Gilbert H. John, Ph.D.

Department of Microbiology & Molecular Genetics

Stillwater, OK

Azoreductase Activity in Intestinal Bacteria

General Research Interests

• Xenobiotic metabolism– Human intestinal bacteria

• Eubacterium facalis, E. facium, Clostridium perfringens.

– Azoreductase

Microbiota

Stuart, Human Physiology:Fourth Edition, 1984.

• colon (109 to 1011 per ml of fecal matter)

• Bacteroides, Bifidobacterium, Eubacterium, Lactobacillus, etc.

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.

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)

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

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

.

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

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/)

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)

Physiology characterization

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

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.

Research Funded

• National Institutes of Health (NIH)

• EPSCoR/NSF

• Kimberly-Clarke

• Recent funding: National Science Foundation (NSF)

Cristee

SumitDr. Punj

Susan

Acknowledgements

• Students– Graduates: Susan Macwana, Cristee Wright and

Sumit Punj.– Undergraduates:Katie Southard, Anna Smith,

Daniel Trobare, John Cooper, Evan Schwenk