Abstract
Despite the extensive study of the biosynthesis of the complex molecule B12 (cobalamin), the mechanism by which the lower ligand 5,6-dimethylbenzimidazole (DMB) is formed has remained something of a mystery. However, recent work has identified and characterized a DMB-synthase (BluB) responsible for the oxygen-dependent, single enzyme conversion of FMN to DMB. In this work, we have identified BluB homologs from the aerobic purple, nonsulfur, photosynthetic bacterium Rhodobacter capsulatus and the aerobic soil bacterium Bacillus megaterium and have demonstrated DMB synthesis by the use of a novel complementation assay in which a B12 deficient strain, substituted with the precursor cobinamide is recovered either by the addition of DMB or by the recombinant expression of a bluB gene. The DMB-synthetic activity of the purified recombinant BluB enzymes was further confirmed in vitro by providing the enzyme with FMNH2 and oxygen and observing the formation of DMB by HPLC. The formation of a 4a-peroxyflavin intermediate, the first step in the oxygen dependent mechanism of DMB biosynthesis, is reported here and is the first intermediate in the enzyme catalysed reaction to be demonstrated experimentally to date. The identification and characterization of an FMN-binding protein found on the cobI operon of B. megaterium, CbiY, is also detailed, revealing an FMN-containing enzyme which is able to stabilize a blue flavin semiquinone upon reduction with a 1-electron donor.
Highlights
BMD_0969, which shall be refered to as BluB(Bm) hereafter, is not located in proximity to any genes known to be involved in cobalamin biosynthesis and displays a higher sequence identity of between 29 to 40% to the other bluB templates (Table S1)
In R. capsulatus the bluB gene is located in a large operon that codes for the majority of genes responsible for the biosynthesis of cobalamin [14]
These findings indicate that the BluB enzyme from B. megaterium, which is known to use the early cobalt insertion pathway for adenosylcobalamin synthesis, has a requirement for oxygen to complete the biosynthesis of cobalamin
Summary
5,6-Dimethylbenzimidazole (DMB), a derivative of benzimidazole, forms the alpha-axial ligand of cobalamin and is the critical distinguishing factor between cobalamin and pseudocobalamin (in which an N7-linked adenine forms the lower ligand) [1,2]. It has long been known that flavin mononucleotide (FMN) is a substrate in the de novo biosynthesis of DMB in B12-producing micro-organisms [5] and that the C2 carbon of DMB is derived from the C19 carbon of the flavin [6] It has only relatively recently been shown that a single protein, BluB, is responsible for the complete conversion of reduced FMN into DMB [1,2]. In the aerobic biosynthesis the formation of DMB from flavin is a complex process involving the contraction of the isoalloxazine ring system and cleavage of the ribityl tail [6,7] This transformation, for which no enzymological precedent exists, sees the destruction of the flavin and production of DMB along with the predicted by-products urea and erythrose-4-phosphate [6,8] and gives rise to the suggestion that BluB could be categorized as a ‘flavin destructase’. We have been able to isolate BluB from each pathway and shown that both are single-enzyme DMB-synthases, and that both require oxygen to catalyze the conversion of FMN into DMB
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