The common threads that join our research interests together are
the topics of bacterial virulence, antibiotic resistance, and
discovery of new leads for antibiotic development, most especially
with respect to Mycobacterium tuberculosis. We have two
enzyme systems on which we are particularly focused: 1) the
multifunctional heme enzyme catalase-peroxidase known as KatG, and
2) shikimate kinase, an enzyme essential for the biosynthesis of
aromatic compounds, most prominently the aromatic amino acids.
KatGs are enzymes found in many bacteria as well as several fungi and protists. As the name suggests, these enzymes have the ability to decompose hydrogen peroxide by two primary mechanisms: catalase and peroxidase. A central component of the defensive response of plants and animals to invading pathogens is the production of copious amounts of hydrogen peroxide. Consequently, KatG figures prominently in the antioxidant defenses of several notorious pathogens. Examples include E. coli O157:H7 (a highly virulent food-borne pathogen), Yersina pestis (the cause of bubonic plague), and Magnaporthe grisea (the cause of rice blast diesease and a major threat to world food security). Interestingly, KatG is the sole catalase-active enzyme produced by M. tuberculosis, an organism that survives and propagates in the phagolysosomes of neutrophils and macrophages. In addition, M. tuberculosis KatG is the enzyme responsible for activating the antitubercular pro-drug isoniazid, one of the front-line agents used to fight tuberculosis. Consequently, mutations affecting the katG gene are a prominent underlying cause for resistance of numerous strains of Tb to isoniazid chemotherapy. Clearly, there are several biomedical benefits to be derived from understanding the connection between KatG structure and function
The bifunctional capability of
catalase-peroxidases is an anomaly. Typical catalases are not
especially robust peroxidases, and canonical peroxidases (e.g.,
cytochrome c peroxidase) are abysmal as catalases.
Interestingly, KatG is a member of the peroxidase-catalase
superfamily along with enzymes like cytochrome c peroxidase.
This is immediately obvious when one compares the active sites of
these enzymes (Figure 1). The active sites of KatG and
cytochrome c peroxidase, one of its closest relatives, are
superimposable. Even the much more distantly related manganese
peroxidase enzyme has essentially the same active site features
with only two active site tryptophan residues replaced by
phenylalanines. Despite the great similarity of their active
sites, KatG is the only member of this superfamily to show
appreciable catalase activity.
What gives KatG the ability to carry out such
robust catalase activity compared to its peroxidase relatives? All
KatGs examined to date show a novel methionine-tyrosine-tryptophan
(MYW) covalent adduct. With substitutions to any of the members of
the adduct, KatG loses all catalase activity but still shows
comparable if not enhanced peroxidase activity. These data suggest
the MYW adduct is a cofactor for KatGs catalase activity.
Together, the distinct peroxidase-like active site (as opposed to
that of a typical catalase) and the presence of a novel cofactor
suggest that KatG operates by novel catalase mechanism. Indeed, a
radical centered on the MYW cofactor, a perhydroxy derivative of
the MYW tryptophan, and a ferri-superoxo heme center have all been
put forward as potential intermediates for this novel mechanism.
In addition, one must ask how KatG manages the interplay of its
two major catalytic activities, catalase and peroxidase. From its
discovery, it has been presumed that the two are mutually
antagonistic, but we have observed that peroxidatic electron
donors actually stimulate KatG catalase activity by several fold.
Our data point to a synergistic cooperation between the two
activities that emerges because of two pathways for intramolecular
electron transfer. What is most striking about this unexpected
synergistic cooperation between catalase and peroxidase
acitivities, is that it expands the capacity and range of KatG to
respond to threats from hydrogen peroxide. This is
particularly important in the context of host innate immune
Plants and microorganisms rely on the shikimate
pathway to produce essential aromatic compounds. These include
ubiquinone and p-aminobenzoic acid, but most importantly,
the aromatic amino acids. Because mammals, including humans,
obtain the aromatic amino acids through the diet, they do not
produce the enzymes of the shikimate pathway, making these
attractive targets for the generation of new antitubercular
agents. With our collaborator in the Harrison School of Pharmacy,
Angela Calderon, we are
generating molecular tools to aid in the identification and
characterization of inhibitors of shikimate kinase (Figure 2), an
enzyme central to the shikimate pathway. Critical to this
endeavor, we must identify mechanistically appropriate
inhibitors. Because the enzyme operates using shikimate, a
metabolite completely absent from human metabolism, and ATP, a
metabolite found throughout human metabolism, it is important to
identify inhibitors that mimic shikimate rather than ATP in their
mechanisms of inhibition. Further, inhibitors which exploit the
known conformational dynamics of the catalytic process are
anticipated to be more effective than those that do not. To this
end, we are using mechanistically-targeted incorporation of
intrinsic protein fluorescence to produce a panel of shikimate
kinase variants that will aid in rapidly determining the mechanism
of action of candidate inhibitors.
Goodwin Lab Pics
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Simithy, J.; Fuanta, N.R.; Kochanowska-Karamayan,
A.; Hobrath, J.V.; Hamann, M.T.; Goodwin, D.C.; Calderon, A.I.
"Mechanism of irreversible inhibition of Mycobacterium
tuberculosis shikimate kinase by ilimaquinone" Biochim.
Biophys. Acta 2018 (in press).
Alturki, M.S.; Fuanta, N.R.; Jarrard, M.A.; Hobrath, J.V.; Goodwin, D.C.; Rants'o, T.A.; Calderon, A.I. "A multifaceted approach to identify non-specific enzyme inhibition: Application to Mycobacterium tuberculosis shikimate kinase" Bioorg. Medicin. Chem. Lett. 2018, 28, 802.
Njuma, O.J.; Davis, I.; Ndontsa, E.N.; Krewall,
J.R.; Liu, A.; Goodwin, D.C. "Mutual synergy between catalase and
peroxidase activities of the bifunctional enzyme KatG is
facilitated by electron-hole hopping within the enzyme" J. Biol.
Chem. 2017, 292, 18408.
McCarty, S.E.; Schellenberger, A.; Goodwin, D.C.;
Fuanta, N.R.; Tekwani, B.L.; Calderon, A.I. "Plasmodium
falciparum thioredoxin reductase (PfTrxR) and its role as a
target for new antimalarial discovery" Molecules 2015,
Gordon, S.; Simithy, J.; Goodwin, D.C.; Calderon,
A.I. "Selective Mycobacterium tuberculosis shikimate
kinase inhibitors as potential antibacterials" Perspect.
Medicin. Chem. 2015, 7, 9.
Huang, J.; Smith, F., Panizzi, J.R.; Goodwin,
D.C.; Panizzi, P. "Inactivation of myeloperoxidase by benzoic acid
hydrazide" Arch. Biochem. Biophys. 2015, 570,
Kudalkar, S.N.; Njuma, O.J.; Li, Y.; Muldowney,
M.; Fuanta, N.R.; Goodwin, D.C. "A role for catalase-peroxidase
large loop 2 revealed by deletion mutagenesis: Control of active
site water and ferric enzyme reactivity" Biochemistry 2015,
Simithy, J.; Gill, G.; Wang, Y.; Goodwin, D.C.;
Calderon, A.I. "Development of an ESI-LC-MS based assay for
kinetic evaluation of M. tuberculosis shikimate kinase" Anal.
Chem. 2015, 87, 2129.
Njuma, O.J.; Ndontsa, E.N.; Goodwin, D.C. "Catalase in peroxidase clothing: Interdependent cooperation of two cofactors in the catalytic versatility of KatG" Arch. Biochem. Biophys. 2014, 544, 27.
Wang, Y.; Goodwin D.C. "Integral role of
the I'-helix in the function of the "inactive" C-terminal domain
of catalase-peroxidase (KatG)" Biochim.
Biophys. Acta 2013,
Kudalkar, S.N.; Campbell, R.A.; Li, Y.; Varnado, C.L.; Prescott, C.; Goodwin, D.C. "Enhancing the peroxidatic activity of KatG by deletion mutagenesis" J. Inorg. Biochem. 2012, 116, 106.
Ndontsa, E.N.; Moore, R.L.; Goodwin, D.C.
"Stimulation of KatG catalase activity by peroxidatic electron
donors" Arch. Biochem.
Biophys. 2012, 105,
Tejero, J.; Biswas, A.; Haque, M.M.; Wang, Z.Q.; Hemann, C.; Varnado, C.L.; Novince, Z.; Hille, R.; Goodwin, D.C.; Stuehr, D.J. "Mesohaem substitution reveals how haem electronic properties can influence the kinetic and catalytic parameters of neuronal NO synthase" Biochem. J. 2011, 433, 163.
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