Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon

Abstract

The structure of phycoviolobilin, the photoactive chromophore of K K-phycoerythrocyanin, is incompatible with a chromophore ligation to the apoprotein via SH-addition (cysteine) to a v v3,3 1 -double bond of the phycobilin. The two putative phycoerythrocyanin lyase genes of Mastigocladus laminosus, pecE and pecF, were overexpressed in Escherichia coli. Their action has been studied on the addition reaction of phycocyanobilin to apo-K K-phycoerythrocyanin (PecA). In the absence of the components of K K-PEC-phycoviolobilin lyase PecE and PecF, or in the presence of only one of them, phycocyanobilin binds covalently to PecA forming a fluorescent chromoprotein with a red-shifted absorption (V V max = 641 nm) and low photoactivity ( 6 10%). In the presence of both PecE and PecF, a chromoprotein forms which by its absorption (V V max = 565 nm) and high photoreversible photochromism (100% type I) has been identified as integral K K-phycoerythrocyanin. We conclude that PecE and PecF jointly catalyze not only the addition of phycocyanobilin to PecA, but also its isomerization to the native phycoviolobilin chromophore.z 2000 Federation of European Biochemical Societies.

Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

“…16 In a R-phycocyanin 34 and phycoerythrin, 35 the isomerization is catalyzed by a lyase. The reaction is similar to the isomerization of PCB to PVB that is lyase-catalyzed in the α-subunit of phycoerythrocyanin, 36,37 and autocatalytic in several CBCRs that contain GAF domains carrying a conserved DXCF motif. 4,[13][14][15][16] For GAFs of group 3, no PEB (PUB) binding could be determined (Fig.…”

Insights into the Biosynthesis and Assembly of Cryptophycean Phycobiliproteins

“…PBP lyases are distinguishable in the clades of E/F-, S/U-, and T-type lyases, and some members of the E/F-type have an additional isomerase function (25)(26)(27).…”

Structure and Mechanism of the Phycobiliprotein Lyase CpcT

“…Moreover, this disulfide bond may be functionally relevant. The CpcT lyase reaction is inhibited by thiols (25), although other lyases, like CpcE/F (13,19) and CpcS (24), tolerate thiols at the concentrations used; and PecE/F (15,21) are even activated by thiols. Activation has also been found for autocatalytic chromophorylation of ApcE (7).…”

“…Last, but not least, it should be noted that deprotonation of the cationic acylimmonium intermediate (Fig. 11) at C2 rather than C5 would result in the conversion of PCB to a phycoviolobilin chromophore; such isomerization occurs in E/F-type lyases (15,16,67) as well as in several cyanobacteriochromes during autocatalytic chromophore attachment (68 -70).…”

“…They are thought to act like chaperones or protective carriers for labile bilin chromophores (6,13,14), but additional functions have been found (15)(16)(17). E/F-type lyases are specific for attaching phycocyanobilin (PCB) to a conserved cysteine (Cys-␣84) in ␣-subunits of phycocyanin (CPC) and phycoerythrocyanin (18,19); homologous CpeY/Z catalyze addition of phycoerythrobilin to the equivalent site in phycoerythrin (20).…”