The end of the cob operon: evidence that the last gene (cobT) catalyzes synthesis of the lower ligand of vitamin B12, dimethylbenzimidazole

Abstract

Salmonella typhimurium makes cobalamin (vitamin B 12 ) during anaerobic growth (17). Most of the cobalamin synthetic genes are organized into a single operon (cob or cbi) mapping at min 41 (3, 18). The genetic map of the cob operon can be divided into three distinct regions, each of which includes mutations with characteristic phenotypes. Part I has 17 genes (cbi) needed for cobinamide (Cbi) synthesis; strains with mutations in this region (CobI) can synthesize vitamin B 12 when Cbi is provided. Part II of the operon includes mutations that prevent vitamin B 12 synthesis unless 5,6-dimethylbenzimidazole (DMB) is provided (CobII), suggesting that this region contains the genes for DMB synthesis. Part III includes genes for joining Cbi and DMB to produce functional adenosyl-cobalamin (Ado-B 12 ); these mutants (CobIII) cannot make vitamin B 12 even when both DMB and Cbi are provided. The standard view of the pathway is depicted in Fig. 1.A fine-structure genetic map of the cob or cbi operon shows that the three types of mutations are ordered as parts I, III, and II in a single operon (3,18). The nucleotide sequences of parts I and III have been determined (27). On the basis of sequence homology to genes of known function in Pseudomonas denitrificans (for a recent review, see reference 2), it has been possible to assign functions to many of the genes in parts I and III of the cob operon. Mutations in the CobIII region have been analyzed genetically and biochemically, demonstrating that the cobU and cobS open reading frames correspond to part III of the genetic map (11,24,35). These two genes have been assigned the functions diagrammed in Fig.

The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli

“…Other open reading frames were disregarded since they were insubstantial in length, exhibited no codon usage bias (codon adaptation index of Ͻ0.2 as determined by the method of Sharp and Li [62]), and did not show the GϩC content bias by codon position (second codon position showing lower percent GϩC) typical of genes with comparable nucleotide contents (43). Three of the inferred open reading frames were clearly homologous to the cobU, cobS, and cobT genes in the S. typhimurium cob operon which provide CobII and CobIII gene functions (11,52). The E. coli genes were termed cobU, cobS, and cobT to indicate their similarity to the Salmonella homologs.…”

“…The restriction map of the cob operon region corresponds to the region from kb 2072 to 2076 on the Kohara map. A homologous asparagine-accepting-tRNA gene (100% identity) is found downstream of the srfK sequence in S. typhimurium (11).…”

“…The fourth open reading frame lay immediately downstream of the E. coli cobT gene and was homologous to the open reading frame located downstream of the S. typhimurium cobT gene (11). In this paper, the Salmonella and E. coli homologs of this gene will be referred to as the srfK and erfK genes, respectively.…”

“…The conversion of uroporphyrinogen III to the complex intermediate cobinamide is referred to as part I of the biosynthetic pathway (hereafter designated CobI). Part II of the pathway (CobII) entails the biosynthesis of dimethylbenzimidazole (DMB) from probable flavin precursors (11,27,39). The covalent linkage of cobinamide, DMB, and a phosphoribosyl group donated by nicotinate mononucleotide is achieved by part III of the pathway (CobIII).…”

Shaping RNA Structures with Metal Ions and Metal Ion Complexes

“…For example, the cob operon of S. typhimurium encodes 20 proteins of the cobalamin biosynthetic pathway and is controlled by the cob riboswitch. [76,77] Instead, the btuB riboswitch regulates a single gene encoding for an outer membrane transporter responsible for the active transport of cobalamins into the periplasmic space. [78][79][80] Both riboswitches are negatively controlled on both the transcriptional and the translational level and the structural rearrangements seem to correlate well.…”