Stout T J, Schellenberger U, Santi D V, Stroud R M
Department of Biochemistry, School of Medicine, University of California, San Francisco 94143-0448, USA.
Biochemistry. 1998 Oct 20;37(42):14736-47. doi: 10.1021/bi981270l.
Unlike all other organisms studied to date, Bacillus subtilis expresses two different thymidylate synthases: bsTS-A and bsTS-B. bsTS-A displays enhanced enzymatic and structural thermal stability uncharacteristic of most TSs. Despite the high level of TS conservation across most species, bsTS-A shares low sequence identity (<40%) with the majority of TSs from other organisms. This TS and the TSs from Lactococcus lactis and phage Phi3T-to which it is most similar-have been of interest for some time since, by structure-based sequence alignment, they appear to lack several key residues shown by mutagenesis to be essential to enzymatic function [Greene, P. J., Yu, P. L., Zhao, J., Schiffer, C. A., and Santi, D. (1994) Protein Sci. 3, 1114-6]. In addition, bsTS-A demonstrates specific activity 2-3-fold higher than TS from Lactobacillus casei or Escherichia coli. We have solved the crystal structure of this unusual TS in four crystal forms to a maximum resolution of 1.7 A. Each of these crystal forms contains either one or two noncrystallographically related dimers. Stabilization of the beta-sheet dimer interface through a dramatic architecture of buttressed internal salt bridges maintains the structural integrity of bsTS-A at elevated temperatures. Melting curves of TSs from L. casei and E. coli are compared to that of TS-A from B. subtilis and correlated with numbers of hydrogen bonds, salt bridges, and the numbers of interactions localized to the dimer interface. Analysis of this structure will shed light on the conservation of function across diversity of sequence, as well as provide insights into the thermal stabilization of a highly conserved enzyme.
与迄今为止研究的所有其他生物体不同，枯草芽孢杆菌表达两种不同的胸苷酸合成酶：bsTS-A和bsTS-B。bsTS-A表现出增强的酶促和结构热稳定性，这在大多数胸苷酸合成酶中并不常见。尽管大多数物种的胸苷酸合成酶具有高度保守性，但bsTS-A与其他生物体的大多数胸苷酸合成酶的序列同一性较低（<40%）。这种胸苷酸合成酶以及与其最相似的乳酸乳球菌和噬菌体Phi3T的胸苷酸合成酶，一段时间以来一直备受关注，因为通过基于结构的序列比对，它们似乎缺少一些经诱变显示对酶功能至关重要的关键残基[格林，P.J.，于，P.L.，赵，J.，希弗，C.A.，和桑蒂，D.（1994年）《蛋白质科学》3，1114 - 1116]。此外，bsTS-A的比活性比干酪乳杆菌或大肠杆菌的胸苷酸合成酶高2 - 3倍。我们已经以四种晶体形式解析了这种不寻常的胸苷酸合成酶的晶体结构，最高分辨率达到1.7埃。这些晶体形式中的每一种都包含一个或两个非晶体学相关的二聚体。通过支撑内部盐桥的显著结构稳定β-折叠二聚体界面，在升高的温度下维持了bsTS-A的结构完整性。将干酪乳杆菌和大肠杆菌的胸苷酸合成酶的熔解曲线与枯草芽孢杆菌的TS-A的熔解曲线进行比较，并与氢键、盐桥的数量以及位于二聚体界面的相互作用数量相关联。对该结构的分析将阐明序列多样性中功能的保守性，以及为一种高度保守的酶的热稳定性提供见解。
