Structural basis of the substrate preference towards CMP for a thymidylate synthase MilA involved in mildiomycin biosynthesis.
Structural basis of the substrate preference towards CMP for a thymidylate synthase MilA involved in mildiomycin biosynthesis.
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米霉素生物合成中胸苷酸合酶 MilA 对 CMP 的底物偏好的结构基础
DOI：
10.1038/srep39675
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发表时间：
2016-12-21
影响因子：
4.6
通讯作者：
He X
中科院分区：
文献类型：
--
作者：
Zhao G;Chen C;Xiong W;Gao T;Deng Z;Wu G;He X
修饰的嘧啶单磷酸，如甲基dCMP（mdCMP）、羟甲基dUMP（hmdUMP）和在某些酶中的hmdCMP，由称为胸苷酸转移酶（TS）的一大组酶合成。胸苷酸是DNA合成所需的核苷酸，因此TS是重要的药物靶标。从龟裂链霉菌ZJU 5119分离的核苷类杀菌剂米多霉素的生物合成途径中，胞苷酸（CMP）羟甲基化酶米拉（MilA）催化CMP转化为5′-羟甲基CMP（hmCMP），其转化效率（kcat/KM）比脱氧胞苷酸（dCMP）快5倍。因此，米拉是TS超家族中第一个偏好CMP而非dCMP的酶。在这里，我们确定了米拉及其与各种基板，包括CMP，dCMP和hmCMP复合物的晶体结构。将这些结构与来自T4噬菌体的dCMP羟甲基化酶（CH）和来自大肠杆菌的TS的结构进行比较，揭示了CH和TS的活性位点中的两个残基，丝氨酸和精氨酸，在米拉中分别被丙氨酸和赖氨酸，Ala 176和Lys 133取代。米拉的A176 S/K133 R突变导致底物偏好从CMP逆转为dCMP。这是第一个研究报告的保守TS在底物选择从DNA代谢到次级核苷生物合成的演变。
Modified pyrimidine monophosphates such as methyl dCMP (mdCMP), hydroxymethyl dUMP (hmdUMP) and hmdCMP in some phages are synthesized by a large group of enzymes termed as thymidylate synthases (TS). Thymidylate is a nucleotide required for DNA synthesis and thus TS is an important drug target. In the biosynthetic pathway of the nucleoside fungicide mildiomycin isolated from Streptomyces rimofaciens ZJU5119, a cytidylate (CMP) hydroxymethylase, MilA, catalyzes the conversion of CMP into 5′-hydroxymethyl CMP (hmCMP) with an efficiency (kcat/KM) of 5-fold faster than for deoxycytidylate (dCMP). MilA is thus the first enzyme of the TS superfamily preferring CMP to dCMP. Here, we determined the crystal structures of MilA and its complexes with various substrates including CMP, dCMP and hmCMP. Comparing these structures to those of dCMP hydroxymethylase (CH) from T4 phage and TS from Escherichia coli revealed that two residues in the active site of CH and TS, a serine and an arginine, are respectively replaced by an alanine and a lysine, Ala176 and Lys133, in MilA. Mutation of A176S/K133R of MilA resulted in a reversal of substrate preference from CMP to dCMP. This is the first study reporting the evolution of the conserved TS in substrate selection from DNA metabolism to secondary nucleoside biosynthesis.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1073/pnas.94.5.1619
发表时间：
1997-03-04
影响因子：
11.1
作者：
Joyce, CM
通讯作者：
Joyce, CM
影响因子：
2.9
作者：
BUTLER, MM;GRAVES, KL;HARDY, LW
通讯作者：
HARDY, LW
DOI：
10.1038/89661
发表时间：
2001-07-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Johansson, K;Ramaswamy, S;Eklund, H
通讯作者：
Eklund, H
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH