Gilbert H. John, Ph.D. Department of Microbiology & Molecular Genetics Stillwater, OK Azoreductase...
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Gilbert H. John, Ph.D.
Department of Microbiology & Molecular Genetics
Stillwater, OK
Azoreductase Activity in Intestinal Bacteria
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General Research Interests
• Xenobiotic metabolism– Human intestinal bacteria
• Eubacterium facalis, E. facium, Clostridium perfringens.
– Azoreductase
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Microbiota
Stuart, Human Physiology:Fourth Edition, 1984.
• colon (109 to 1011 per ml of fecal matter)
• Bacteroides, Bifidobacterium, Eubacterium, Lactobacillus, etc.
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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.
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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)
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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
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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
.
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N = NN N=NH2NH2 OHOH
R1
R2R3
R4
R1
R2R3
R4 ?
NH2H2N
?
azoreductase
Toxic amines orNon toxic products
Proposed Mechanism
Nonenzymaticreduction
Gene(s)
Other function
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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/)
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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)
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Physiology characterization
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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
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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.
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Research Funded
• National Institutes of Health (NIH)
• EPSCoR/NSF
• Kimberly-Clarke
• Recent funding: National Science Foundation (NSF)
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Cristee
SumitDr. Punj
Susan
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Acknowledgements
• Students– Graduates: Susan Macwana, Cristee Wright and
Sumit Punj.– Undergraduates:Katie Southard, Anna Smith,
Daniel Trobare, John Cooper, Evan Schwenk