Wagner Wendi, Breksa Andrew P, Monzingo Arthur F, Appling Dean R, Robertus Jon D
Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, USA.
Biochemistry. 2005 Oct 4;44(39):13163-71. doi: 10.1021/bi051038x.
5,10-Methylenetetrahydrofolate dehydrogenase (MTD) catalyzes the reversible oxidation of 5,10-methylenetetrahydrofolate to 5,10-methenyltetrahydrofolate. This reaction is critical for the supply of one-carbon units at the required oxidation states for the synthesis of purines and dTMP. For most MTDs, dehydrogenase activity is co-located with a methenyl-THF cyclohydrolase activity as part of bifunctional or trifunctional enzyme. The yeast Saccharomyces cerevisiae contains a monofunctional NAD(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase (yMTD). Kinetic, crystallographic, and mutagenesis studies were conducted to identify critical residues in order to gain further insight into the reaction mechanism of this enzyme and its apparent lack of cyclohydrolase activity. Hydride transfer was found to be rate-limiting for the oxidation of methylenetetrahydrofolate by kinetic isotope experiments (V(H)/V(D) = 3.3), and the facial selectivity of the hydride transfer to NAD(+) was determined to be Pro-R (A-specific). Model building based on the previously solved structure of yMTD with bound NAD cofactor suggested a possible role for three conserved amino acids in substrate binding or catalysis: Glu121, Cys150, and Thr151. Steady-state kinetic measurements of mutant enzymes demonstrated that Glu121 and Cys150 were essential for dehydrogenase activity, whereas Thr151 allowed some substitution. Our results are consistent with a key role for Glu121 in correctly binding the folate substrate; however, the exact role of C150 is unclear. Single mutants Thr57Lys and Tyr98Gln and double mutant T57K/Y98Q were prepared to test the hypothesis that the lack of cyclohydrolase activity in yMTD was due to the substitution of a conserved Lys/Gln pair found in bifunctional MTDs. Each mutant retained dehydrogenase activity, but no cyclohydrolase activity was detected.
5,10-亚甲基四氢叶酸脱氢酶（MTD）催化5,10-亚甲基四氢叶酸可逆氧化为5,10-次甲基四氢叶酸。该反应对于为嘌呤和dTMP合成提供所需氧化态的一碳单位至关重要。对于大多数MTD而言，脱氢酶活性与次甲基-THF环水解酶活性共同存在于双功能或三功能酶中。酿酒酵母含有一种单功能的NAD(+)依赖性5,10-亚甲基四氢叶酸脱氢酶（yMTD）。进行了动力学、晶体学和诱变研究以确定关键残基，以便进一步深入了解该酶的反应机制及其明显缺乏环水解酶活性的原因。通过动力学同位素实验发现，氢化物转移是亚甲基四氢叶酸氧化的限速步骤（V(H)/V(D)=3.3），并且确定氢化物向NAD(+)转移的面选择性为Pro-R（A特异性）。基于先前解析的结合NAD辅因子的yMTD结构进行的模型构建表明，三个保守氨基酸在底物结合或催化中可能起作用：Glu121、Cys150和Thr151。突变酶的稳态动力学测量表明，Glu121和Cys150对脱氢酶活性至关重要，而Thr151允许一些取代。我们的结果与Glu121在正确结合叶酸底物中起关键作用一致；然而，C150的确切作用尚不清楚。制备了单突变体Thr57Lys和Tyr98Gln以及双突变体T57K/Y98Q，以检验yMTD缺乏环水解酶活性是由于双功能MTD中发现的保守Lys/Gln对被取代这一假设。每个突变体都保留了脱氢酶活性，但未检测到环水解酶活性。
