荚膜红细菌尿卟啉原Ⅲ转甲基酶的纯化及其酶学性质

Purification and characterization of S-adenosyl-L-methionine:uroporphyrinogen Ⅲ methyltransferase from Rhodobacter capsulatus SB1003

摘要:

S-腺苷-L-甲硫氨酸依赖型尿卟啉原Ⅲ转甲基酶 (S-adenosy-L-methionine uroprophyrinogen Ⅲ methyltransferase, SUMT) 催化尿卟啉原Ⅲ (Uroprophyrinogen Ⅲ, urogen Ⅲ)的中心碳原子C-2和C-7位上甲基化生成前咕啉-2，是维生素B12生物合成途径中的一步关键酶，但大部分SUMT受其底物urogen Ⅲ和副产物S-腺苷同型半胱氨酸 (S-adenosy-L-homocysteine, SAH) 的抑制作用。为了挖掘能耐受高浓度urogen Ⅲ的转甲基酶，文中从荚膜红细菌Rhodobacter capsulatus SB1003中克隆2个SUMT基因 (RCcobA1, RCcobA2)，经表达与纯化后，检测发现RCcobA1和RCcobA2的酶活分别为27.3 U/mg和68.9 U/mg，后者比VB12工业生产菌株脱氮假单胞菌Pseudomonas denitrificans中内源的SUMT (PDcobA，27.9 U/mg)高2.4倍，并且当urogen Ⅲ浓度高达70 μmol/L时都几乎不受抑制作用。因此，RCcobA2的发现可以为解除VB12合成途径的瓶颈以及提高VB12产量提供理论支持和方向指导。

Abstract:

Biosynthesis of vitamin B12 (VB12) requires the methylation at positions C-2 and C-7 of the precursor uroporphyrinogen Ⅲ (urogen Ⅲ) to precorrin-2 by S-adenosyl-L-methionine uroporphyrinogen Ⅲ methyltransferase (SUMT), which is a potential bottleneck step. Most of SUMTs are inhibited by urogenⅢand by-product S-adenosyl-L-homocysteine (SAH). In order to mine an SUMT that lacks such an inhibitory property allowing greater flux through the VB12 biosynthetic pathway, we cloned two SUMT genes (RCcobA1, RCcobA2) from Rhodobacter capsulatus SB1003 and expressed them in Escherichia coli BL21 (DE3). Thereafter, the two enzymes were purified and their specific activity of 27.3 U/mg, 68.9 U/mg were determined respectively. The latter was 2.4 times higher than PDcobA (27.9 U/mg) from Pseudomonas denitrifican. Additionally, RCcobA2 could tolerate over 70 μmol/L of urogen Ⅲ, which has never been reported before. Hence, RCcobA2 can be used as an efficient enzyme to regulate the VB12 metabolic pathway and enhance VB12 production in industrial strains.