Smith Clyde A, Cross Jennifer A, Bognar Andrew L, Sun Xiaolin
Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA, USA.
Acta Crystallogr D Biol Crystallogr. 2006 May;62(Pt 5):548-58. doi: 10.1107/S0907444906009796. Epub 2006 Apr 19.
Based upon the three-dimensional structure of Lactobacillus casei folylpolyglutamate synthetase (FPGS), site-directed mutagenesis studies were performed on three residues associated with the ATPase site: Gly51, Ser52 and Ser73. Gly51 and Ser52 are at the end of the P-loop, which is involved in triphosphate binding. A G51S mutant enzyme and a G51S/S52T double-mutant enzyme were made in order to alter the FPGS P-loop to more closely resemble the sequences found in other ATPase and GTPase enzymes. Ser73 is on a neighboring loop (the Omega-loop) and precedes a proline residue found to be in a cis conformation. The carbonyl O atom of Ser73 is one of the protein ligands for the essential Mg(2+) ion involved in ATP binding and hydrolysis and the Omega-loop is involved in binding the folate substrate 5,10-methylenetetrahydrofolate. The serine residue was mutated to alanine and this is the only one of the three mutants which retains some FPGS activity. The structures of the G51S, G51S/S52T and S73A mutant proteins have been solved to high resolution, along with the structure of the apo wild-type FPGS. The P-loop in both the G51S and G51S/S52T mutant proteins remains unaltered, yet both structures show a large conformational rearrangement of the Omega-loop in which a cis-Pro residue has switched conformation to a trans-peptide. The structure of the Omega-loop is severely disrupted and as a consequence structural rearrangements are observed in the peptide linker joining the two domains of the enzyme. Magnesium binding in the active site is also disrupted by the presence of the serine side chain at position 51 and by the repositioning of the carbonyl O atom of Ser73 and a water molecule is bound in place of the Mg(2+) ion. The S73A mutant protein retains the cis-Pro configuration in the Omega-loop and the Mg(2+) site remains intact. The cis-Pro is also observed in the structure of the substrate-free form of FPGS (apoFPGS), maintained in the absence of Mg(2+) by a hydrogen-bonding network involving water molecules in the active site. It is only in the complete absence of water or Mg(2+) in the binding site that the cis-Pro switches to the trans conformation.
基于干酪乳杆菌叶酰聚谷氨酸合成酶（FPGS）的三维结构，对与ATP酶位点相关的三个残基进行了定点诱变研究：甘氨酸51、丝氨酸52和丝氨酸73。甘氨酸51和丝氨酸52位于P环末端，该环参与三磷酸结合。构建了G51S突变酶和G51S/S52T双突变酶，以改变FPGS的P环，使其更类似于其他ATP酶和GTP酶中的序列。丝氨酸73位于相邻的环（Ω环）上，在一个处于顺式构象的脯氨酸残基之前。丝氨酸73的羰基氧原子是参与ATP结合和水解的必需镁离子（Mg(2+)）的蛋白质配体之一，且Ω环参与结合叶酸底物5,10-亚甲基四氢叶酸。该丝氨酸残基突变为丙氨酸，这是三个突变体中唯一保留一定FPGS活性的突变体。已解析出G51S、G51S/S52T和S73A突变蛋白的高分辨率结构，以及无辅基野生型FPGS的结构。G51S和G51S/S52T突变蛋白中的P环保持不变，但两种结构均显示Ω环发生了较大的构象重排，其中一个顺式脯氨酸残基转变为反式肽段。Ω环的结构受到严重破坏，因此在连接酶两个结构域的肽连接区观察到结构重排。活性位点中的镁结合也因51位丝氨酸侧链的存在以及丝氨酸73羰基氧原子的重新定位而受到破坏，并且一个水分子取代镁离子（Mg(2+)）与之结合。S73A突变蛋白在Ω环中保留了顺式脯氨酸构型，且镁离子位点保持完整。在无底物形式的FPGS（无辅基FPGS）结构中也观察到了顺式脯氨酸，在活性位点中通过涉及水分子的氢键网络在无镁离子（Mg(2+)）的情况下得以维持。只有在结合位点完全无水或无镁离子（Mg(2+)）时，顺式脯氨酸才转变为反式构象。
