RESUMO
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.
Assuntos
Genes Bacterianos/genética , Metiltransferases/genética , Propionibacterium/genética , Uroporfirinogênios/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Isótopos de Carbono , Clonagem Molecular , Escherichia coli/genética , Heme/análogos & derivados , Heme/biossíntese , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Propionibacterium/enzimologia , Proteínas Recombinantes/biossíntese , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Uroporfirinas/metabolismo , Vitamina B 12/biossínteseRESUMO
Despite the widespread use of Bin 19 as a vector for plant transformation, detailed sequence information on its T-DNA region has only recently become available. We now show that the non-T-DNA region, like the T-DNA region, contains several superfluous insertions and find that some functional elements may not contain optimal sequences. Knowledge of the complete 11,777 bp sequence will aid in the construction of exceptionally efficient derivative vectors of approximately half this size. Precise knowledge of restriction sites and removal of unnecessary sequences will facilitate plasmid manipulations and plant transformation.
