Purification and properties of NADH-dependent 5,10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli
Purification and properties of NADH-dependent 5,10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli
复制标题
DOI：
10.1128/jb.181.3.718-725.1999
复制
发表时间：
1999-02-01
影响因子：
3.2
通讯作者：
Matthews, RG
中科院分区：
文献类型：
--
作者：
Sheppard, CA;Trimmer, EE;Matthews, RG
构建了过量产生亚甲基四氢叶酸还原酶（MetF）的大肠杆菌K-12菌株，并将该酶纯化至表观均一性。指定MetF的质粒;在C末端添加六个组氨酸残基的方法已经用于通过在镍-琼脂糖上的亲和层析在单一步骤中将组氨酸标记的MetF纯化至均一，产生具有与未修饰的酶相当的比活性的制剂。天然蛋白质包含四个相同的33-kDa亚基，每个亚基含有一个非共价结合的黄素腺嘌呤二核苷酸（FAB）分子，未检测到其他辅因子或金属。纯化的酶以NADH为还原剂催化亚甲基四氢叶酸还原为甲基四氢叶酸。在15 ℃和pH7.2条件下用停流分光光度计测定了动力学参数;对NADH的Km为13 μ M，对CH 2-H-4叶酸的Km为0.8 μ M，在V-max条件下对反应估计的周转数为1，800 mol NADH氧化min（-1）（酶结合FAD的mol）（-1）。NADPH也用作还原剂，但表现出高得多的K-m。MetF还催化甲基四氢叶酸在甲萘醌存在下氧化为亚甲基四氢叶酸，甲萘醌作为电子受体。来自大肠杆菌的MetF的性质不同于从同型产乙酸梭菌分离的铁氧还蛋白依赖性亚甲基四氢叶酸还原酶的性质，并且更接近于来自Peptostreptococcus productus的NADH依赖性酶和来自真核生物的NADPH依赖性酶的性质。
A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF; with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAB), No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant, Kinetic parameters have been determined at 15 degrees C and pH 7.2 in a stopped-flow spectrophotometer; the K-m for NADH is 13 mu M, the K-m for CH2-H-4 folate is 0.8 mu M, and the turnover number under V-max conditions estimated for the reaction is 1,800 mol of NADH oxidized min(-1) (mol of enzyme-bound FAD)(-1). NADPH also serves as a reductant, but exhibits a much higher K-m. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E, coli differ from those of the ferredoxin dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.