Catalytic Mechanism of S-type Phycobiliprotein Lyase

Abstract

We now show that CpcS1 binds PCB and PEB rapidly with bi-exponential kinetics (38/119 and 12/8300 ms, respectively). Chromophore binding to the lyase is reversible and much faster than the spontaneous, but low fidelity chromophore addition to the apo-protein in the absence of the lyase. This indicates kinetic control by the enzyme, which then transfers the chromophore to the apo-protein in a slow (tens of minutes) but stereo-and regioselectively corrects the reaction. This mode of action is reminiscent of chaperones but does not require ATP. The amino acid residues Arg-18 and Arg-149 of the lyase are essential for chromophore attachment in vitro and in Escherichia coli, mutations of His-21, His-22, Trp-75, Trp-140, and Arg-147 result in reduced activity (<30% of wild type in vitro). Mutants R147Q and W69M were active but had reduced capacity for PCB binding; additionally, with W69M there was loss of fidelity in chromophore attachment. Imidazole is a noncompetitive inhibitor, supporting a bilin-binding function of histidine. Evidence was obtained that CpcS1 also catalyzes exchange of C-␤84-bound PCB in biliproteins by PEB.Phycobiliproteins are a homologous family of light-harvesting proteins present in cyanobacteria, red algae, and cryptophytes that absorb light in the spectral region between the chlorophyll absorption maxima at ϳ430 and ϳ680 nm: they contain linear tetrapyrroles (phycobilins) of which 1-4 are covalently attached to the subunits by thioether bonds to conserved cysteines (1-4). The chromophores are derived from heme by oxidative ring-opening and subsequent reduction (5) and then attached post-translationally. The correct attachment of most chromophores is catalyzed by lyases. Three phylogenetically unrelated types of lyases have been characterized in cyanobacteria (6). They are specific for certain binding sites and chromophores (7-16), but the reaction mechanisms are still unclear. A chaperone-like action has been proposed for E/Ftype lyases that catalyze chromophore attachment to Cys-84 of ␤-subunits of phyco(erythro)cyanins (17, 18). A more highly evolved function is suggested, however, by concomitant isomerizing reactions catalyzed by certain lyases (12,19,20), and by the finding that most lyases can bind the chromophore and then transfer it to the acceptor protein (15,21).Chromophore binding is particularly pronounced with the S-type lyases. CpcS1 from Nostoc PCC7120 6 rapidly forms an adduct with phycocyanobilin (PCB), 7 and the latter can be transferred in a much slower reaction to cysteine 84 of the ␤-subunits of phycocyanin (CpcB) or phycoerythrocyanin (PecB), suggesting that PCB-CpcS1 is an intermediate of the enzymatic reaction (21). Model reactions of PCB with nucleophiles resulted in the formation of addition products in which the chromophore is isomerized and which are also substrates for a CpcS1-catalzed chromophore transfer to the apo-proteins (20). We now report chromophore binding kinetics obtained by stopped-flow techniques, and identification of functional amino acid residues f...

CpeS Is a Lyase Specific for Attachment of 3Z-PEB to Cys82 of β-phycoerythrin from Prochlorococcus marinus MED4

“…The resulting K d value of the CpeS⅐3E-PEB complex was 0.7 M, which represents tight binding and which is driven by both favorable enthalpy (⌬H 0 ϭ Ϫ6.1 Ϯ 0.5 kcal/mol) as well as entropy (⌬S 0 ϭ 7.3 Ϯ 1.3 cal/mol/deg) changes upon complex formation. The K d determined for the CpeS⅐3E-PEB complex is in range with values obtained for other lyases (8,39,41,42).…”

“…However, DHBV-transferring lyases are at least feasible for cryptophytes with DHBV-containing phycobiliproteins (47). As lyases are also able to detach bilins (8,41,48), CpeS might remove incorrectly bound DHBV from CpeB, but this could not yet be verified in preliminary studies (data not shown).…”

“…PCC7120 and CpcE/CpcF from Synechococcus sp. PCC7002 bind both PEB and PCB (8,35,41). One might argue that these species synthesize only PCB but not PEB, so that discrimination between these two bilins is unnecessary.…”

“…Because members of the S/U type lyase family are characterized by a low bilin specificity (6,8), we tested CpeS for binding of all bilins synthesized by MED4. We included 3Z-and 3E-isomers of PCB and PEB to address the largely unstudied bilin stereoselectivity of lyases.…”

“…Subsequent bilin attachment to phycobiliproteins is in most cases supported by members of the three known phycobiliprotein lyase families (E/F, S/U, and T) (6). These proteins are supposed to guide the reaction in a chaperone-like manner probably by conformational control of the bilin (7,8). In addition, some lyases act as isomerases and generate phycoviolobilin or phycourobilin upon attachment to the phycobiliprotein (9 -11).…”