title: Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center.
abstract: The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.
authors.affiliation: Protein Science Group, Department of Biosciences, University of Kent, Canterbury, Kent CT27NJ, United Kingdom.
authors.forename: Susanne
authors.initials: S
authors.lastname: Schroeder
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Lawrence
authors.forename: Rebekka
authors.initials: R
authors.lastname: Biedendieck
authors.forename: Ruth-Sarah
authors.initials: RS
authors.lastname: Rose
authors.forename: Evelyne
authors.initials: E
authors.lastname: Deery
authors.forename: Ross M
authors.initials: RM
authors.lastname: Graham
authors.forename: Kirsty J
authors.initials: KJ
authors.lastname: McLean
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Munro
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Warren
mesh_terms: D000332:Aerobiosis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D017347:Brucella melitensis / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain / Q000502:physiology; D003038:Cobamides / Q000737:chemistry* / Q000235:genetics; D007501:Iron / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000737:chemistry; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D013455:Sulfur / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D003038:Cobamides; D009569:Nitric Oxide; D013455:Sulfur; D007501:Iron; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C083406:CobG protein, Pseudomonas denitrificans