Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans.
S-Adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT), the enzyme of the cobalamin biosynthetic pathway which catalyzes C methylation of uroporphyrinogen III, was purified about 150-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans derived from a cobalamin-overproducing strain by ammonium sulfate fractionation, anion-exchange chromatography, and hydroxyapatite chromatography. The purified protein has an isoelectric point of 6.4 and molecular weights of 56,500 as estimated by gel filtration and 30,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer. It does not contain a chromophoric prosthetic group and does not seem to require metal ions or cofactors for activity. SUMT catalyzes the two successive C-2 and C-7 methylation reactions involved in the conversion of uroporphyrinogen III to precorrin-2 via the intermediate formation of precorrin-1. In vitro studies suggest that the intermediate monomethylated product (precorrin-1) is released from the protein and then added back to the enzyme for the second C-methylation reaction. The pH optimum was 7.7, the Km values for S-adenosyl-L-methionine and uroporphyrinogen III were 6.3 and 1.0 microM, respectively, and the turnover number was 38 h-1. The enzyme activity was shown to be completely insensitive to feedback inhibition by cobalamin and corrinoid intermediates tested at physiological concentration. At uroporphyrinogen III concentrations above 2 microM, SUMT exhibited a substrate inhibition phenomenon. It is suggested that this property might play a regulatory role in cobalamin biosynthesis in the cobalamin-overproducing strain studied.
Blanche
F
F
Département de Chimie Analytique, Centre de Recherche de Vitry, Vitry-sur-Seine, France.
Debussche
L
L
Thibaut
D
D
Crouzet
J
J
Cameron
B
B
D000596:Amino Acids / Q000032:analysis; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D005796:Genes; D006882:Hydroxyapatites; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics
D000596:Amino Acids; D006882:Hydroxyapatites; D017886:Durapatite; D008780:Methyltransferases; C060508:uroporphyrin-III C-methyltransferase