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

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

3. Microbiota Stuart, Human Physiology:Fourth Edition, 1984. colon (10 9 to 10 11 per ml of fecal matter) Bacteroides, Bifidobacterium, Eubacterium, Lactobacillus, etc.

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 10 5 tons produced annually worldwide –Metabolites are potential carcinogens - bladder cancer in humans and liver nodules in experimental animals (Dillion et al. 1994)

6. Azoreduction 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 Intestinal bacteria 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. N = NNN= NH 2 OH R1 R2 R3 R4 R1 R2 R3 R4 ? NH 2 H2NH2N ? azoreductase Toxic amines or Non toxic products Proposed Mechanism Nonenzymatic reduction Gene(s) Other function

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. Cristee Sumit Dr. Punj Susan

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