Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii)

Abstract

We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B 12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B 12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri-and tetra-methylated compounds that are not part of the vitamin B 12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B 12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.Uroporphyrinogen (urogen) III methyltransferase, a key enzyme in the biosynthetic pathways of vitamin B 12 and siroheme, catalyzes the S-adenosyl-L-methionine (SAM)-dependent bismethylation of its substrate, urogen III, resulting in the formation of dihydrosirohydrochlorin (known as precorrin-2 in the vitamin B 12 pathway). In the biosynthesis of vitamin B 12 in the anaerobe Propionibacterium freudenreichii, labeling experiments have indicated that cobalt is inserted soon after the formation of precorrin-2 (3, 20), and the recent isolation of a cobalt-containing tetramethylated corphinoid, possibly an intermediate, from this organism supports these observations (3). Cobalt insertion in the vitamin B 12 pathway of the aerobe Pseudomonas denitrificans, however, occurs at a much later stage with insertion into hydrogenobyrinic acid diamide (6).Urogen III methyltransferase has been purified to homogeneity from several different organisms, and the nucleotide sequences of the corresponding genes have been determined, revealing that the enzyme exists in at least two forms. One form, encoded by the cysG gene, is required for siroheme and, thus, for cysteine synthesis in Escherichia coli (14,31,35) and siroheme and vitamin B 12 synthesis in Salmonella typhimurium (8, 10). The ...

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

“…Thus, in vivo at least, it would appear that CobA P. denitrificans is able to overmethylate precorrin-2 in a manner similar to that of CysG (34,46) and P. freudenreichii CobA (33). The spectrum of the material isolated from ER182 was found to be very similar to the spectrum of a cobalt isobacteriochlorin (cobalt sirohydrochlorin) (1).…”

Vitamin B 12 - Structure, Biosynthesis , Functions, and Methods of Determination

“…Badania molekularne dotyczące zwiększenia efektywności biosyntezy witaminy B 12 przez drobnoustroje doprowadziły do wyizolowania szeregu enzymów odpowiedzialnych za tworzenie się tego związku [62]. Szlaki metaboliczne na których syntetyzowana jest witamina B 12 zostały scharakteryzowane w przypadku bakterii Pseudomonas denitrificans [6], Salmonella typhimurium [76] oraz Propionibacterium freudenreichii [73,79]. Geny odpowiedzialne za biosyntezę witaminy B 12 lub jej prekursorów zostały wyizolowane także dla rodzaju Rhodobacter [57,63,64].…”

Evidence that the CysG protein catalyzes the first reaction specific to B12 synthesis in Salmonella typhimurium, insertion of cobalt

“…typhimurium enzyme is inferred to be responsible for siroheme synthesis, ring oxidation, and iron insertion (43) and, based on the presented results, for cobalt insertion in B 12 synthesis by S. typhimurium. The UroIII methylase from P. shermanii (early cobalt insertion) also corresponds only to the C-terminal region of the Salmonella CysG enzyme (35), suggesting that it performs only the methyl transfer reactions. Thus, in this organism, early cobalt insertion may be catalyzed by a polypeptide distinct from the UroIII methylase.…”