Thibaut D, Couder M, Crouzet J, Debussche L, Cameron B, Blanche F
Département de Chimie Analytique, Centre de Recherche de Vitry, Rhône-Poulenc Santé, Vitry-sur-Seine, France.
J Bacteriol. 1990 Nov;172(11):6245-51. doi: 10.1128/jb.172.11.6245-6251.1990.
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.
S-腺苷-L-甲硫氨酸：前咕啉-2甲基转移酶（SP2MT）催化前咕啉-2的C-20甲基化生成前咕啉-3，该酶从源自钴胺素高产菌株的反硝化假单胞菌重组菌株提取物中纯化至同质。硫酸铵分级分离后，通过DEAE-三乙醇胺琼脂糖凝胶、羟基磷灰石和Mono Q HR柱层析将该酶纯化了约110倍，产率为28%。为了进行酶的纯化和特性鉴定，开发了一种偶联酶测定法，该方法能原位从δ-氨基乙酰丙酸生成对氧高度敏感的底物前咕啉-2。有证据表明，纯SP2MT可将化学还原形式的二氢卟吩（二氢四吡咯）在C-20处甲基化生成前咕啉-3。未检测到前咕啉-3随后发生的依赖SP2MT的甲基化反应。通过凝胶过滤法估计，天然酶的表观分子量为53,000，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计，它由两个分子量为26,000的相同亚基组成。逐步的埃德曼降解法确定了前17个氨基酸的N端序列。
