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 urogen Ⅲ and by-product S-adenosyl-L-homocysteine (SAH). In order to mine an SUMT that lacks such an inhibitory property to drive 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 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.
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。.
Keywords: Rhodobacter capsulatus; tandem-enzyme reaction; uroporphyrinogen Ⅲ; uroporphyrinogen Ⅲ methyltransferase; vitamin B₁₂.