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. 2003 Mar 15;370(Pt 3):945–952. doi: 10.1042/BJ20021510

Active-site-mutagenesis study of rat liver betaine-homocysteine S-methyltransferase.

Beatriz González 1, Nuria Campillo 1, Francisco Garrido 1, María Gasset 1, Juliana Sanz-Aparicio 1, María A Pajares 1
PMCID: PMC1223237  PMID: 12487625

Abstract

A site-directed-mutagenesis study of putative active-site residues in rat liver betaine-homocysteine S-methyltransferase has been carried out. Identification of these amino acids was based on data derived from a structural model of the enzyme. No alterations in the CD spectra or the gel-filtration chromatography elution pattern were observed with the mutants, thus suggesting no modification in the secondary structure content or in the association state of the proteins. All the mutants obtained showed a reduction of the enzyme activity, the most dramatic effect being that of Glu(159), followed by Tyr(77) and Asp(26). Changes in affinity for either of the substrates, homocysteine or betaine, were detected when substitutions were performed of Glu(21), Asp(26), Phe(74) and Cys(186). Interestingly, Asp(26), postulated to be involved in homocysteine binding, has a strong effect on affinity for betaine. The relevance of these results is discussed in the light of very recent structural data obtained for the human enzyme.

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Selected References

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  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Avila M. A., Berasain C., Torres L., Martín-Duce A., Corrales F. J., Yang H., Prieto J., Lu S. C., Caballería J., Rodés J. Reduced mRNA abundance of the main enzymes involved in methionine metabolism in human liver cirrhosis and hepatocellular carcinoma. J Hepatol. 2000 Dec;33(6):907–914. doi: 10.1016/s0168-8278(00)80122-1. [DOI] [PubMed] [Google Scholar]
  3. Bose N, Momany C Crystallization and preliminary X-ray crystallographic studies of recombinant human betaine-homocysteine S-methyltransferase. Acta Crystallogr D Biol Crystallogr. 2001 Mar;57(Pt 3):431–433. doi: 10.1107/s0907444900020576. [DOI] [PubMed] [Google Scholar]
  4. Breksa A. P., 3rd, Garrow T. A. Recombinant human liver betaine-homocysteine S-methyltransferase: identification of three cysteine residues critical for zinc binding. Biochemistry. 1999 Oct 19;38(42):13991–13998. doi: 10.1021/bi991003v. [DOI] [PubMed] [Google Scholar]
  5. Cantoni G. L. Biological methylation: selected aspects. Annu Rev Biochem. 1975;44:435–451. doi: 10.1146/annurev.bi.44.070175.002251. [DOI] [PubMed] [Google Scholar]
  6. Coelho-Sampaio T., Ferreira S. T., Castro Júnior E. J., Vieyra A. Betaine counteracts urea-induced conformational changes and uncoupling of the human erythrocyte Ca2+ pump. Eur J Biochem. 1994 May 1;221(3):1103–1110. doi: 10.1111/j.1432-1033.1994.tb18830.x. [DOI] [PubMed] [Google Scholar]
  7. Cuff J. A., Clamp M. E., Siddiqui A. S., Finlay M., Barton G. J. JPred: a consensus secondary structure prediction server. Bioinformatics. 1998;14(10):892–893. doi: 10.1093/bioinformatics/14.10.892. [DOI] [PubMed] [Google Scholar]
  8. Delgado-Reyes C. V., Wallig M. A., Garrow T. A. Immunohistochemical detection of betaine-homocysteine S-methyltransferase in human, pig, and rat liver and kidney. Arch Biochem Biophys. 2001 Sep 1;393(1):184–186. doi: 10.1006/abbi.2001.2474. [DOI] [PubMed] [Google Scholar]
  9. Dixon M. M., Huang S., Matthews R. G., Ludwig M. The structure of the C-terminal domain of methionine synthase: presenting S-adenosylmethionine for reductive methylation of B12. Structure. 1996 Nov 15;4(11):1263–1275. doi: 10.1016/s0969-2126(96)00135-9. [DOI] [PubMed] [Google Scholar]
  10. Drennan C. L., Huang S., Drummond J. T., Matthews R. G., Lidwig M. L. How a protein binds B12: A 3.0 A X-ray structure of B12-binding domains of methionine synthase. Science. 1994 Dec 9;266(5191):1669–1674. doi: 10.1126/science.7992050. [DOI] [PubMed] [Google Scholar]
  11. Evans John C., Huddler Donald P., Jiracek Jiri, Castro Carmen, Millian Norman S., Garrow Timothy A., Ludwig Martha L. Betaine-homocysteine methyltransferase: zinc in a distorted barrel. Structure. 2002 Sep;10(9):1159–1171. doi: 10.1016/s0969-2126(02)00796-7. [DOI] [PubMed] [Google Scholar]
  12. Finkelstein J. D., Cello J. P., Kyle W. E. Ethanol-induced changes in methionine metabolism in rat liver. Biochem Biophys Res Commun. 1974 Nov 27;61(2):525–531. doi: 10.1016/0006-291x(74)90988-7. [DOI] [PubMed] [Google Scholar]
  13. Finkelstein J. D., Harris B. J., Kyle W. E. Methionine metabolism in mammals: kinetic study of betaine-homocysteine methyltransferase. Arch Biochem Biophys. 1972 Nov;153(1):320–324. doi: 10.1016/0003-9861(72)90451-1. [DOI] [PubMed] [Google Scholar]
  14. Finkelstein J. D., Harris B. J., Martin J. J., Kyle W. E. Regulation of hepatic betaine-homocysteine methyltransferase by dietary methionine. Biochem Biophys Res Commun. 1982 Sep 16;108(1):344–348. doi: 10.1016/0006-291x(82)91872-1. [DOI] [PubMed] [Google Scholar]
  15. Finkelstein J. D., Martin J. J. Methionine metabolism in mammals. Adaptation to methionine excess. J Biol Chem. 1986 Feb 5;261(4):1582–1587. [PubMed] [Google Scholar]
  16. Finkelstein J. D., Martin J. J. Methionine metabolism in mammals. Distribution of homocysteine between competing pathways. J Biol Chem. 1984 Aug 10;259(15):9508–9513. [PubMed] [Google Scholar]
  17. Finkelstein J. D., Mudd S. H. Trans-sulfuration in mammals. The methionine-sparing effect of cystine. J Biol Chem. 1967 Mar 10;242(5):873–880. [PubMed] [Google Scholar]
  18. Fischer D., Barret C., Bryson K., Elofsson A., Godzik A., Jones D., Karplus K. J., Kelley L. A., MacCallum R. M., Pawowski K. CAFASP-1: critical assessment of fully automated structure prediction methods. Proteins. 1999;Suppl 3:209–217. doi: 10.1002/(sici)1097-0134(1999)37:3+<209::aid-prot27>3.3.co;2-p. [DOI] [PubMed] [Google Scholar]
  19. Fischer D. Hybrid fold recognition: combining sequence derived properties with evolutionary information. Pac Symp Biocomput. 2000:119–130. [PubMed] [Google Scholar]
  20. Forestier M., Reichen J., Solioz M. Application of mRNA differential display to liver cirrhosis: reduced fetuin expression in biliary cirrhosis in the rat. Biochem Biophys Res Commun. 1996 Aug 14;225(2):377–383. doi: 10.1006/bbrc.1996.1183. [DOI] [PubMed] [Google Scholar]
  21. Gasset M., Saiz J. L., Laynez J., Sanz L., Gentzel M., Töpper-Petersen E., Calvete J. J. Conformational features and thermal stability of bovine seminal plasma protein PDC-109 oligomers and phosphorylcholine-bound complexes. Eur J Biochem. 1997 Dec 15;250(3):735–744. doi: 10.1111/j.1432-1033.1997.00735.x. [DOI] [PubMed] [Google Scholar]
  22. González Beatriz, Pajares María A., Too Heng Phon, Garrido Francisco, Blundell T. L., Sanz-Aparicio Julia. Crystallization and preliminary X-ray study of recombinant betaine-homocysteine S-methyltransferase from rat liver. Acta Crystallogr D Biol Crystallogr. 2002 Aug 23;58(Pt 9):1507–1510. doi: 10.1107/s0907444902011885. [DOI] [PubMed] [Google Scholar]
  23. Goormaghtigh E., Cabiaux V., Ruysschaert J. M. Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy. III. Secondary structures. Subcell Biochem. 1994;23:405–450. doi: 10.1007/978-1-4615-1863-1_10. [DOI] [PubMed] [Google Scholar]
  24. Heil S. G., Lievers K. J., Boers G. H., Verhoef P., den Heijer M., Trijbels F. J., Blom H. J. Betaine-homocysteine methyltransferase (BHMT): genomic sequencing and relevance to hyperhomocysteinemia and vascular disease in humans. Mol Genet Metab. 2000 Nov;71(3):511–519. doi: 10.1006/mgme.2000.3078. [DOI] [PubMed] [Google Scholar]
  25. Jones D. T., Taylor W. R., Thornton J. M. A new approach to protein fold recognition. Nature. 1992 Jul 2;358(6381):86–89. doi: 10.1038/358086a0. [DOI] [PubMed] [Google Scholar]
  26. KLEE W. A., RICHARDS H. H., CANTONI G. L. The synthesis of methionine by enzymic transmethylation. VII. Existence of two separate homocysteine methylpherases on mammalian liver. Biochim Biophys Acta. 1961 Nov 25;54:157–164. doi: 10.1016/0006-3002(61)90948-9. [DOI] [PubMed] [Google Scholar]
  27. Lee K. H., Cava M., Amiri P., Ottoboni T., Lindquist R. N. Betaine:homocysteine methyltransferase from rat liver: purification and inhibition by a boronic acid substrate analog. Arch Biochem Biophys. 1992 Jan;292(1):77–86. doi: 10.1016/0003-9861(92)90053-y. [DOI] [PubMed] [Google Scholar]
  28. Lüthy R., Bowie J. U., Eisenberg D. Assessment of protein models with three-dimensional profiles. Nature. 1992 Mar 5;356(6364):83–85. doi: 10.1038/356083a0. [DOI] [PubMed] [Google Scholar]
  29. Mato J. M., Alvarez L., Ortiz P., Pajares M. A. S-adenosylmethionine synthesis: molecular mechanisms and clinical implications. Pharmacol Ther. 1997;73(3):265–280. doi: 10.1016/s0163-7258(96)00197-0. [DOI] [PubMed] [Google Scholar]
  30. McGuffin L. J., Bryson K., Jones D. T. The PSIPRED protein structure prediction server. Bioinformatics. 2000 Apr;16(4):404–405. doi: 10.1093/bioinformatics/16.4.404. [DOI] [PubMed] [Google Scholar]
  31. Medrano F. J., Gasset M., López-Zúmel C., Usobiaga P., García J. L., Menéndez M. Structural characterization of the unligated and choline-bound forms of the major pneumococcal autolysin LytA amidase. Conformational transitions induced by temperature. J Biol Chem. 1996 Nov 15;271(46):29152–29161. doi: 10.1074/jbc.271.46.29152. [DOI] [PubMed] [Google Scholar]
  32. Millian N. S., Garrow T. A. Human betaine-homocysteine methyltransferase is a zinc metalloenzyme. Arch Biochem Biophys. 1998 Aug 1;356(1):93–98. doi: 10.1006/abbi.1998.0757. [DOI] [PubMed] [Google Scholar]
  33. Mingorance J., Alvarez L., Sánchez-Góngora E., Mato J. M., Pajares M. A. Site-directed mutagenesis of rat liver S-adenosylmethionine synthetase. Identification of a cysteine residue critical for the oligomeric state. Biochem J. 1996 May 1;315(Pt 3):761–766. doi: 10.1042/bj3150761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mizuguchi K., Deane C. M., Blundell T. L., Johnson M. S., Overington J. P. JOY: protein sequence-structure representation and analysis. Bioinformatics. 1998;14(7):617–623. doi: 10.1093/bioinformatics/14.7.617. [DOI] [PubMed] [Google Scholar]
  35. Motulsky A. G. Nutritional ecogenetics: homocysteine-related arteriosclerotic vascular disease, neural tube defects, and folic acid. Am J Hum Genet. 1996 Jan;58(1):17–20. [PMC free article] [PubMed] [Google Scholar]
  36. Rao P. V., Garrow T. A., John F., Garland D., Millian N. S., Zigler J. S., Jr Betaine-homocysteine methyltransferase is a developmentally regulated enzyme crystallin in rhesus monkey lens. J Biol Chem. 1998 Nov 13;273(46):30669–30674. doi: 10.1074/jbc.273.46.30669. [DOI] [PubMed] [Google Scholar]
  37. Refsum H., Ueland P. M., Nygård O., Vollset S. E. Homocysteine and cardiovascular disease. Annu Rev Med. 1998;49:31–62. doi: 10.1146/annurev.med.49.1.31. [DOI] [PubMed] [Google Scholar]
  38. Rost B. PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol. 1996;266:525–539. doi: 10.1016/s0076-6879(96)66033-9. [DOI] [PubMed] [Google Scholar]
  39. Sali A., Blundell T. L. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993 Dec 5;234(3):779–815. doi: 10.1006/jmbi.1993.1626. [DOI] [PubMed] [Google Scholar]
  40. Sali A., Blundell T. L. Definition of general topological equivalence in protein structures. A procedure involving comparison of properties and relationships through simulated annealing and dynamic programming. J Mol Biol. 1990 Mar 20;212(2):403–428. doi: 10.1016/0022-2836(90)90134-8. [DOI] [PubMed] [Google Scholar]
  41. Sandu C., Nick P., Hess D., Schiltz E., Garrow T. A., Brandsch R. Association of betaine-homocysteine S-methyltransferase with microtubules. Biol Chem. 2000 Jul;381(7):619–622. doi: 10.1515/BC.2000.080. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Skiba W. E., Wells M. S., Mangum J. H., Awad W. M., Jr Betaine-homocysteine S-methyltransferase (human). Methods Enzymol. 1987;143:384–388. doi: 10.1016/0076-6879(87)43067-x. [DOI] [PubMed] [Google Scholar]
  44. Sowden M. P., Collins H. L., Smith H. C., Garrow T. A., Sparks J. D., Sparks C. E. Apolipoprotein B mRNA and lipoprotein secretion are increased in McArdle RH-7777 cells by expression of betaine-homocysteine S-methyltransferase. Biochem J. 1999 Aug 1;341(Pt 3):639–645. [PMC free article] [PubMed] [Google Scholar]
  45. Sunden S. L., Renduchintala M. S., Park E. I., Miklasz S. D., Garrow T. A. Betaine-homocysteine methyltransferase expression in porcine and human tissues and chromosomal localization of the human gene. Arch Biochem Biophys. 1997 Sep 1;345(1):171–174. doi: 10.1006/abbi.1997.0246. [DOI] [PubMed] [Google Scholar]
  46. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Trikha J., Theil E. C., Allewell N. M. High resolution crystal structures of amphibian red-cell L ferritin: potential roles for structural plasticity and solvation in function. J Mol Biol. 1995 May 19;248(5):949–967. doi: 10.1006/jmbi.1995.0274. [DOI] [PubMed] [Google Scholar]
  48. Uerre J. A., Miller C. H. Preparation of L-homocysteine from L-homocysteine thiolactone. Anal Biochem. 1966 Nov;17(2):310–315. doi: 10.1016/0003-2697(66)90209-0. [DOI] [PubMed] [Google Scholar]
  49. WILLIAMS J. N., Jr, MONSON W. J., SREENIVASAN A., DIETRICH L. S., HARPER A. E., ELVEHJEM C. A. Effects of a vitamin B12 deficiency on liver enzymes in the rat. J Biol Chem. 1953 May;202(1):151–156. [PubMed] [Google Scholar]
  50. Wang Z., Quiocho F. A. Complexes of adenosine deaminase with two potent inhibitors: X-ray structures in four independent molecules at pH of maximum activity. Biochemistry. 1998 Jun 9;37(23):8314–8324. doi: 10.1021/bi980324o. [DOI] [PubMed] [Google Scholar]
  51. Wilson D. K., Quiocho F. A. A pre-transition-state mimic of an enzyme: X-ray structure of adenosine deaminase with bound 1-deazaadenosine and zinc-activated water. Biochemistry. 1993 Feb 23;32(7):1689–1694. doi: 10.1021/bi00058a001. [DOI] [PubMed] [Google Scholar]
  52. Wilson D. K., Rudolph F. B., Quiocho F. A. Atomic structure of adenosine deaminase complexed with a transition-state analog: understanding catalysis and immunodeficiency mutations. Science. 1991 May 31;252(5010):1278–1284. doi: 10.1126/science.1925539. [DOI] [PubMed] [Google Scholar]

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