A role for Salmonella typhimurium cbiK in cobalamin (vitamin B12) and siroheme biosynthesis

Abstract

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 302⌬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 Co 2؉ than for Fe 2؉

Characterization of a Novel Outer Membrane Hemin-Binding Protein of Porphyromonas gingivalis

“…CbiK is conformationally similar to the PPIX ferrochelatase of B. subtilis, although there is little sequence identity (1,40). CbiK has also been shown to function as a ferrochelatase, as well as a cobalt chelatase (35). Both CbiK and the PPIX ferrochelatase are bilobal in structure, consisting of two similar domains that each contain a four-stranded parallel ␤-sheet flanked by ␣-helices.…”

“…6A) exhibited significant similarity to only one other known protein, CbiK from S. typhimurium (37.6% identity). CbiK is an anaerobic cobalt chelatase that inserts cobalt into the tetrapyrrole ring of precorrin and has been proposed to be part of the cobalamin biosynthesis pathway in S. typhimurium (35,40). CbiK is a single-subunit, ATP-independent enzyme that, on the basis of biochemical characterization and sequence similarity, has been proposed to be a member of a class of enzymes including the PPIX ferrochelatase (HemZ), sirohydrochlorin ferrochelatases (CysG and Met8P), and the other known anaerobic cobalt chelatase (CbiX) (40).…”

Identification of a Dichelobacter nodosus Ferric Uptake Regulator and Determination of Its Regulatory Targets

“…CbiK is a cobalt chelatase that is involved in the anaerobic pathway of vitamin B 12 (cobalamin) synthesis and in some species has been shown to invoke a human immune response (45,49). Since CbiK from S. enterica serovar Typhimurium has been shown in vivo to act as a ferrochelatase by binding to Fe 2ϩ (46), it is possible that in D. nodosus the CbiK homologue is a metal ion acquisition protein that may have a role in iron acquisition or iron and/or other metal ion homeostasis.…”

An in Vitro Reducing System for the Enzymic Conversion of Cobalamin to Adenosylcobalamin

“…Initially, the cob(II)alamin substrate was generated using NAD(P)H:flavin oxidoreductase H 6 Fre enzyme, NADH, and FMN (20). Once the Fpr protein was isolated, it was used in lieu of H 6 Fre to reduce HOCbl to cob(II)alamin. Elimination of the H 6 Fre protein simplified the reaction conditions.…”