surface and ribbon representation of KatG

Laboratory of

Doug Goodwin

Associate Professor

Department of Chemistry and Biochemistry

179 Chemistry Building
Auburn University, AL 36849-5312
Phone: (334) 844-6992
FAX: (334) 844-6959
E mail: goodwdc  (at)  auburn  (dot)  edu

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Division: Biochemistry

Research Interests

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.

KatG and CcP OverlaidCatalase-peroxidase (KatG)

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 responses.

Shikimate Kinase ConformationsShikimate Kinase

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, Dr. 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.


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Goodwin Lab Pics



McCarty, S.E.; Schellenberger, A.; Goodwin, D.C.; Fuanta, N.R.; Tekwani, B.L.; Calderón, A.I. "Plasmodium falciparum thioredoxin reductase (PfTrxR) and its role as a target for new antimalarial discovery" Molecules 2015, 20, 11459.

Gordon, S.; Simithy, J.; Goodwin, D.C.; Calderón, 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, 14.

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, 54, 1648.

Simithy, J.; Gill, G.; Wang, Y.; Goodwin, D.C.; Calderón, 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, 1834, 362.

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, 215.

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.

You can view a listing of all our publications here.

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