CRYSTAL-STRUCTURES OF A UNIQUE THERMAL-STABLE THYMIDYLATE SYNTHASE FROM BACILLUS-SUBTILIS

Unlike all other organisms studied to date, Bacillus subtilis expresse s two different thymidylate synthases: bsTS-A and bsTS-B. bsTS-A displ ays enhanced enzymatic and structural thermal stability uncharacterist ic 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 lact is and phage Phi 3T-to which it is most similar-have been of interest for some time since, by structure-based sequence alignment, they appea r 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 Lactobacil lus 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 BL Each of these crystal forms contains either one or two noncrystallo graphically related dimers, Stabilization of the beta-sheet dimer inte rface through a dramatic architecture of buttressed internal salt brid ges maintains the structural integrity of bsTS-A at elevated temperatu res. Melting curves of TSs from L, casei and E. coli are compared to t hat of TS-A from B. subtilis and correlated with numbers of hydrogen b onds, salt bridges, and the numbers of interactions localized to the d imer interface. Analysis of this structure will shed light on the cons ervation of function across diversity of sequence, as well as provide insights into the thermal stabilization of a highly conserved enzyme.