In vitro reversible dehydration in C3-substituents of zinc chlorophyll analogs by BchF and BchV enzymes: Stereoselectivity and substrate specificity in the dehydration

Abstract

In the biosynthetic pathway of bacteriochlorophyll(BChl)-a/b/c/d/e molecules, BchF and BchV enzymes catalyze the hydration of a C3-vinyl to C3-1-hydroxyethyl group. In this study, the in vitro reactions catalyzed by BchF and BchV partially afforded a C3¹-epimeric mixture of the hydrated products (secondary alcohols), with the primary recovery of the C3-vinylated substrate. The stereoselectivity and substrate specificity for the in vitro reverse enzymatic dehydration were examined using zinc chlorophyll analogs as model substrates by BchF and BchV, which were obtained from extracts of Escherichia coli overexpressing the respective genes from Chlorobaculum tepidum and used without further purification. Both BchF and BchV preferred dehydration of the (3¹R)-epimers over the (3¹S)-epimers. The (3¹R)-epimer was directly dehydrated by BchF and BchV to give the C3-vinylated product. By contrast, two reaction pathways for BchF and BchV dehydrations of the (3¹S)-epimer were proposed: (1) the (3¹S)-epimer would be directly dehydrated to C3-vinyl group. (2) the (3¹S)-epimer would be epimerized to the (3¹R)-epimer, and the resulting epimer was dehydrated. The results indicated that both BchF and BchV did function as a hydratase/dehydratase and could play a role in the C3¹-epimerization. An increase in the alkyl size at the C8-position gradually suppressed the BchF and BchV-catalyzed dehydration in vitro, while the C12¹- and C20-methylation only slightly affected the reaction. Using the BchF dehydration, a large amount of 3-vinyl-bacteriochlorophyllide-a was successfully prepared, with the retention of the chemically labile, central magnesium atom.

Keywords: Biosynthesis; Epimer; Pigment; Stereospecificity; Substitution effect.