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. 1999 Mar 1;18(5):1104–1113. doi: 10.1093/emboj/18.5.1104

Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex.

H K Song 1, S H Sohn 1, S W Suh 1
PMCID: PMC1171202  PMID: 10064578

Abstract

Bacteriophage T4 deoxycytidylate hydroxymethylase (EC 2.1.2.8), a homodimer of 246-residue subunits, catalyzes hydroxymethylation of the cytosine base in deoxycytidylate (dCMP) to produce 5-hydroxymethyl-dCMP. It forms part of a phage DNA protection system and appears to function in vivo as a component of a multienzyme complex called deoxyribonucleoside triphosphate (dNTP) synthetase. We have determined its crystal structure in the presence of the substrate dCMP at 1.6 A resolution. The structure reveals a subunit fold and a dimerization pattern in common with thymidylate synthases, despite low (approximately 20%) sequence identity. Among the residues that form the dCMP binding site, those interacting with the sugar and phosphate are arranged in a configuration similar to the deoxyuridylate binding site of thymidylate synthases. However, the residues interacting directly or indirectly with the cytosine base show a more divergent structure and the presumed folate cofactor binding site is more open. Our structure reveals a water molecule properly positioned near C-6 of cytosine to add to the C-7 methylene intermediate during the last step of hydroxymethylation. On the basis of sequence comparison and crystal packing analysis, a hypothetical model for the interaction between T4 deoxycytidylate hydroxymethylase and T4 thymidylate synthase in the dNTP-synthesizing complex has been built.

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Selected References

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  1. Agarwalla S., LaPorte S., Liu L., Finer-Moore J., Stroud R. M., Santi D. V. A novel dCMP methylase by engineering thymidylate synthase. Biochemistry. 1997 Dec 16;36(50):15909–15917. doi: 10.1021/bi971873h. [DOI] [PubMed] [Google Scholar]
  2. Allen J. R., Lasser G. W., Goldman D. A., Booth J. W., Mathews C. K. T4 phage deoxyribonucleotide-synthesizing enzyme complex. Further studies on enzyme composition and regulation. J Biol Chem. 1983 May 10;258(9):5746–5753. [PubMed] [Google Scholar]
  3. Barton G. J. ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng. 1993 Jan;6(1):37–40. doi: 10.1093/protein/6.1.37. [DOI] [PubMed] [Google Scholar]
  4. Butler M. M., Graves K. L., Hardy L. W. Evidence from 18O exchange studies for an exocyclic methylene intermediate in the reaction catalyzed by T4 deoxycytidylate hydroxymethylase. Biochemistry. 1994 Aug 30;33(34):10521–10526. doi: 10.1021/bi00200a038. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Cohen F. E., Sternberg M. J., Taylor W. R. Analysis of the tertiary structure of protein beta-sheet sandwiches. J Mol Biol. 1981 May 25;148(3):253–272. doi: 10.1016/0022-2836(81)90538-6. [DOI] [PubMed] [Google Scholar]
  7. Cox G. S., Conway T. W. Template properties of glucose-deficient T-even bacteriophage DNA. J Virol. 1973 Dec;12(6):1279–1287. doi: 10.1128/jvi.12.6.1279-1287.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elcock A. H., Potter M. J., Matthews D. A., Knighton D. R., McCammon J. A. Electrostatic channeling in the bifunctional enzyme dihydrofolate reductase-thymidylate synthase. J Mol Biol. 1996 Sep 27;262(3):370–374. doi: 10.1006/jmbi.1996.0520. [DOI] [PubMed] [Google Scholar]
  9. Fauman E. B., Rutenber E. E., Maley G. F., Maley F., Stroud R. M. Water-mediated substrate/product discrimination: the product complex of thymidylate synthase at 1.83 A. Biochemistry. 1994 Feb 15;33(6):1502–1511. doi: 10.1021/bi00172a029. [DOI] [PubMed] [Google Scholar]
  10. Finer-Moore J. S., Liu L., Birdsall D. L., Brem R., Apfeld J., Santi D. V., Stroud R. M. Contributions of orientation and hydrogen bonding to catalysis in Asn229 mutants of thymidylate synthase. J Mol Biol. 1998 Feb 13;276(1):113–129. doi: 10.1006/jmbi.1997.1495. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Gommers-Ampt J. H., Borst P. Hypermodified bases in DNA. FASEB J. 1995 Aug;9(11):1034–1042. doi: 10.1096/fasebj.9.11.7649402. [DOI] [PubMed] [Google Scholar]
  13. Gommers-Ampt J. H., Van Leeuwen F., de Beer A. L., Vliegenthart J. F., Dizdaroglu M., Kowalak J. A., Crain P. F., Borst P. beta-D-glucosyl-hydroxymethyluracil: a novel modified base present in the DNA of the parasitic protozoan T. brucei. Cell. 1993 Dec 17;75(6):1129–1136. doi: 10.1016/0092-8674(93)90322-h. [DOI] [PubMed] [Google Scholar]
  14. Graves K. L., Butler M. M., Hardy L. W. Roles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis. Biochemistry. 1992 Oct 27;31(42):10315–10321. doi: 10.1021/bi00157a020. [DOI] [PubMed] [Google Scholar]
  15. Grindley H. M., Artymiuk P. J., Rice D. W., Willett P. Identification of tertiary structure resemblance in proteins using a maximal common subgraph isomorphism algorithm. J Mol Biol. 1993 Feb 5;229(3):707–721. doi: 10.1006/jmbi.1993.1074. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Holm L., Sander C. Protein structure comparison by alignment of distance matrices. J Mol Biol. 1993 Sep 5;233(1):123–138. doi: 10.1006/jmbi.1993.1489. [DOI] [PubMed] [Google Scholar]
  18. Horton N., Lewis M. Calculation of the free energy of association for protein complexes. Protein Sci. 1992 Jan;1(1):169–181. doi: 10.1002/pro.5560010117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jackson R. M., Gabb H. A., Sternberg M. J. Rapid refinement of protein interfaces incorporating solvation: application to the docking problem. J Mol Biol. 1998 Feb 13;276(1):265–285. doi: 10.1006/jmbi.1997.1519. [DOI] [PubMed] [Google Scholar]
  20. Janin J., Rodier F. Protein-protein interaction at crystal contacts. Proteins. 1995 Dec;23(4):580–587. doi: 10.1002/prot.340230413. [DOI] [PubMed] [Google Scholar]
  21. Jones S., Thornton J. M. Principles of protein-protein interactions. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):13–20. doi: 10.1073/pnas.93.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Lamm N., Wang Y., Mathews C. K., Rüger W. Deoxycytidylate hydroxymethylase gene of bacteriophage T4. Nucleotide sequence determination and over-expression of the gene. Eur J Biochem. 1988 Mar 15;172(3):553–563. doi: 10.1111/j.1432-1033.1988.tb13925.x. [DOI] [PubMed] [Google Scholar]
  25. Liu L., Santi D. V. Exclusion of 2'-deoxycytidine 5'-monophosphate by asparagine 229 of thymidylate synthase. Biochemistry. 1993 Sep 14;32(36):9263–9267. doi: 10.1021/bi00087a001. [DOI] [PubMed] [Google Scholar]
  26. Mathews C. K., Wheeler L. J., Ungermann C., Young J. P., Ray N. B. Enzyme interactions involving T4 phage-coded thymidylate synthase and deoxycytidylate hydroxymethylase. Adv Exp Med Biol. 1993;338:563–570. doi: 10.1007/978-1-4615-2960-6_115. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. McGaughey K. M., Wheeler L. J., Moore J. T., Maley G. F., Maley F., Mathews C. K. Protein-protein interactions involving T4 phage-coded deoxycytidylate deaminase and thymidylate synthase. J Biol Chem. 1996 Sep 20;271(38):23037–23042. doi: 10.1074/jbc.271.38.23037. [DOI] [PubMed] [Google Scholar]
  30. Merritt E. A., Murphy M. E. Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D Biol Crystallogr. 1994 Nov 1;50(Pt 6):869–873. doi: 10.1107/S0907444994006396. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  33. Perry K. M., Carreras C. W., Chang L. C., Santi D. V., Stroud R. M. Structures of thymidylate synthase with a C-terminal deletion: role of the C-terminus in alignment of 2'-deoxyuridine 5'-monophosphate and 5,10-methylenetetrahydrofolate. Biochemistry. 1993 Jul 20;32(28):7116–7125. doi: 10.1021/bi00079a007. [DOI] [PubMed] [Google Scholar]
  34. Rice P. A., Steitz T. A. Model for a DNA-mediated synaptic complex suggested by crystal packing of gamma delta resolvase subunits. EMBO J. 1994 Apr 1;13(7):1514–1524. doi: 10.2210/pdb1gdr/pdb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rutenber E. E., Stroud R. M. Binding of the anticancer drug ZD1694 to E. coli thymidylate synthase: assessing specificity and affinity. Structure. 1996 Nov 15;4(11):1317–1324. doi: 10.1016/s0969-2126(96)00139-6. [DOI] [PubMed] [Google Scholar]
  36. Rüger W. Transcription of bacteriophage T4 DNA in vitro: selective initiation with dinucleotides. Eur J Biochem. 1978 Jul 17;88(1):109–117. doi: 10.1111/j.1432-1033.1978.tb12427.x. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Schellenberger U., Livi L. L., Santi D. V. Cloning, expression, purification, and characterization of 2'-deoxyuridylate hydroxymethylase from phage SPO1. Protein Expr Purif. 1995 Aug;6(4):423–430. doi: 10.1006/prep.1995.1057. [DOI] [PubMed] [Google Scholar]
  39. Shapiro L., Fannon A. M., Kwong P. D., Thompson A., Lehmann M. S., Grübel G., Legrand J. F., Als-Nielsen J., Colman D. R., Hendrickson W. A. Structural basis of cell-cell adhesion by cadherins. Nature. 1995 Mar 23;374(6520):327–337. doi: 10.1038/374327a0. [DOI] [PubMed] [Google Scholar]
  40. Story R. M., Weber I. T., Steitz T. A. The structure of the E. coli recA protein monomer and polymer. Nature. 1992 Jan 23;355(6358):318–325. doi: 10.1038/355318a0. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Stroud R. M. An electrostatic highway. Nat Struct Biol. 1994 Mar;1(3):131–134. doi: 10.1038/nsb0394-131. [DOI] [PubMed] [Google Scholar]
  43. Stroud R. M., Finer-Moore J. S. Stereochemistry of a multistep/bipartite methyl transfer reaction: thymidylate synthase. FASEB J. 1993 May;7(8):671–677. doi: 10.1096/fasebj.7.8.8500692. [DOI] [PubMed] [Google Scholar]
  44. Terwilliger T. C., Berendzen J. Correlated phasing of multiple isomorphous replacement data. Acta Crystallogr D Biol Crystallogr. 1996 Jul 1;52(Pt 4):749–757. doi: 10.1107/S0907444996000832. [DOI] [PubMed] [Google Scholar]
  45. Thylén C. Expression and DNA sequence of the cloned bacteriophage T4 dCMP hydroxymethylase gene. J Bacteriol. 1988 Apr;170(4):1994–1998. doi: 10.1128/jb.170.4.1994-1998.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vrielink A., Rüger W., Driessen H. P., Freemont P. S. Crystal structure of the DNA modifying enzyme beta-glucosyltransferase in the presence and absence of the substrate uridine diphosphoglucose. EMBO J. 1994 Aug 1;13(15):3413–3422. doi: 10.1002/j.1460-2075.1994.tb06646.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Warren R. A. Modified bases in bacteriophage DNAs. Annu Rev Microbiol. 1980;34:137–158. doi: 10.1146/annurev.mi.34.100180.001033. [DOI] [PubMed] [Google Scholar]
  48. Waterbury P. G., Lane M. J. Generation of lambda phage concatemers for use as pulsed field electrophoresis size markers. Nucleic Acids Res. 1987 May 11;15(9):3930–3930. doi: 10.1093/nar/15.9.3930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wheeler L. J., Ray N. B., Ungermann C., Hendricks S. P., Bernard M. A., Hanson E. S., Mathews C. K. T4 phage gene 32 protein as a candidate organizing factor for the deoxyribonucleoside triphosphate synthetase complex. J Biol Chem. 1996 May 10;271(19):11156–11162. doi: 10.1074/jbc.271.19.11156. [DOI] [PubMed] [Google Scholar]
  50. Wheeler L., Wang Y., Mathews C. K. Specific associations of T4 bacteriophage proteins with immobilized deoxycytidylate hydroxymethylase. J Biol Chem. 1992 Apr 15;267(11):7664–7670. [PubMed] [Google Scholar]
  51. Wilhelm K., Rüger W. Deoxyuridylate-hydroxymethylase of bacteriophage SPO1. Virology. 1992 Aug;189(2):640–646. doi: 10.1016/0042-6822(92)90587-f. [DOI] [PubMed] [Google Scholar]
  52. Young J. P., Mathews C. K. Interactions between T4 phage-coded deoxycytidylate hydroxymethylase and thymidylate synthase as revealed with an anti-idiotypic antibody. J Biol Chem. 1992 May 25;267(15):10786–10790. [PubMed] [Google Scholar]

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