunits. TeCpcS-III ligates phycocyanobilin, phycoerythrobilin and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of TeCpcS-III is a dimer, which is consistent with the structure observed in the crystal. Using the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and TeCpcS was produced.

Insights into the Biosynthesis and Assembly of Cryptophycean Phycobiliproteins

“…Binding studies with these GtCPES variants and different phycobilins revealed a participation of Glu-136 and Arg-146 in phycobilin binding. The importance of Arg-146 or homologous amino acid residues for phycobilin binding has previously been confirmed for TeCpcS and NCpcS-III (where N is Nostoc) (23,68). Interestingly, the TeCpcS-R151G variant still bound enough PCB to confer transfer to the apoprotein CpcB in E. coli (68).…”

“…The importance of Arg-146 or homologous amino acid residues for phycobilin binding has previously been confirmed for TeCpcS and NCpcS-III (where N is Nostoc) (23,68). Interestingly, the TeCpcS-R151G variant still bound enough PCB to confer transfer to the apoprotein CpcB in E. coli (68). For Glu-136, the carboxylic function might be involved in positioning of the substrate by interaction with the tetrapyrrole nitrogens, as found for bilin-binding in FDBRs (51,73,74).…”

“…Furthermore, the structure of the PBP lyase TeCpcS was determined within the National Institutes of Health Protein Structure Initiative in 2007 and published by Kronfel et al (68). TeCpcS is a homodimeric universal PBP lyase capable of binding a wide range of phycobilins like PCB, PEB, and P⌽B, transferring them to residue Cys-84 of diverse ␣ and ␤ apo-PBPs (68). Utilizing the similarity to UnaG, which has been crystallized with bound bilirubin, a docking model for an extended PCB molecule in TeCpcS was described, which buries the bilin's D-ring in the barrel and exposes the A-ring on the barrel mouth.…”

“…Previous studies focused on PBP lyases like TeCpcS and NCpcS-III, which bind and transfer a broad range of phycobilins (PCB, PEB, and P⌽B) (23,68). In contrast, GtCPES is only capable of binding phycobilins with a reduced C15ϭC16 double bond (DHBV and PEB).…”

Structure and Mechanism of the Phycobiliprotein Lyase CpcT

“…CpcS transiently binds bilins such as PCB (14). The crystal structures of a cyanobacterial CpcS (PDB code 3BDR) 5 (11,28) and a cryptophytes CPES (29) have been determined in the absence of a chromophore. They both adopt a ␤-barrel structure similar to those of fatty acid-binding proteins (FABP), a subfamily of the calycin superfamily (Pfam0116) that binds a variety of small, mostly lipophilic molecules with high selectivity (30 -32).…”