cobyric-acid and siroheme

The Escherichia coli CysG protein (sirohaem synthase) catalyses four separate reactions that are required for the transformation of uroporphyrinogen III into sirohaem, initially two S-adenosyl-l-methionine-dependent transmethylations at positions 2 and 7, mediated through the C-terminal, or CysGA, catalytic domain of the protein, and subsequently a ferrochelation and dehydrogenation, mediated through the N-terminal, or CysGB, catalytic domain of the enzyme. This report describes how the deletion of the NAD+-binding site of CysG, located within the first 35 residues of the N-terminus, is detrimental to the activity of CysGB but does not affect the catalytic activity of CysGA, whereas the mutation of a number of phylogenetically conserved residues within CysGA is detrimental to the transmethylation reaction but does not affect the activity of CysGB. Further studies have shown that CysGB is not essential for cobalamin biosynthesis because the presence of the Salmonella typhimurium CobI operon with either cysGA or the Pseudomonas denitrificans cobA are sufficient for the synthesis of cobyric acid in an E. coli cysG deletion strain. Evidence is also presented to suggest that a gene within the S. typhimurium CobI operon might act as a chelatase that, at low levels of cobalt, is able to aid in the synthesis of sirohaem.

Topics: Amino Acid Sequence; Consensus Sequence; Escherichia coli; Genetic Complementation Test; Heme; Methyltransferases; Molecular Sequence Data; Point Mutation; S-Adenosylmethionine; Salmonella typhimurium; Sequence Alignment; Sequence Homology, Amino Acid; Vitamin B 12

The role of cbiK, a gene found encoded within the Salmonella typhimurium cob operon, has been investigated by studying its in vivo function in Escherichia coli. First, it was found that cbiK is not required for cobalamin biosynthesis in the presence of a genomic cysG gene (encoding siroheme synthase) background. Second, in the absence of a genomic cysG gene, cobalamin biosynthesis in E. coli was found to be dependent upon the presence of cobA(P. denitrificans) (encoding the uroporphyrinogen III methyltransferase from Pseudomonas denitrificans) and cbiK. Third, complementation of the cysteine auxotrophy of the E. coli cysG deletion strain 302delta a could be attained by the combined presence of cobA(P. denitrificans) and the S. typhimurium cbiK gene. Collectively these results suggest that CbiK can function in fashion analogous to that of the N-terminal domain of CysG (CysG(B)), which catalyzes the final two steps in siroheme synthesis, i.e., NAD-dependent dehydrogenation of precorrin-2 to sirohydrochlorin and ferrochelation. Thus, phenotypically CysG(B) and CbiK have very similar properties in vivo, although the two proteins do not have any sequence similarity. In comparison to CysG, CbiK appears to have a greater affinity for Co2+ than for Fe2+, and it is likely that cbiK encodes an enzyme whose primary role is that of a cobalt chelatase in corrin biosynthesis.

Topics: Cobalt; Corrinoids; Cysteine; Escherichia coli; Gene Deletion; Genes, Bacterial; Genetic Complementation Test; Heme; Methyltransferases; Mutagenesis, Site-Directed; Porphyrins; Salmonella typhimurium; Species Specificity; Spectrophotometry, Ultraviolet; Vitamin B 12