Chromophore attachment in phycocyanin. Functional amino acids of phycocyanobilin--alpha-phycocyanin lyase and evidence for chromophore binding.
Covalent attachment of phycocyanobilin (PCB) to the alpha-subunit of C-phycocyanin, CpcA, is catalysed by the heterodimeric PCB : CpcA lyase, CpcE/F [Fairchild CD, Zhao J, Zhou J, Colson SE, Bryant DA & Glazer AN (1992) Proc Natl Acad Sci USA89, 7017-7021]. CpcE and CpcF of the cyanobacterium, Mastigocladus laminosus PCC 7603, form a 1 : 1 complex. Lyase-mutants were constructed to probe functional domains. When in CpcE (276 residues) the N terminus was truncated beyond the R33YYAAWWL motif, or the C terminus beyond amino acid 237, the enzyme became inactive. Activity decreases to 20% when C-terminal truncations went beyond L275, which is a key residue: the K(m) of CpcE(L275D) and (L276D) increased by 61% and 700%, k(cat)/K(m) decreased 3- and 83-fold, respectively. The enzyme also lost activity when in CpcF (213 residues) the 20 N-terminal amino acids were truncated; truncation of 53 C-terminal amino acids inhibited complex formation with CpcE, possibly due to misfolding. According to chemical modifications, one accessible arginine and one accessible tryptophan are essential for CpcE activity, and one carboxylate for CpcF. Both subunits bind PCB, as assayed by Ni2+ affinity chromatography, SDS/PAGE and Zn2+-induced fluorescence. The bound PCB could be transferred to CpcA to yield alpha-CPC. The PCB transfer capacity correlates with the activity of the lyase, indicating that PCB bound to CpcE/F is an intermediate of the enzymatic reaction. A catalytic mechanism is proposed, in which a CpcE/F complex binds PCB and adjusts via a salt bridge the conformation of PCB, which is then transferred to CpcA.
Zhao
Kai-Hong
KH
College of Life Science and Technology, Huazhong University of Science and Technology, Hubei, China. kaihongzhao@tom.com
Wu
Dong
D
Zhang
Ling
L
Zhou
Ming
M
Böhm
Stephan
S
Bubenzer
Claudia
C
Scheer
Hugo
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D000458:Cyanobacteria; D008190:Lyases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D052980:Phycobilins; D045524:Phycobilisomes; D010798:Phycocyanin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010860:Pigments, Biological; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D045725:Tetrapyrroles / Q000378:metabolism*
D000596:Amino Acids; D052980:Phycobilins; D045524:Phycobilisomes; D010860:Pigments, Biological; D045725:Tetrapyrroles; D010798:Phycocyanin; C011069:phycocyanobilin; D008190:Lyases; C075919:phycocyanin alpha-subunit phycocyanobilin lyase