179 Chemistry Building
Auburn University, AL 36849-5312
Phone: (334) 844-6992
FAX: (334) 844-6959
E mail: goodwdc@auburn.edu
The primary focus of our laboratory is the evaluation of structure/function relationships in bacterial enzymes termed catalase-peroxidases. Catalase-peroxidase from Mycobacterium tuberculosis figures prominently in the mechanism of action of the antitubercular agent isoniazid. A consequence of this is that a large proportion of M. tuberculosis strains resistant to isoniazid carry mutations that alter the structure/function of the catalase-peroxidases. Moreover, in some other bacterial species, catalase-peroxidase may serve as a virulence factor. In particular, a periplasmic form of catalase-peroxidase has been identified in E. coli O157:H7 (food borne illness), Yersinia pestis (bubonic plague), and Legionella pneumophila (Legionnaires' disease). In these pathogens, the periplasmic catalase-peroxidase is associated with other virulence factors. Furthermore, nonpathogenic relatives of these organisms do not carry the periplasmic enzyme. Much remains to be determined regarding the role of catalase-peroxidase in mechanisms of antibiotic resistance and pathogenesis. Clearly, understanding the structure and function of these enzymes has great biomedical relevance.
As the name suggests, a striking feature of the
catalase-peroxidases is their substantial catalase activity. They
are able to convert two equivalents of hydrogen peroxide to two
equivalents of water and one equivalent of molecular oxygen at rates in
the neighborhood of 5,000 times per second. Strangely, this is
accomplished with an active site that has little resemblance to typical
catalase enzymes (i.e., monofunctional catalases). On the other
hand, the biofunctional catalase-peroxidases bear a striking
resemblance to typical peroxidases (i.e., monofunctional
peroxidases). Indeed, in Figure 1 shown below, the active site of
a typical peroxidase (cytochrome c peroxidase [CCP]) and a
catalase-peroxidase are nearly superimposable. Interestingly,
peroxidases like CCP have little, if any, catalase activity.
How does the catalase-peroxidase use a
monofunctional peroxidase active site to perform such substantial
peroxidase activity? One may begin to answer that question by
observing the features unique to catalase-peroxidases that are absent
from the monofunctional peroxidases like CCP. The
catalase-peroxidase structure shown in Figure 2 indicates three
structural components that monofunctional peroxidases do not have: a
300-amino acid C-terminal domain, and two interhelical insertions of
about 35 amino acids a piece. All three of these structural
components are necessary for the function of
catalase-peroxidases. Both interhelical insertions are necessary
to for catalase (but not peroxidase) activity. Others have shown
that the DE insertion supplies a strictly conserved tyrosine that is
part of a unique Trp-Tyr-Met crosslink. The Trp involved is Trp
95 (or its equivalent) found in the catalase-peroxidase active
site. We have shown that the FG insertion is also required for
catalase activity, but the mechanism by which this occurs is
unknown.
Interestingly, catalase-peroxidase structure appears
to be the result of gene duplication and fusion of a predecessor
monofunctional peroxidase. The N-terminal domain bears the
heme-dependent active site, but the C-terminal domain has since lost
its ability to bind heme or catalyze any reaction. Nevertheless,
despite its >30 Angstrom distance from the N-terminal domain active
site, the C-terminal domain is essential for both catalase AND
peroxidase activity. Our data indicate that one of the functions
of the C-terminal domain is to maintain the catalase-peroxidase active
site by preventing His106 from coordinating to the heme iron. We
are currently investigating how structures a great distance from the
active site serve to support and fine-tune its function. This is
typically a poorly understood aspect of enzyme structure and
function. Progress in this area is expected to help address
unresolved questions surrounding catalase-peroxidases in antibiotic
resistance and bacterial virulence as well as open doors to
capitalizing on these highly versatile catalysts as a starting point
for enzyme engineering.
These investigations involve the use of a variety of molecular biological and biophysical techniques ranging from site-directed mutagenesis and large-scale protein expression to electron paramagnetic resonance (EPR) spectroscopy, magnetic circular dichroism spectroscopy, and stopped-flow kinetic analysis.
Figure 1. Side by side comparison of the typical
peroxidase active site and the catalase-peroxidase active site.*
Figure 2. Three features unique to
catalase-peroxidases that are absent from monofunctional peroxidases: a
C-terminal domain and two interhelical insertions.
SELECTED PUBLICATIONS:
Moore, R.L.; Goodwin, D.C. "Peroxidatic reducing substrates
broaden the catalase activity pH range of E. coli catalase-peroxidase" J. Bacteriol. 2009, (submitted)
Cook, C.O.; Moore, R.L.; Goodwin, D.C. "The effect of R117 and D597 interdomain residue substitutions on the reactivation of Escherichia coli catalase-peroxidase" NOBCChE Proceedings 2008, 35 (in press).
Moore, R.L.; Cook, C.O.; Williams, R.: Goodwin,
D.C. "Substitution of strictly conserved Y111 in
catalase-peroxidase: Impact of remote interdomain contacts on
active site structure and catalytic performance" J. Inorg. Biochem. 2008, 102, 1819.
Moore, R.L.; Powell, L.J.; Goodwin, D.C.
“The kinetic properties producing the perfunctory pH profiles of
catalase-peroxidases” Biochim.
Biophys. Acta 2008, 1784, 900.
Trostchansky, A.; O’Donnell, V.B.; Goodwin, D.C.;
Landino, L.M.; Marnett, L.J.; Radi, R.; Rubbo, H. “PGHS-1 in turnover
is inactivated by peroxynitrite-derived radicals: Differential effect
of ·NO on peroxidase and cyclooxygenase activities” Free Rad. Biol. Med. 2007, 42, 1029.
Baker, R.D.; Cook, C.O.; Goodwin, D.C.
"Catalase-peroxidase active site restructuring by a distant and
'inactive' domain" Biochemistry
2006, 45, 7113. (Selected as a Biochemistry
"hot"
article for June 2006).
Goodwin, D.C.; Laband, K.A.; Hertwig, K.M. 2005.
Oxidation of capsaicinoids by peroxidases: kinetic, structural,
and physiological considerations. In Phenolics in
Foods and Natural Health Products. Shahidi, F., and Ho, C. T.,
Eds. ACS Symposium Series, Vol. 909, pp. 161 - 174.
Baker, R.D.; Cook, C.O.; Goodwin, D.C. "Properties of catalase-peroxidase lacking its C-terminal domain" Biochem. Biophys. Res. Comm. 2004, 320, 833.
Li, Y.; Goodwin, D.C. "Vital Roles of an
interhelical insertion
in catalase-peroxidase bifunctionality" Biochem. Biophys. Res. Comm.
2004, 318, 970.
Varnado, C.L.; Goodwin, D.C. "System for the
expression of recombinant hemoproteins in Escherichia coli" Protein
Expr. Purif. 2004, 35, 76.
Varnado, C.L.; Hertwig, K.M.; Thomas, R.; Roberts,
J.K.; Goodwin, D.C. "Properties of a novel periplasmic
catalase-peroxidase from Escherichia coli O157:H7" Arch.
Biochem. Biophys.
2004, 421, 166.
Goodwin, D.C.; Hertwig, K.M. "Peroxidase-catalyzed
oxidation of capsaicinoids: steady-state and transient-state kinetic
studies" Arch. Biochem. Biophys. 2003, 417, 18.
Goodwin, D.C.; Rowlinson, S.W; Marnett, L.J. "Substitution of tyrosine for the proximal histidine ligand to the heme of prostaglandin endoperoxide synthase-2: Implications for mechanism of cyclooxygenase activation and catalysis" Biochemistry 2000, 39, 5422.
Goodwin, D.C.; Landino, L.M.; Marnett, L.J. "Effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis" FASEB J. 1999, 13, 1121.
Goodwin, D.C.; Gunther, M.R.; Hsi, L.C.; Crews, B.C.; Eling, T.E.; Mason, R.P.; Marnett, L.J. "Nitric oxide trapping of tyrosyl radicals generated during prostaglandin endoperoxide synthase turnover: detection of the radical derivative of tyrosine 385" J. Biol. Chem. 1998, 273, 8903.
Goodwin, D.C.; Grover, T.A.; Aust, S.D. "Roles of efficient substrates in peroxidase-catalyzed oxidations" Biochemistry 1997, 36, 139.
Goodwin, D.C.; Grover, T.A.; Aust, S.D. "Redox mediation in the peroxidase-catalyzed oxidation of aminopyrine: possible implications for drug-drug interactions" Chem. Res. Toxicol. 1996, 9, 476.
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