title: Assay and purification of S-adenosyl-L-methionine:precorrin-2 methyltransferase from Pseudomonas denitrificans.
abstract: S-Adenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT), which catalyzes the C-20 methylation of precorrin-2 to precorrin-3, was purified to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans derived from a cobalamin-overproducing strain. Ammonium sulfate fractionation followed by chromatography on DEAE-Trisacryl, hydroxyapatite, and Mono Q HR purified the enzyme about 110-fold, with a 28% yield. For enzyme purification and characterization, a coupled-enzyme assay was developed which generated in situ the highly oxygen-sensitive substrate, precorrin-2, from delta-aminolevulinic acid. Evidence is given that the chemically reduced form of sirohydrochlorin (dihydrosirohydrochlorin) is methylated at C-20 to precorrin-3 by pure SP2MT. No subsequent SP2MT-dependent methylation reaction of precorrin-3 was detected. The native enzyme has an apparent molecular weight of 53,000, as estimated by gel filtration, and consists of two identical subunits of Mr 26,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Stepwise Edman degradation provided the N-terminal sequence of the first 17 amino acids.
authors.affiliation: Département de Chimie Analytique, Centre de Recherche de Vitry, Rhône-Poulenc Santé, Vitry-sur-Seine, France.
authors.forename: D
authors.initials: D
authors.lastname: Thibaut
authors.forename: M
authors.initials: M
authors.lastname: Couder
authors.forename: J
authors.initials: J
authors.lastname: Crouzet
authors.forename: L
authors.initials: L
authors.lastname: Debussche
authors.forename: B
authors.initials: B
authors.lastname: Cameron
authors.forename: F
authors.initials: F
authors.lastname: Blanche
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D017886:Durapatite; D004926:Escherichia coli / Q000235:genetics; D006882:Hydroxyapatites; D007700:Kinetics; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics
chemical_list: D000596:Amino Acids; D006882:Hydroxyapatites; D017886:Durapatite; D008780:Methyltransferases; C066231:precorrin-2 O-methyltransferase