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dc.contributor.authorWiesselmann, Milan
dc.contributor.authorHebecker, Stefanie
dc.contributor.authorBorrero-de Acuña, José M
dc.contributor.authorNIMTZ, MANFRED
dc.contributor.authorBollivar, David
dc.contributor.authorJänsch, Lothar
dc.contributor.authorMoser, Jürgen
dc.contributor.authorJahn, Dieter
dc.date.accessioned2021-01-13T15:04:28Z
dc.date.available2021-01-13T15:04:28Z
dc.date.issued2020-11-19
dc.identifier.citationBiochem J. 2020 Dec 11;477(23):4635-4654. doi: 10.1042/BCJ20200761.en_US
dc.identifier.pmid33211085
dc.identifier.doi10.1042/BCJ20200761
dc.identifier.urihttp://hdl.handle.net/10033/622676
dc.description.abstractDuring 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.isoenen_US
dc.publisherPortland Pressen_US
dc.rightsAttribution-NonCommercial-ShareAlike 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.subjectRhodobacter capsulatusen_US
dc.subjectBchEen_US
dc.subjectMg-protoporphyrin IX monomethyl ester cyclaseen_US
dc.subjectchlorophyllen_US
dc.subjectradical SAM enzymeen_US
dc.titleMg-protoporphyrin IX monomethyl ester cyclase from Rhodobacter capsulatus: radical SAM-dependent synthesis of the isocyclic ring of bacteriochlorophylls.en_US
dc.typeArticleen_US
dc.identifier.eissn1470-8728
dc.contributor.departmentHZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.en_US
dc.identifier.journalThe Biochemical journalen_US
dc.source.volume477
dc.source.issue23
dc.source.beginpage4635
dc.source.endpage4654
dc.source.journaltitleThe Biochemical journal
dc.source.countryEngland


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