Novel Inhibitor of E. coli DNA Adenine Methyltransferase (Ecodam)

Authors: H. Elsawy, A. Jeltsch

Abstract:

EcoDam is an adenine-N6 DNA methyltransferase that methylates the GATC sites in the Escherichia coli genome. DNA-adenine methylation is not present in higher eukaryotes including humans. These observations raise the possibility that dam inhibitors may be used as anti-microbial agents. Polyphosphate (Poly(P)) is an important metabolite and signaling molecule in prokaryotes and eukaryotes. Here, by using gel retardation experiments to investigate the competition of DNA binding by EcoDam in the presence of polyphosphate, we found that Poly (P) strongly interferes with DNA binding by EcoDam, while same concentration of monophosphate does not. In addition, we demonstrated that Poly (P) binding inhibits the activity of EcoDam and our results suggest that Poly (P) led to strong inhibition of the EcoDam catalytic activity, while monophosphate had only moderate effect.

Keywords: Antibacterial drugs, EcoDam inhibitors, Polyphosphate.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1062462

References:

[1] Cassell, G. H. and Mekalanos, J. (2001) Development of antimicrobial agents in the Era of new reemerging infectious diseases and increasing antibiotic resistance. J. Am. Med. Assoc. 285, 601-605.
[2] Nakao, M. (2001) Epigenetics: interaction of DNA methylation and chromatin. Gene. 278, 25-31.
[3] Robertson, K. D. and Wolffe, A. P. (2000) DNA methylation in health and disease. Nat. Rev. Genet. 1, 11-19.
[4] Jeltsch, A. (2002) Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases. ChemBioChem. 3, 274-293.
[5] Zweiger, G., Marczynski, G. and Shapiro, L. (1994) A Caulobacter DNA methyltransferase that functions only in the predivisional cell. J. Mol. Biol. 235, 472-485.
[6] Mahan, M. J., Heithoff, D. M., Sinsheimer, R. L. and Low, D. A. (2000) Assessment of bacterial pathogenesis by analysis of gene expression in the host. Ann. Rev. Genet. 34, 139-164.
[7] Mahan, M. J. and Low, D. A. (2001) DNA methylation regulates bacterial gene expression and virulence. ASM News. 67, 356-361.
[8] Taylor, V. L., Titball, R. W. and Oyston, P. C. F. (2005) Oral immunization with a dam mutant of Yersinia pseudotuberculosis protects against plague. Microbiology. 151, 1919-1926.
[9] Waston, M. E., Jarisch, J. and Smith, A. L. (2004) Inactivation of deoxyadenosine methyltransferase (dam) attenuates Haemophilus influenzae virulence. Mol. Microbiol. 55, 651-654.
[10] Chen, L., Paulsen, D. B., Scruggs, D. W., Banes, M. M., Reeks, B. Y. and Lawrence, M. L. (2003) Alteration of DNA adenine methylase (Dam) activity in Pasteurella multocida causes increased spontaneous mutation frequency and attenuation in mice. Microbiology. 149, 2283- 2290.
[11] Julio, S. M., Heithoff, D. M., Sinsheimer, R. L., Low, D. A. and Mahan, M. J. (2002) DNA adenine methylase overproduction in Yersinia pseudotuberculosis alters YopE expression and secretion and host immune responses to infection. Infect. Immun. 70, 1006-1009.
[12] Honma, Y., Fernandez, R. E. and Maurelli, A. T. (2004) A DNA adenine methylase mutant of Shigella flexneri shows no significant attenuation of virulence. Microbiology. 150, 1073-1078.
[13] Horton, J. R., Liebert, K., Bekes, M., Jeltsch, A. and Cheng, X. (2006) Structure and substrate recognition of the Escherichia coli DNA Adenine methyltransferase. J. Mol. Biol. 358, 559-570.
[14] Pace, C. N., Vajdos, F., Fee, L., Grimsley, G. and Gray, T. (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4 (11), 2411-2423.
[15] Roth, M. & Jeltsch, A. (2000) Biotin-Avidin microtiter plate assay for quantitative analysis of DNA methylation by DNA methyltransferases. Biol. Chem. 381, 269-272.
[16] Elsawy, H., Podobinschi, S., Chahar, S. and Jeltsch, A. (2009) Transition from EcoDam to T4Dam DNA recognition mechanism without loss of activity and specificity, ChemBioChem. 10, 2488-2493.