Synthesis, remodeling, and salvaging of the lower ligand of coenzyme B12.

摘要

Cobamides (e.g. adenosylcobalamin [AdoCbl, coenzyme B12]), are complex cobalt-containing cyclic tetrapyrrole cofactors which have unique structural properties, including both upper and lower axial ligands coordinating the central cobalt ion. The lower ligand of AdoCbl is 5,6-dimethylbenzimidazole (DMB), which is not found in any other known biological molecule. The lower ligands of cobamides are tethered to the tetrapyrrole ring by a nucleotide-like loop structure, which contains an alpha-linkage between ribose and the nucleotide base analog (e.g. DMB). All other known natural nucleotides contain beta-linkages. This dissertation focuses on the aerobic synthesis of DMB, on the ability of some bacteria to remove the nucleotide loop from cobamides and replace it with a different lower ligand, and on the salvaging of alpha-ribosides for incorporation as the nucleotide loop of cobamides.;The BIuB enzyme of Rhodospirillum rubrum was shown to catalyze the O2-dependent conversion of dihydroflavin mononucleotide to DMB in vitro, and the consequences of loss of the bluB gene on photosynthetic growth of R. rubrum were explored. The CbiZ corrinoid amidohydrolase (cobyric acid-producing) enzyme of Rhodobacter sphaeroides was shown by in vivo and in vitro analysis to be responsible for removing the adenine-containing lower ligand nucleotide loop of pseudocobalamin, allowing R. sphaeroides to rebuild the nucleotide loop with a BluB-synthesized DMB lower ligand. Phylogenetic analysis suggested that CbiZ is a corrinoid-salvaging enzyme of archaeal origin which has been horizontally transferred to several bacterial lineages. The CblT and CblS enzymes of Listeria innocua were shown in vitro and in vivo to constitute a pathway for salvage of the DMB-containing alpha-riboside alpha-ribazole, which this organism cannot synthesize de novo. CblT was shown to be a specific alpha-ribazole transporter, while CbIS had alpha-ribazole kinase activity, activating alpha-ribazole for incorporation as the lower ligand of AdoCbl. The phylogenetic distribution of BluB, CbiZ, CblT, and CblS homologs suggested that bacteria have varied and complex mechanisms for constructing and remodeling the lower ligands of cobamide cofactors.