Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls. Issue 23 (11th December 2020)

• Record Type: Journal Article
• Authors: Wiesselmann, Milan; Hebecker, Stefanie; Borrero-de Acuña, José M.; Nimtz, Manfred; Bollivar, David; Jänsch, Lothar; Moser, Jürgen; Jahn, Dieter
• Is Part Of: Biochemical journal. Volume 477:Issue 23(2020)
• Page Start: 4635
• Page End: 4654
• Publication Date: 2020-12-11
• Subjects: BchE -- chlorophyll -- Mg-protoporphyrin IX monomethyl ester cyclase -- radical SAM enzyme -- Rhodobacter capsulatus; Biochemistry -- Periodicals; 572
• DOI: 10.1042/BCJ20200761
• Languages: English
• ISSNs: 0264-6021

Abstract

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 H2 18 O isotope label transfer onto the carbonyl-group (C-13 1 -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 (Cys 204, Cys 208, Cys 211 ), of SAM (Phe 210, Glu 308 and Lys 320 ) and of the proposed cobalamin cofactor (Asp 248, Glu 249, Leu 29, Thr 71, Val 97 ) were identified.