Transcript Research Interests - Montana State University

Azoreductase Activity in Intestinal Bacteria
Gilbert H. John, Ph.D.
Department of Microbiology &
Molecular Genetics
Stillwater, OK
General Research Interests
• Xenobiotic metabolism
– Human intestinal bacteria
• Eubacterium facalis, E. facium, Clostridium
perfringens.
– Azoreductase
Microbiota
• colon (109 to 1011 per ml of
fecal matter)
• Bacteroides, Bifidobacterium,
Eubacterium, Lactobacillus, etc.
Stuart, Human Physiology:Fourth Edition, 1984.
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
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
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
.
Proposed Mechanism
NH2
OH
OH
R1
N= N
R2
R4
N= N
?
R1
R4
R2
NH2
Gene(s)
Other function
azoreductase
Nonenzymatic
reduction
R3
R3
H2N
NH2
Toxic amines or
Non toxic products
?
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
Sumit
Dr. Punj
Susan
Acknowledgements
• Students
– Graduates: Susan Macwana, Cristee Wright and
Sumit Punj.
– Undergraduates:Katie Southard, Anna Smith,
Daniel Trobare, John Cooper, Evan Schwenk