Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Molecular Cloning and Characterization of Bacillus cereus O-Methyltransferase

  • Lee Hyo-Jung (Division of Bioscience & Biotechnology, Bio/Molecular Informatics Center, Konkuk University) ;
  • Kim Bong-Gyu (Division of Bioscience & Biotechnology, Bio/Molecular Informatics Center, Konkuk University) ;
  • Ahn Joong-Hoon (Division of Bioscience & Biotechnology, Bio/Molecular Informatics Center, Konkuk University)
  • Published : 2006.04.01
Download PDF
⟨ Previous Next ⟩

Abstract

Biotransformation is a good tool to synthesize regioselective compounds. It could be performed with diverse sources of genes, and microorganisms provide a myriad of gene sources for biotransformation. We were interested in modification of flavonoids, and therefore, we cloned a putative O-methyltransferase from Bacillus cereus, BcOMT-2. It has a 668-bp open reading frame that encodes a 24.6-kDa protein. In order to investigate the modification reaction mediated by BcOMT-2, it was expressed in E. coli as a His-tag fusion protein and purified to homogeneity. Several substrates such as naringenin, luteolin, kaempferol, and quercetin were tested and reaction products were analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). BcOMT-2 could transfer a methyl group to substrates that have a 3' functional hydroxyl group, such as luteolin and quercetin. Comparison of the HPLC retention time and UV spectrum of the quercetin reaction product with corresponding authentic 3'-methylated and 4'-methylated compounds showed that the methylation position was at either the 3'-hydroxyl or 4'-hydroxyl group. Thus, BcOMT-2 transfers a methyl group either to the 3'-hydroxyl or 4'-hydroxyl group of flavonoids when both hydroxyl groups are available. Among several flavonoids that contain a 3'- and 4'-hydroxyl group, fisetin was the best substrate for the BcOMT-2.

Keywords

References

  1. Dixon, A. B. and M. J. Harrison. 1999. Activation structure and organization of genes involved in microbial defense in plants. Adv. Genet. 28: 165-234
  2. García-Granados, A., A. Fernández, M. C. Gutiérrez, A. Martínez, R. Quirós, F. Rivas, and J. M. Arias. 2004. Biotransformation of ent-13-epi-manoyl oxides difunctionalized at C-3 and C-12 by filamentous fungi. Phytochemistry 65: 107-115 https://doi.org/10.1016/j.phytochem.2003.09.017
  3. Holland, H. L. and H. K. Weber. 2000. Enzymatic hydroxylation reactions. Curr. Opin. Biotechnol. 11: 547-553 https://doi.org/10.1016/S0958-1669(00)00142-7
  4. Ibdah, M., X.-H. Zhang, J. Schnot, and T. Vogt. 2003. A novel $Mg^{2+}$-dependent O-methyltransferase in the phenylpropanoid metabolism of Mesembryanthemum crystallinum. J. Biol. Chem. 278: 43961-43972 https://doi.org/10.1074/jbc.M304932200
  5. Ivanova, N., A. Sorokin, I. Anderson, N. Galleron, B. Candelon, V. Kapatral, A. Bhattacharyya, G. Reznik, N. Mikhailova, A. Lapidus, L. Chu, M. Mazur, E. Goltsman, N. Larsen, M. D'Souza, T. Walunas, Y. Grechkin, G. Pusch, R. Haselkorn, M. Fonstein, D. S. D. Ehrlich, R. Overbeek, and N. Kyrpides. 2003. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423: 87-91 https://doi.org/10.1038/nature01582
  6. Kim, H.-A., D.-Y. Yoon, S.-M. Lee, S.-H. Baek, G.-H. Han, Y.-H. Kho, and C.-H. Lee. 2005. Screening and biotransformation of interleukin-1$\beta$converting enzyme production inhibitors from Arctii fructus. J. Microbiol. Biotechnol. 15: 269-273
  7. Kim, Y. M., K. Park, J.-H. Choi, J.-E. Kim, and I.-K. Rhee. 2004. Biotransformation of the fungicide chlorothalonil by bacterial glutathione S-transferase. J. Microbiol. Biotechnol. 14: 938-943
  8. Kim, D. H., B. G. Kim, Y. Lee, J. Y. Ryu, Y. Lim, H.-G. Hur, and J.-H. Ahn. 2005. Regiospecific methylation of naringenin to ponciretin by soybean O-methyltransferase expressed in Escherichia coli. J. Biotechnol. 119: 155-162 https://doi.org/10.1016/j.jbiotec.2005.04.004
  9. Lim, E. K., D. A. Ashford, B. Hou, R. G. Jackson, and D. J. Bowles. 2004. Arabidopsis glycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides. Biotechnol. Bioeng. 87: 623-631 https://doi.org/10.1002/bit.20154
  10. Middleton, E. Jr. and C. Kandaswami. 1994. The impact of plant flavonoids on mammalian biology: Implications for immunity, inflammation and cancer, pp. 619-652. In Harborne, J. B. (ed.). The Flavonoids: Advances in Research Since 1986. Chapman & Hall, London
  11. Morishige, T., K. B. Choi, and F. Sato. 2004. In vivo bioconversion of tetrahydroisoquinoline by recombinant coclaurine N-methyltransferase. Biosci. Biotechnol. Biochem. 68: 939-941 https://doi.org/10.1271/bbb.68.939
  12. Park, M.-K., K.-H. Liu, Y. Lim, Y.-H. Lee, H.-G. Hur, and J.-H. Kim. 2003. Biotransformation of a fungicide ethaboxam by soil fungus Cunninghamella elegans. J. Microbiol. Biotechnol. 13: 43-49
  13. Raadt, A. D. and H. Griengl. 2002. The use of substrate engineering in biohydroxylation. Curr. Opin. Biotechnol. 13: 537-542 https://doi.org/10.1016/S0958-1669(02)00358-0
  14. Suenaga, H., M. Mitsuoka, Y. Ura, T. Watanabe, and K. Furukawa. 2001. Directed evolution of biphenyl dioxygenase: Emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes. J. Bacteriol. 183: 5441-5444 https://doi.org/10.1128/JB.183.18.5441-5444.2001