Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii).
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 B12 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 B12 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 B12 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 B12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.
Sattler
I
I
Center for Biological NMR, Chemistry Department, Texas A&M University, College Station 77843.
Roessner
C A
CA
Stolowich
N J
NJ
Hardin
S H
SH
Harris-Haller
L W
LW
Yokubaitis
N T
NT
Murooka
Y
Y
Hashimoto
Y
Y
Scott
A I
AI
D000595:Amino Acid Sequence; D001483:Base Sequence; D002247:Carbon Isotopes; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D006418:Heme / Q000031:analogs & derivatives / Q000096:biosynthesis; D009682:Magnetic Resonance Spectroscopy; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D011424:Propionibacterium / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014577:Uroporphyrinogens / Q000378:metabolism*; D014578:Uroporphyrins / Q000378:metabolism; D014805:Vitamin B 12 / Q000096:biosynthesis
D002247:Carbon Isotopes; D011994:Recombinant Proteins; D014577:Uroporphyrinogens; D014578:Uroporphyrins; D006418:Heme; C009272:siroheme; C063209:15,23-dihydrosirohydrochlorin; D008780:Methyltransferases; C060508:uroporphyrin-III C-methyltransferase; D014805:Vitamin B 12