In Vitro Hydrolysis of Zinc Chlorophyllide a Homologues by a BciC Enzyme

Abstract

Chlorosomes in green photosynthetic bacteria are the largest and most efficient light-harvesting antenna systems of all phototrophs. The core part of chlorosomes consists of bacteriochlorophyll c, d, or e molecules. In their biosynthetic pathway, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. In this study, the in vitro enzymatic reactions of chlorophyllide a analogues, C132-methylene- and ethylene-inserted zinc complexes, were examined using a BciC protein from Chlorobaculum tepidum. As the products, their hydrolyzed free carboxylic acids were observed without the corresponding demethoxycarbonylated compounds. The results showed that the in vivo demethoxycarbonylation of chlorophyllide a by an action of the BciC enzyme would occur via two steps: (1) an enzymatic hydrolysis of a methyl ester at the C132-position, followed by (2) a spontaneous (nonenzymatic) decarboxylation in the resulting carboxylic acid.

Predicted Structure of the BciC Enzyme Catalyzing the Removal of the C132-Methoxycarbonyl Group for Biosynthesis of Chlorosomal Chlorophylls: A Mechanism for Dual Catalytic Functions of Hydrolysis and Decarboxylation inside Its Active Site

“…The H137A mutant regio-and stereoselectively hydrolyzed the C13 2 -COOCH 3 ester of Zn-Me-Pheide-a to give the corresponding (13 2 R)-COOH as the major product. Although the BciC enzymatic reactions of Chl derivatives with modified functional groups on the E ring (Figure S1) have already afforded Chl derivatives possessing a free carboxy group in the C13 2 -substituent, 33,34 the β-keto-carboxylic acid was first detected in this study.…”

“… ,,, However, no C13 2 -COOH intermediate bearing the natural E ring had been detected, except in the BciC enzymatic reactions of Chl analogues possessing the 13 1 -hydroxy, 13 2 -methylene, or 13 2 -ethylene group (Figure S1). , Hence, the former mechanism is preferable where the C13 2 -COOH product was decarboxylated through the conformational rotation inside the active site of the BciC enzyme.…”

“…A BciC enzyme was speculated to branch the biosynthetic pathways of the former two pigments and chlorosomal BChl- c molecules. , The BciC enzyme catalyzes the removal of a methoxycarbonyl group at the C13 2 -position in Chlide- a to pyroChlide- a (Figure ). It has been speculated that the in vivo BciC-catalyzed reaction mechanism occurs through the following two sequential steps: , (1) enzymatic hydrolysis of the C13 2 -methyl ester on the E ring [Figure (i)], followed by (2) spontaneous (non-enzymatic) decarboxylation in the resulting carboxylic acid due to its chemically labile β-keto-carboxylic acid [Figure (ii)]. The BciC-catalyzed demethoxycarbonylation mechanism was supported by the detection of C13 2 -COOH intermediates using model substrates with modified functional groups on the E ring (Figure S1).…”

“…The BciC-catalyzed demethoxycarbonylation mechanism was supported by the detection of C13 2 -COOH intermediates using model substrates with modified functional groups on the E ring (Figure S1). , No intermediates bearing the natural E ring have yet been observed (Figure , middle). In this study, the three-dimensional structure of the BciC enzyme and its active site were predicted by AlphaFold2, , and in vitro enzymatic reactions using BciC, which mutated the amino acid residues in the expected active site, were examined.…”