Abstract
Thymidylate synthase (TS) converts dUMP to dTMP by reductive methylation, where 5,10-methylenetetrahydrofolate is the source of both the methylene group and reducing equivalents. The mechanism of this reaction has been extensively studied, mainly using the enzyme from Escherichia coli. Bacillus subtilis contains two genes for TSs, ThyA and ThyB. The ThyB enzyme is very similar to other bacterial TSs, but the ThyA enzyme is quite different, both in sequence and activity. In ThyA TS, the active site histidine is replaced by valine. In addition, the B. subtilis enzyme has a 2.4-fold greater k(cat) than the E. coli enzyme. The structure of B. subtilis thymidylate synthase in a ternary complex with 5-fluoro-dUMP and 5,10-methylenetetrahydrofolate has been determined to 2.5 A resolution. Overall, the structure of B. subtilis TS (ThyA) is similar to that of the E. coli enzyme. However, there are significant differences in the structures of two loops, the dimer interface and the details of the active site. The effects of the replacement of histidine by valine and a serine to glutamine substitution in the active site area, and the addition of a loop over the carboxy terminus may account for the differences in k(cat) found between the two enzymes.
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- Brünger A. T. Crystallographic refinement by simulated annealing. Application to a 2.8 A resolution structure of aspartate aminotransferase. J Mol Biol. 1988 Oct 5;203(3):803–816. doi: 10.1016/0022-2836(88)90211-2. [DOI] [PubMed] [Google Scholar]
- Carreras C. W., Santi D. V. The catalytic mechanism and structure of thymidylate synthase. Annu Rev Biochem. 1995;64:721–762. doi: 10.1146/annurev.bi.64.070195.003445. [DOI] [PubMed] [Google Scholar]
- Dev I. K., Yates B. B., Atashi J., Dallas W. S. Catalytic role of histidine 147 in Escherichia coli thymidylate synthase. J Biol Chem. 1989 Nov 15;264(32):19132–19137. [PubMed] [Google Scholar]
- Evans S. V. SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. J Mol Graph. 1993 Jun;11(2):134-8, 127-8. doi: 10.1016/0263-7855(93)87009-t. [DOI] [PubMed] [Google Scholar]
- Finer-Moore J. S., Maley G. F., Maley F., Montfort W. R., Stroud R. M. Crystal structure of thymidylate synthase from T4 phage: component of a deoxynucleoside triphosphate-synthesizing complex. Biochemistry. 1994 Dec 27;33(51):15459–15468. doi: 10.1021/bi00255a028. [DOI] [PubMed] [Google Scholar]
- Hardy L. W., Finer-Moore J. S., Montfort W. R., Jones M. O., Santi D. V., Stroud R. M. Atomic structure of thymidylate synthase: target for rational drug design. Science. 1987 Jan 23;235(4787):448–455. doi: 10.1126/science.3099389. [DOI] [PubMed] [Google Scholar]
- Hyatt D. C., Maley F., Montfort W. R. Use of strain in a stereospecific catalytic mechanism: crystal structures of Escherichia coli thymidylate synthase bound to FdUMP and methylenetetrahydrofolate. Biochemistry. 1997 Apr 15;36(15):4585–4594. doi: 10.1021/bi962936j. [DOI] [PubMed] [Google Scholar]
- Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
- Kenny E., Atkinson T., Hartley B. S. Nucleotide sequence of the thymidylate synthetase gene (thyP3) from the Bacillus subtilis phage phi 3T. Gene. 1985;34(2-3):335–342. doi: 10.1016/0378-1119(85)90142-8. [DOI] [PubMed] [Google Scholar]
- Knighton D. R., Kan C. C., Howland E., Janson C. A., Hostomska Z., Welsh K. M., Matthews D. A. Structure of and kinetic channelling in bifunctional dihydrofolate reductase-thymidylate synthase. Nat Struct Biol. 1994 Mar;1(3):186–194. doi: 10.1038/nsb0394-186. [DOI] [PubMed] [Google Scholar]
- Lorenson M. Y., Maley G. F., Maley F. The purification and properties of thymidylate synthetase from chick embryo extracts. J Biol Chem. 1967 Jul 25;242(14):3332–3344. [PubMed] [Google Scholar]
- Maley G. F., Maley F. An anomaly in the active site region of thymidylate synthase. Adv Enzyme Regul. 1989;29:181–187. doi: 10.1016/0065-2571(89)90100-3. [DOI] [PubMed] [Google Scholar]
- Matthews D. A., Appelt K., Oatley S. J., Xuong N. H. Crystal structure of Escherichia coli thymidylate synthase containing bound 5-fluoro-2'-deoxyuridylate and 10-propargyl-5,8-dideazafolate. J Mol Biol. 1990 Aug 20;214(4):923–936. doi: 10.1016/0022-2836(90)90346-N. [DOI] [PubMed] [Google Scholar]
- Matthews D. A., Villafranca J. E., Janson C. A., Smith W. W., Welsh K., Freer S. Stereochemical mechanism of action for thymidylate synthase based on the X-ray structure of the covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate. J Mol Biol. 1990 Aug 20;214(4):937–948. doi: 10.1016/0022-2836(90)90347-O. [DOI] [PubMed] [Google Scholar]
- Montfort W. R., Perry K. M., Fauman E. B., Finer-Moore J. S., Maley G. F., Hardy L., Maley F., Stroud R. M. Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate. Biochemistry. 1990 Jul 31;29(30):6964–6977. doi: 10.1021/bi00482a004. [DOI] [PubMed] [Google Scholar]
- Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
- Neuhard J., Price A. R., Schack L., Thomassen E. Two thymidylate synthetases in Bacillus subtilis. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1194–1198. doi: 10.1073/pnas.75.3.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pedersen-Lane J., Maley G. F., Chu E., Maley F. High-level expression of human thymidylate synthase. Protein Expr Purif. 1997 Jul;10(2):256–262. doi: 10.1006/prep.1997.0750. [DOI] [PubMed] [Google Scholar]
- Sage C. R., Rutenber E. E., Stout T. J., Stroud R. M. An essential role for water in an enzyme reaction mechanism: the crystal structure of the thymidylate synthase mutant E58Q. Biochemistry. 1996 Dec 17;35(50):16270–16281. doi: 10.1021/bi961269r. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schiffer C. A., Clifton I. J., Davisson V. J., Santi D. V., Stroud R. M. Crystal structure of human thymidylate synthase: a structural mechanism for guiding substrates into the active site. Biochemistry. 1995 Dec 19;34(50):16279–16287. doi: 10.1021/bi00050a007. [DOI] [PubMed] [Google Scholar]
- Stout T. J., Schellenberger U., Santi D. V., Stroud R. M. Crystal structures of a unique thermal-stable thymidylate synthase from Bacillus subtilis. Biochemistry. 1998 Oct 20;37(42):14736–14747. doi: 10.1021/bi981270l. [DOI] [PubMed] [Google Scholar]
- Tam N. H., Borriss R. The thyA gene from Bacillus subtilis exhibits similarity with the phage phi 3T thymidylate synthase gene. Microbiology. 1995 Feb;141(Pt 2):291–297. doi: 10.1099/13500872-141-2-291. [DOI] [PubMed] [Google Scholar]