Chromophore Attachment to Biliproteins:  Specificity of PecE/PecF, a Lyase-Isomerase for the Photoactive 3¹-Cys-α84-phycoviolobilin Chromophore of Phycoerythrocyanin

Abstract

PecE and PecF, the products of two phycoerythrocyanin lyase genes (pecE and pecF) of Mastigocladus laminosus (Fischerella), catalyze two reactions: (1) the regiospecific addition of phycocyanobilin (PCB) to Cys-alpha 84 of the phycoerythrocyanin alpha-subunit (PecA), and (2) the Delta 4-->Delta 2 isomerization of the PCB to the phycoviolobilin (PVB)-chromophore [Zhao et al. (2000) FEBS Lett. 469, 9-13]. The alpha-apoprotein (PecA) as well PecE and PecF were overexpressed from two strains of M. laminosus, with and without His-tags. The products of the spontaneous addition of PCB to PecA, and that of the reaction catalyzed by PecE/F, were characterized by their photochemistry and by absorption, fluorescence, circular dichroism of the four states obtained by irradiation with light (15-Z/E isomers of the chromophore) and/or modification of Cys-alpha 98/99 with thiol-directed reagents. The spontaneous addition leads to a 3(1)-Cys-PCB adduct, which is characteristic of allophycocyanins and phycocyanins, while the addition catalyzed by PecE and PecF leads to a 3(1)-Cys-PVB adduct which after purification was identical to alpha-PEC. The specificity and kinetics of the chromophore additions were investigated with respect to the structure of the bilin substrate: The 3-ethylidene-bilins, viz., PCB, its 18-vinyl analogue phytochromobilin, phycoerythrobilin and its dimethylester, react spontaneously to yield the conventional addition products (3-H, 3(1)-Cys), while the 3-vinyl-substituted bilins, viz., bilirubin and biliverdin, were inactive. Only phycocyanobilin and phytochromobilin are substrates to the addition-isomerization reaction catalyzed by PecE/F. The slow spontaneous addition of phycoerythrobilin is not influenced, and there is in particular no catalyzed isomerization to urobilin.

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.…”

Phycoerythrin-specific bilin lyase–isomerase controls blue-green chromatic acclimation in marineSynechococcus

“…This enzyme is unique among phycobilin lyase-isomerases described so far because it chromophorylates phycoerythrin, specifically PEII (MpeA). The only other such enzymes known are PecE/PecF, which bind phycocyanobilin at C84 of the phycoerythrocyanin α-subunit and isomerize it to a phycoviolobilin (21,22), and RpcG, which ligates PEB at C84 of a phycocyanin α-subunit and isomerizes it to PUB (8). All three enzymes belong to the E/F clan of phycobilin lyases, characterized by the presence of an α/α-superhelix fold and Armadillo repeat motifs (23)(24)(25), although MpeZ is only distantly related to PecE (27-32% identity and 45-47% homology) and the N terminus of RpcG (23-25% identity and 42-43% homology) and matches just a short part of PecF (29% identity and 53-55% homology over 57 residues) (Fig.…”

Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits

“…PEB was the generous gift of Dr. Hugo Scheer and Dr. Max Storf (University of Munich, Germany) [15].…”

“…Non-enzymatic (autocatalytic) attachment of phycobilins to genetically expressed aposubunits of phycobiliproteins is also possible since phycobilins can be isolated and purified from phycobiliproteins after reflux in methanol [14,15]. Attachment of phycobilins to recombinant apo-phycobiliproteins from cyanobacteria has been investigated in vitro.…”

“…Products of in vitro attachment of PEB to apo-alpha subunit of C-phycoerythrin (C-PE) include covalently bound PEB, DBV, and urobilin (UB) [19]. After prolonged sonication of apo-subunits located in inclusion bodies, incubation of apo-alpha subunit of phycoerythrocyanin with PCB and PEB yielded PCB and PEB adducts, respectively [15].…”