Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls.

Milan Wiesselmann,David Bollivar,Stefanie Hebecker,Manfred Nimtz,José M Borrero-De Acuña,Dieter Jahn,Jürgen Moser,Lothar Jänsch

doi:10.1042/bcj20200761

Abstract

During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.

Highlights

The fundamental pathways for the synthesis of chlorophyll a or bacteriochlorophyll a (BChl a) are well described in the literature [1], but the enzymatic formation of the isocyclic ring moiety of these pigments still remains an obstacle [2,3,4]
The assay was verified by HPLC and isotope label transfer experiments and revealed that the oxygen atom of the C-131-oxo group is derived from a water molecule
This was confirmed since BchE activity was stimulated by increasing concentrations of SAM or NADPH and clear inhibition in the presence of the SAM analog S-adenosylhomocysteine was shown

Summary

Introduction

The fundamental pathways for the synthesis of chlorophyll a or bacteriochlorophyll a (BChl a) are well described in the literature [1], but the enzymatic formation of the isocyclic ring moiety of these pigments still remains an obstacle [2,3,4]. The respective acsF gene was expressed in E. coli and in vivo cyclase activity was observed when the cell culture was fed with Mg-PME, which diffuses across the bacterial membrane Based on this important experiment, the overall chlorophyll biosynthetic pathway was successfully reconstituted in E. coli [4]. Different cobalamin-dependent radical SAM mechanisms for the non-oxygenating conversion performed by OxsB and for the catalysis of BchE have been discussed [3]. The H2O-dependentoxygenationof a substrate in the presence of a cobalamin-cofactor has been never exemplified for a radical SAM enzyme so far Up to this point, the absence of a reliable in vitro activity assay was hampering the further investigation of the sophisticated BchE catalysis.

Experimental procedures

Results

Discussion

Literature