Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls.
dc.contributor.author | Wiesselmann, Milan | |
dc.contributor.author | Hebecker, Stefanie | |
dc.contributor.author | Borrero-de Acuña, José M | |
dc.contributor.author | NIMTZ, MANFRED | |
dc.contributor.author | Bollivar, David | |
dc.contributor.author | Jänsch, Lothar | |
dc.contributor.author | Moser, Jürgen | |
dc.contributor.author | Jahn, Dieter | |
dc.date.accessioned | 2021-01-13T15:04:28Z | |
dc.date.available | 2021-01-13T15:04:28Z | |
dc.date.issued | 2020-11-19 | |
dc.identifier.citation | Biochem J. 2020 Dec 11;477(23):4635-4654. doi: 10.1042/BCJ20200761. | en_US |
dc.identifier.pmid | 33211085 | |
dc.identifier.doi | 10.1042/BCJ20200761 | |
dc.identifier.uri | http://hdl.handle.net/10033/622676 | |
dc.description.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. | en_US |
dc.language.iso | en | en_US |
dc.publisher | Portland Press | en_US |
dc.rights | Attribution-NonCommercial-ShareAlike 4.0 International | * |
dc.rights.uri | http://creativecommons.org/licenses/by-nc-sa/4.0/ | * |
dc.subject | Rhodobacter capsulatus | en_US |
dc.subject | BchE | en_US |
dc.subject | Mg-protoporphyrin IX monomethyl ester cyclase | en_US |
dc.subject | chlorophyll | en_US |
dc.subject | radical SAM enzyme | en_US |
dc.title | Mg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls. | en_US |
dc.type | Article | en_US |
dc.identifier.eissn | 1470-8728 | |
dc.contributor.department | HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. | en_US |
dc.identifier.journal | The Biochemical journal | en_US |
dc.source.volume | 477 | |
dc.source.issue | 23 | |
dc.source.beginpage | 4635 | |
dc.source.endpage | 4654 | |
dc.source.journaltitle | The Biochemical journal | |
dc.source.country | England |