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.
Highlights
Vitamin B12 is the antipernicious anemia factor [1], whose isolation was first described over 60 years ago [2, 3], and whose subsequent structure determination by x-ray crystallography revealed that it is a cobalt-containing modified tetrapyrrole [4]
There has been some considerable interest in the biosynthesis of vitamin B12 as there are no chemical or biological parallels for the removal and extrusion of the integral meso carbon atom that is required for the contraction of the macrocycle [6]
Cobalamin biosynthesis is further complicated by the presence of two similar though biochemically distinct pathways that are referred to as the aerobic and anaerobic routes [7,8,9,10]
Summary
Ment is eventually lost as acetic acid. Despite the characterization of the reaction catalyzed by CobG, comparatively little is known about the enzyme itself. Sulfite reductase, which catalyzes the six-electron reduction of sulfite to sulfide, requires both a [4Fe-4S] cluster and siroheme for activity. In this unusual enzyme, the two cofactors are covalently linked through a shared cysteine residue [17,18,19]. The cysteine residues required for Fe-S formation are confirmed and the presence of the Fe-S center is shown to be essential for catalytic activity of the enzyme
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