Purification, Characterization, and Cloning of Trimethylamine Dehydrogenase from Methylophaga sp. Strain SK1

  • Kim, Hee-Gon (Department of Biomaterials Engineering, Chosun University) ;
  • Kim, Yan (Department of Biomaterials Engineering, Chosun University) ;
  • Lim, Heon-Man (Department of Biology, Chungnam National University) ;
  • Shin, Hyun-Jae (Department of Chemical Engineering, Chosun University) ;
  • Kim, Si-Wouk (Department of Biomaterials Engineering, Chosun University)
  • Published : 2006.08.30

Abstract

Trimethylamine dehydrogenase (TMADH, EC 1.5.99.7), an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde, was purified from Methylophaga sp. strain SK1. The active TMADH was purified 12.3-fold through three purification steps. The optimal pH and temperature for enzyme activity was determined to be 8.5 and $55^{\circ}C$, respectively. The $V_{max}\;and\;K_m$ values were 7.9 nmol/min/mg protein and 1.5 mM. A genomic DNA of 2,983 bp from Methylophaga sp. strain SK1 was cloned, and DNA sequencing revealed the open reading frame (ORF) of the gene coding for TMADH. The ORF contained 728 amino acids with extensive identity (82%) to that of Methylophilus methylotrophus $W_3A_1$.

Keywords

References

  1. Guest, I. and D. R. Varma (1992) Teratogenic and macromolecular synthesis inhibitory effects of trimethylamine on mouse embryos in culture. J. Toxicol. Environ. Health 36: 27-41
  2. Colby, J. and L. J. Zatman (1973) Trimethylamine metabolism in obligate and facultative methylotrophs. Biochem. J. 132: 101-112 https://doi.org/10.1042/bj1320101
  3. Colby, J. and L. J. Zatman (1975) Enzymological aspects of the pathways for trimethylamine oxidation and C1 assimilation in obligate methylotrophs and restricted facultative methylotrophs. Biochem. J. 148: 513-520 https://doi.org/10.1042/bj1480513
  4. Sutcliffe, M. J. and N. S. Scrutton (2000) Enzymology takes a quantum leap forward. Philos. Trans. R. Soc. Lond. A 358: 367-386 https://doi.org/10.1098/rsta.2000.0536
  5. Loginova, N. V. and Y. A. Trotsenko (1978) Carbon metabolism in methylotrophic bacteria isolated from activated sludge. Mikrobiologiia 47: 939-946
  6. Colby, J. and L. J. Zatman (1974) Purification and properties of the trimethylamine dehydrogenase of bacterium 4B6. Biochem. J. 143: 555-567
  7. Lim, L. W., F. S. Mathews, and D. J. Steenkamp (1982) Crystallographic study of the iron-sulfur flavoprotein trimethylamine dehydrogenase from the bacterium $W_3A_1$. J. Mol. Biol. 162: 869-876 https://doi.org/10.1016/0022-2836(82)90551-4
  8. Lim, L. W., N. Shamala, F. S. Mathews, D. J. Steenkamp, R. Hamlin, and N. H. Xuong (1986) Three-dimensional structure of the iron-sulfur flavoprotein trimethylamine dehydrogenase at 2.4-A resolution. J. Biol. Chem. 261: 15140-15146
  9. Koh, M. J., C. S. Kim, Y. A. Kim, H. S. Choi, E. H. Cho, E. B. Kim, Y. M. Kim, and S. W. Kim (2002) Properties of electron carriers in the process of methanol oxidation in a new restricted facultative marine methylotrophic bacterium, Methylophaga sp. MP. J. Microbiol. Biotechnol. 12: 476-482
  10. Park, J. H., J. H. Lee, Y. S. Kim, Y. K. Hong, and I. S. Kong (2001) Molecular cloning and expression of a sodium- driven flagella motor component gene (motX) from Vibrio fluvialis. J. Microbiol. Biotechnol. 11: 973-978
  11. Ryou, C., T. Chung, and M. Kwon (2001) Molecular cloning and hyperexpression of a Bt gene, cryIAc, in Escherichia coli DH5$\alpha$: Production and usage of anti-cryIAc antibody. J. Microbiol. Biotechnol. 11: 1093-1098
  12. Shin, J. H., D. H. Roh, G. Y. Heo, G. J. Joo, and I. K. Rhee (2001) Purification and characterization of a regulatory protein XylR in the D-xylose operon from Escherichia coli. J. Microbiol. Biotechnol. 11: 1002-1010
  13. Jeong, Y. S., H. J. Yoo, S. D. Kim, D. H. Nam, and Y. H. Khang (2005) Cloning and sequencing of a novel glutaryl acylase $\beta$-subunit gene of Pseudomonas cepacia BY21 from bioinformatics. Biotechnol. Bioprocess Eng. 10: 510-515 https://doi.org/10.1007/BF02932286
  14. Lee, J. H., M. H. Choi, J. Y. Park, H. K. Kang, H. W. Ryu, C. S. Sunwo, Y. J. Wee, K. D. Park, D. W. Kim, and D. Kim (2004) Cloning and characterization of the lactate dehydrogenase genes from Lactobacillus sp. RKY2. Biotechnol. Bioprocess Eng. 9: 318-322 https://doi.org/10.1007/BF02942351
  15. Dower, W. J., J. F. Miller, and C. W. Ragsdale (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16: 6127-6145 https://doi.org/10.1093/nar/16.13.6127
  16. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  17. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  18. Kim, H. G. and S. W. Kim (2006) Purification and characterization of a methanol dehydrogenase derived from Methylomicrobium sp. HG-1 cultivated using a compulsory circulation diffusion system. Biotechnol. Bioprocess Eng. 11: 134-139 https://doi.org/10.1007/BF02931897
  19. Goldberg, J. B. and D. E. Ohman (1984) Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J. Bacteriol. 158: 1115-1121
  20. Birnboim, H. C. and J. Doly (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7: 1513-1523 https://doi.org/10.1093/nar/7.6.1513
  21. Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402 https://doi.org/10.1093/nar/25.17.3389
  22. Kasprzak, A. A. and D. J. Steenkamp (1983) Localization of the major dehydrogenases in two methylotrophs by radiochemical labeling. J. Bacteriol. 156: 348-353
  23. Boyd, G., F. S. Mathews, L. C. Packman, and N. S. Scrutton (1992) Trimethylamine dehygrogenase of bacterium $W_3A_1$. Molecular cloning, sequence determination and over-expression of the gene. FEBS Lett. 308: 271-276 https://doi.org/10.1016/0014-5793(92)81291-S
  24. Barber, M. J., P. J. Neame, L. W. Lim, S. White, and F. S. Matthews (1992) Correlation of x-ray deduced and experimental amino acid sequences of trimethylamine dehydrogenase. J. Biol. Chem. 267: 6611-6619
  25. Steenkamp, D. J., W. S. McIntire, and W. C. Kenney (1978) Structure of the covalently bound coenzyme of trimethylamine dehydrogenase. Evidence for a 6-substituted flavin. J. Biol. Chem. 253: 2818-2824
  26. Steenkamp, D. J., W. C. Kenney, and T. P. Singer (1978) A novel type of covalently bound coenzyme in trimethylamine dehydrogenase. J. Biol. Chem. 253: 2812-2817
  27. Kasprzak, A. A., E. J. Papas, and D. J. Steenkamp (1983) Identity of the subunits and the stoicheiometry of prosthetic groups in trimethylamine dehydrogenase and dimethylamine dehydrogenase. Biochem. J. 211: 535-541 https://doi.org/10.1042/bj2110535
  28. Mewies, M., J. Basran, L. C. Packman, R. Hille, and N. S. Scrutton (1997) Involvement of a flavin iminoquinone methide in the formation of 6-hydroxyflavin mononucleotide in trimethylamine dehydrogenase: A rationale for the existence of 8$\alpha$-methyl and C6-linked covalent flavoproteins. Biochemistry 36: 7162-7168 https://doi.org/10.1021/bi970621d
  29. Yang, C. C., L. C. Packman, and N. S. Scrutton (1995) The primary structure of Hyphomicrobium X dimethylamine dehydrogenase. Relationship to trimethylamine dehydrogenase and implications for substrate recognition. Eur. J. Biochem. 232: 264-271 https://doi.org/10.1111/j.1432-1033.1995.tb20808.x