Szebenyi D M, Liu X, Kriksunov I A, Stover P J, Thiel D J
Department of Molecular Biology and Genetics and Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, USA.
Biochemistry. 2000 Nov 7;39(44):13313-23. doi: 10.1021/bi000635a.
Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single carbon units for purine, thymidine, and methionine biosynthesis. The enzyme is a homotetramer comprising two obligate dimers and four pyridoxal phosphate-bound active sites. The mammalian enzyme is present in cells in both catalytically active and inactive forms. The inactive form is a ternary complex that results from the binding of glycine and 5-formyltetrahydrofolate polyglutamate, a slow tight-binding inhibitor. The crystal structure of a close analogue of the inactive form of murine cytoplasmic SHMT (cSHMT), lacking only the polyglutamate tail of the inhibitor, has been determined to 2.9 A resolution. This first structure of a ligand-bound mammalian SHMT allows identification of amino acid residues involved in substrate binding and catalysis. It also reveals that the two obligate dimers making up a tetramer are not equivalent; one can be described as "tight-binding" and the other as "loose-binding" for folate. Both active sites of the tight-binding dimer are occupied by 5-formyltetrahydrofolate (5-formylTHF), whose N5-formyl carbon is within 4 A of the glycine alpha-carbon of the glycine-pyridoxal phosphate complex; the complex appears to be primarily in its quinonoid form. In the loose-binding dimer, 5-formylTHF is present in only one of the active sites, and its N5-formyl carbon is 5 A from the glycine alpha-carbon. The pyridoxal phosphates appear to be primarily present as geminal diamine complexes, with bonds to both glycine and the active site lysine. This structure suggests that only two of the four catalytic sites on SHMT are catalytically competent and that the cSHMT-glycine-5-formylTHF ternary complex is an intermediate state analogue of the catalytic complex associated with serine and glycine interconversion.
丝氨酸羟甲基转移酶（SHMT）是一种依赖磷酸吡哆醛的酶，它催化丝氨酸和四氢叶酸可逆转化为甘氨酸和亚甲基四氢叶酸。该反应为嘌呤、胸苷和甲硫氨酸的生物合成产生单碳单位。该酶是一个同四聚体，由两个必需二聚体和四个结合磷酸吡哆醛的活性位点组成。哺乳动物的这种酶以催化活性和非活性两种形式存在于细胞中。非活性形式是一种三元复合物，由甘氨酸和5-甲酰基四氢叶酸多聚谷氨酸（一种缓慢紧密结合的抑制剂）结合形成。已确定小鼠细胞质SHMT（cSHMT）非活性形式的一种紧密类似物的晶体结构，该类似物仅缺少抑制剂的多聚谷氨酸尾巴，分辨率为2.9埃。这种结合配体的哺乳动物SHMT的首个结构使得能够鉴定参与底物结合和催化的氨基酸残基。它还揭示了构成四聚体的两个必需二聚体并不等同；对于叶酸而言，一个可描述为“紧密结合”，另一个为“松散结合”。紧密结合二聚体的两个活性位点均被5-甲酰基四氢叶酸（5-formylTHF）占据，其N5-甲酰基碳距离甘氨酸-磷酸吡哆醛复合物的甘氨酸α-碳在4埃以内；该复合物似乎主要以醌型形式存在。在松散结合二聚体中，5-甲酰基四氢叶酸仅存在于一个活性位点，其N5-甲酰基碳距离甘氨酸α-碳5埃。磷酸吡哆醛似乎主要以偕二胺复合物的形式存在，与甘氨酸和活性位点赖氨酸均有键合。该结构表明，SHMT的四个催化位点中只有两个具有催化活性，并且cSHMT-甘氨酸-5-甲酰基四氢叶酸三元复合物是与丝氨酸和甘氨酸相互转化相关的催化复合物的中间态类似物。
