title: Structure and mechanism of the phycobiliprotein lyase CpcT.
abstract: Pigmentation of light-harvesting phycobiliproteins of cyanobacteria requires covalent attachment of open-chain tetrapyrroles, bilins, to the apoproteins. Thioether formation via addition of a cysteine residue to the 3-ethylidene substituent of bilins is mediated by lyases. T-type lyases are responsible for attachment to Cys-155 of phycobiliprotein β-subunits. We present crystal structures of CpcT (All5339) from Nostoc (Anabaena) sp. PCC 7120 and its complex with phycocyanobilin at 1.95 and 2.50 Å resolution, respectively. CpcT forms a dimer and adopts a calyx-shaped β-barrel fold. Although the overall structure of CpcT is largely retained upon chromophore binding, arginine residues at the opening of the binding pocket undergo major rotameric rearrangements anchoring the propionate groups of phycocyanobilin. Based on the structure and mutational analysis, a reaction mechanism is proposed that accounts for chromophore stabilization and regio- and stereospecificity of the addition reaction. At the dimer interface, a loop extending from one subunit partially shields the opening of the phycocyanobilin binding pocket in the other subunit. Deletion of the loop or disruptions of the dimer interface significantly reduce CpcT lyase activity, suggesting functional relevance of the dimer. Dimerization is further enhanced by chromophore binding. The chromophore is largely buried in the dimer, but in the monomer, the 3-ethylidene group is accessible for the apophycobiliprotein, preferentially from the chromophore α-side. Asp-163 and Tyr-65 at the β- and α-face near the E-configured ethylidene group, respectively, support the acid-catalyzed nucleophilic Michael addition of cysteine 155 of the apoprotein to an N-acylimmonium intermediate proposed by Grubmayr and Wagner (Grubmayr, K., and Wagner, U. G. (1988) Monatsh. Chem. 119, 965-983).
authors: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors: Wei
authors: W
authors: Zhou
authors: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors: Wen-Long
authors: WL
authors: Ding
authors: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors: Xiao-Li
authors: XL
authors: Zeng
authors: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors: Liang-Liang
authors: LL
authors: Dong
authors: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors: Bin
authors: B
authors: Zhao
authors: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors: Ming
authors: M
authors: Zhou
authors: Department of Biologie I, Universität München, Menzinger Str. 67, D-80638 München, Germany.
authors: Hugo
authors: H
authors: Scheer
authors: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China,. Electronic address: khzhao@163.com.
authors: Kai-Hong
authors: KH
authors: Zhao
authors: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, and; Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607. Electronic address: xiaojing@uic.edu.
authors: Xiaojing
authors: X
authors: Yang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008190:Lyases / Q000737:chemistry* / Q000235:genetics; D046937:Nostoc / Q000201:enzymology* / Q000235:genetics; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D008190:Lyases; C093743:phycoerythrocyanin alpha-subunit phycobiliviolin lyase protein, bacteria
keywords: Crystal Structure; Cyanobacteria; Lyase; Photosynthetic Pigment; Phycobiliprotein; Stereoselectivity; X-ray Crystallography