Crystal Structures of a Unique Thermal-Stable Thymidylate Synthase from Bacillus subtilis
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- 作者：Thomas J. Stout ; Ute Schellenberger ; Daniel V. Santi ; and Robert M. Stroud
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：October 20, 1998
- 年：1998
- 卷：37
- 期：42
- 页码：14736 - 14747
- 全文大小：701K
- 年卷期：v.37,no.42(October 20, 1998)
- ISSN：1520-4995
文摘
Unlike all other organisms studied to date, Bacillus subtilis expresses two different thymidylatesynthases: bsTS-A and bsTS-B. bsTS-A displays enhanced enzymatic and structural thermal stabilityuncharacteristic of most TSs. Despite the high level of TS conservation across most species, bsTS-Ashares low sequence identity (<40%) with the majority of TSs from other organisms. This TS and theTSs from Lactococcus lactis and phage 
3T-to which it is most similar-have been of interest for sometime since, by structure-based sequence alignment, they appear to lack several key residues shown bymutagenesis to be essential to enzymatic function [Greene, P. J., Yu, P. L., Zhao, J., Schiffer, C. A., andSanti, D. (1994) Protein Sci. 3, 1114-6]. In addition, bsTS-A demonstrates specific activity 2-3-foldhigher than TS from Lactobacillus casei or Escherichia coli. We have solved the crystal structure of thisunusual TS in four crystal forms to a maximum resolution of 1.7 Å. Each of these crystal forms containseither one or two noncrystallographically related dimers. Stabilization of the 
-sheet dimer interfacethrough a dramatic architecture of buttressed internal salt bridges maintains the structural integrity ofbsTS-A at elevated temperatures. Melting curves of TSs from L. casei and E. coli are compared to thatof TS-A from B. subtilis and correlated with numbers of hydrogen bonds, salt bridges, and the numbersof interactions localized to the dimer interface. Analysis of this structure will shed light on the conservationof function across diversity of sequence, as well as provide insights into the thermal stabilization of ahighly conserved enzyme.
3T-to which it is most similar-have been of interest for sometime since, by structure-based sequence alignment, they appear to lack several key residues shown bymutagenesis to be essential to enzymatic function [Greene, P. J., Yu, P. L., Zhao, J., Schiffer, C. A., andSanti, D. (1994) Protein Sci. 3, 1114-6]. In addition, bsTS-A demonstrates specific activity 2-3-foldhigher than TS from Lactobacillus casei or Escherichia coli. We have solved the crystal structure of thisunusual TS in four crystal forms to a maximum resolution of 1.7 Å. Each of these crystal forms containseither one or two noncrystallographically related dimers. Stabilization of the -sheet dimer interfacethrough a dramatic architecture of buttressed internal salt bridges maintains the structural integrity ofbsTS-A at elevated temperatures. Melting curves of TSs from L. casei and E. coli are compared to thatof TS-A from B. subtilis and correlated with numbers of hydrogen bonds, salt bridges, and the numbersof interactions localized to the dimer interface. Analysis of this structure will shed light on the conservationof function across diversity of sequence, as well as provide insights into the thermal stabilization of ahighly conserved enzyme.