Gilbert H. John, Ph.D.

1. Azoreductase Activity in Intestinal Bacteria Gilbert H. John, Ph.D. Department of Microbiology & Molecular Genetics Stillwater, OK

2. General Research Interests • Xenobiotic metabolism • Human intestinal bacteria • Eubacterium facalis, E. facium, Clostridium perfringens. • Azoreductase

3. Microbiota • colon (109 to 1011 per ml of fecal matter) • Bacteroides, Bifidobacterium, Eubacterium, Lactobacillus, etc. Stuart, Human Physiology:Fourth Edition, 1984.

4. Microbiota engage in xenobiotic metabolism • Human intestinal microorganisms • CTAB (detergent)- John, GH, et al. Microbial Ecology in Health and Disease (MEHD), 2001; 13:229-233. • Phenobarbital (drug to treat epilepsy)- John, GH, et al., 2006, MEHD, 18:32-37. • Classification of azoreductase – John, GH, et al., 2007, SJI (J Biology), Vol. 1, Issue 1. • Enterococcus faecalis – Physiological characterization of Enterococcus faecalis during azoreductase activity – Punj, S and John, GH., 2008, MEDH • Enteroccoccus faecium -Azo dye metabolism and azoreductase gene isolation and characterization – MacWana and John, et al., 2008 submitted, FEMS Letters.

5. Azoreductase reduces Azo dyes • Azo dyes • Used as synthetic colorants (>2000 dyes) • Food, Pharmaceuticals, Textiles, Cosmetics, tatoos, hair dyes, etc. • 7 x 105 tons produced annually worldwide • Metabolites are potential carcinogens - bladder cancer in humans and liver nodules in experimental animals (Dillion et al. 1994)

6. Azoreduction Intestinal bacteria • Azoreductase catalyze the reductive cleavage of azo compounds to their corresponding amines. • Both hepatic and intestinal bacterial azoreductases are capable of this process or P450 Liver

7. Azoreductase in Intestinal Bacteria • Discovered in 1981 • First gene was identified in 2001 (E. coli) and 2003 (E. faecalis). • Low homology at the primary sequence level– nucleotide and amino acid, • Higher homology at the 3-D structure level • Broad and narrow specificity for different azo dyes • Azoreductase Family • Type 1 • FMN-dependent NADH-azoreductase • Type 2 • FMN-dependent NADPH-azoreductase • Type 3 • Both NADH and NADPH .

8. Proposed Mechanism NH2 OH NH2 OH N = N N = N R1 R1 Nonenzymatic reduction ? R4 R4 R2 R2 R3 R3 Toxic amines or Non toxic products H2N NH2 ? Gene(s) Other function azoreductase

9. Enterococcus faecalis • Gram positive, facultative anaerobe • Resistant to antibiotics (vancomycin • Opportunistic pathogen – nosocomial infections • Commensal - present in the human intestine CDC website(http://www.cdc.gov/)

10. Human intestinal bacteria containing azoreductase • AzoA from E. faecalis has been characterized (Chen, et al. Prot Exp & Purific, 34:302-310 (2004) • AzoM from E. faecium(Macwan, S. and John, GH, FEMS Let, submitted, 2008) • AzoC from Clostridium perfringens (Wright, C., Trobare, D., and John, G.H.., In preparation, 2008)

11. Physiology characterization

14. Conclusion • Azoreductase is present in human intestinal bacteria • Broad substrate specificity for dye and cofactors (NADH and NADPH) • Azoreductase activity during the lag and log phase

15. How is this important for pathogens? • Recent publication “Enhancing survival of Escherichia coli by expression of azoreductase AZR possessing quinone reductase activity (Guangfei, l. et al. Appl Microbiol Biotechnol. (2008) 80:409-416). • Azoreductase may be involved in overcoming heat shock and oxidative stress.

16. Research Funded • National Institutes of Health (NIH) • EPSCoR/NSF • Kimberly-Clarke • Recent funding: National Science Foundation (NSF)

17. Sumit Dr. Punj Cristee Susan

18. Acknowledgements • Students • Graduates: Susan Macwana, Cristee Wright and Sumit Punj. • Undergraduates:Katie Southard, Anna Smith, Daniel Trobare, John Cooper, Evan Schwenk