Isoniazid-resistance conferring mutations in Mycobacterium tuberculosis KatG: Catalase, peroxidase, and INH-NADH adduct formation activities Christine E. Cade,1 Adrienne C. Dlouhy,1 Katalin F. Medzihradszky,2 Saida Patricia Salas-Castillo,2 and Reza A. Ghiladi1* 1Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204 2Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158-2517 Received 27 August 2009 Revised 30 November 2009 Accepted 11 December 2009 DOI: 10.1002/pro.324 Published online 6 January 2010 proteinscience.org Abstract: Mycobacterium tuberculosis catalase-peroxidase (KatG) is a bifunctional hemoprotein that has been shown to activate isoniazid (INH), a pro-drug that is integral to frontline antituberculosis treatments. The activated species, presumed to be an isonicotinoyl radical, couples to NAD1/NADH forming an isoniazid-NADH adduct that ultimately confers anti-tubercular activity. To better understand the mechanisms of isoniazid activation as well as the origins of KatG-derived INH-resistance, we have compared the catalytic properties (including the ability to form the INH-NADH adduct) of the wild-type enzyme to 23 KatG mutants which have been associated with isoniazid resistance in clinical M. tuberculosis isolates. Neither catalase nor peroxidase activities, the two inherent enzymatic functions of KatG, were found to correlate with isoniazid resistance. Furthermore, catalase function was lost in mutants which lacked the Met-Tyr- Trp crosslink, the biogenic cofactor in KatG which has been previously shown to be integral to this activity. The presence or absence of the crosslink itself, however, was also found to not correlate with INH resistance. The KatG resistance-conferring mutants were then assayed for their ability to generate the INH-NADH adduct in the presence of peroxide (t-BuOOH and H2O2), superoxide, and no exogenous oxidant (air-only background control). The results demonstrate that residue location plays a critical role in determining INH-resistance mechanisms associated with INH activation however, different mutations at the same location can produce vastly different reactivities that are oxidant-specific. Furthermore, the data can be interpreted to suggest the presence of a second mechanism of INH-resistance that is not correlated with the formation of the INH-NADH adduct. Keywords: catalase-peroxidase KatG isoniazid INH heme crosslink Introduction Multi-drug resistant tuberculosis (MDR-TB), defined as strains which are resistant to more than one of the frontline antibiotics used in TB treatment, has been recorded at its highest levels ever, comprising about 5% of the 9 million incident cases of TB in 2006.1 MDR-TB is particularly difficult to treat, and has a high death rate of 50���80% within 4 months of diagnosis.1 Extensively drug resistant (XDR) TB, which comprises about 7% of all MDR cases, is virtu- ally untreatable.1,2 Thus, as the prevalence and severity of drug-resistant tuberculosis (TB) are on the rise, new efforts to understand the fundamental molecular basis of drug resistance are needed. It is now well-established that the catalase-per- oxidase (KatG) enzyme of Mycobacterium tuberculo- sis (Mtb) is responsible for activating the pro-drug Additional Supporting Information may be found in the online version of this article. Grant sponsor: NIH/NIAID Grant number: N01 AI-75320. *Correspondence to: Reza A. Ghiladi, Department of Chemistry, Dabney Hall, North Carolina State University, Campus Box 8204, Raleigh, NC 27695-8204. E-mail: reza_ghiladi@ncsu.edu 458 PROTEIN SCIENCE 2010 VOL 19:458���474 Published by Wiley-Blackwell. V C 2010 The Protein Society

isoniazid (INH). Although the details of this chemi- cal transformation are still the subject of ongoing investigations, it is hypothesized that the activation of INH leads to an isonicotinoyl acyl radical (Fig. 1) that then combines with NAD��/NADH to form what has been termed the isoniazid-NADH adduct (INH- NADH).3���5 This species was found to be a potent in- hibitor of InhA, an enoyl acyl-carrier protein reduc- tase involved in the production of mycolic acids, which are the key structural components of the mycobacterial cell wall.6���9 Although the phenomeno- logical observation is that mutations in KatG can give rise to INH-resistance due to their inability to activate the INH pro-drug,10���13 an increased under- standing of the interplay between an INH-conferring mutation and its consequences on the mechanism of isoniazid activation is necessary so that novel drug therapies can be developed to target these drug- resistant TB strains. Several mechanisms have been proposed for INH activation.3,14,15 As KatG belongs to the Class I family of peroxidases,16 the enzyme is thus capable of utilizing hydrogen peroxide (or alkyl hydroperox- ides) to catalyze the oxidation of various substrates via upwards of two consecutive one-electron oxida- tion steps, and it has been proposed that KatG oxi- dizes isoniazid in a similar manner (Fig. 2).15,17 Both, a two-electron oxidized KatG intermediate termed Compound I (ferryl porphyrin p-cation radi- cal) and a one-electron oxidized form called Com- pound II (evidence suggests a ferric heme coupled with a protein radical in KatG rather than the tradi- tional ferryl intermediate found in the monofunc- tional peroxidases, although this is the subject of current debate), have been observed in wild-type (WT) KatG,18,19 whereas an iron(IV)-oxo Compound II intermediate has been identified in KatGs con- taining active site mutations.19���22 Both, Compound I and the ferryl Compound II, species are intermedi- ates in the traditional peroxidase cycle, whereas Compound I is the key intermediate in the tradi- tional catalase cycle, which is the other major enzy- matic activity of KatG. Additional catalytic activities for KatG, such as NADH-oxidase,23 peroxynitri- tase,24 and Mn2��-dependent peroxidase,25 have also been reported. The heme species, Compound III, has also been proposed as a critical intermediate in KatG linked to INH activation and drug susceptibility.14,26���28 Such an oxyferrous intermediate may be formed in vivo upon the binding of superoxide to the active site heme-iron of resting (ferric) KatG, by the addition of dioxygen to the ferrous form of the enzyme, or by the addition of a large excess of hydrogen peroxide to the ferric heme center (Fig. 2). Previous in vitro studies correlated the attenuation of INH-NADH adduct formation when catalyzed by KatG in the presence of superoxide to several mutants that were known to give rise to drug-resistance in vivo.14 Fur- thermore, it was observed in earlier pulse radiolysis studies that oxyferrous WT KatG underwent reac- tion with isoniazid, whereas the oxyferrous form of the resistance mutant KatG(S315T) was unable to do so.28 Additional evidence in support of an oxyfer- rous mechanism for INH activation came from superoxide consumption studies which showed that superoxide was consumed by WT KatG in the pres- ence of INH and NADH, but not by those KatG active site mutants that were linked to drug-resist- ance in clinical Mtb isolates.14 To date, no systematic study exists in which KatG mutations that confer isoniazid-resistance are correlated to three variables: enzymatic activity, strain fitness, and TB transmission. As the first step towards such a larger and more comprehensive informatics-styled interdisciplinary study in which isoniazid-resistance is fully correlated to those three factors, the principal goal of this current investiga- tion is to generate a more extensive library of mutants that have been shown clinically to be related to INH resistance (Table I and Fig. 3), and identify Figure 1. Schematic representation of INH-NADH adduct formation as catalyzed by KatG via a putative isonicotinoyl radical. Figure 2. Proposed reactions and putative intermediates of KatG involved in the oxidation of isoniazid. Cade et al. PROTEIN SCIENCE VOL 19:458���474 459

potential trends between mutant residue location, en- zymatic activity, INH-NADH adduct formation as a function of heme intermediate (oxidant), and INH re- sistance. To probe the role of key heme intermediates in INH oxidation, the ability to form the INH-NADH adduct was assayed in the presence of peroxide (t- BuOOH and the H2O2-generating system glucose/glu- cose oxidase) and superoxide (xanthine/xanthine oxi- dase), and compared to air-only background controls (no exogenously added oxidant). In addition, peroxi- dase and catalase activities of each mutant were also assayed and correlated with the above INH-NADH adduct formation studies. Finally, we also investigated whether the presence of the structural feature unique to KatG, namely the Met-Tyr-Trp crosslink in which three non-sequential residues M255, Y229, and W107 are covalently linked together through their side chains (Fig. 4), can be correlated with INH-resistance. As will be demonstrated, the results of this study strongly suggest multiple mutation-specific pathways that, either separately or in combination, can affect INH-NADH adduct formation. Interestingly, several resistance-conferring INH mutations were identified which do not alter INH-NADH adduct formation, pos- sibly suggesting a yet-to-be identified mechanism for INH-resistance in TB. Results Site-directed mutagenesis and overexpression of KatG mutants The plasmid encoding wild-type KatG with an N-ter- minal poly-His tag (pMRLB11) was obtained from Colorado State University under the TB Research Materials and Vaccine Testing Contract (NIH, NIAID NO1 AI-75320). PCR amplification of pMRLB11 using mutagenic primers yielded the mu- tant plasmid(s) as confirmed by DNA sequencing. Hemin (30 mg L 1, dissolved in 10 mL 0.1M NaOH) was added to the culture medium before autoclaving. In this fashion, no insoluble hemin was observed. The addition of hemin (or, alternatively, aminolevu- linic acid, ALA18,29) assures stoichiometric incorpo- ration of the heme cofactor during overexpression in Escherichia coli for maximal holoenzyme isolation.30 Purification using immobilized metal affinity chro- matography yielded KatG with an acceptable optical purity ratio (Reinheitzahl or Rz, defined as ASoret/ Figure 3. Active site of Mtb KatG showing the heme prosthetic group, as well as the Met-Tyr-Trp crosslink. Coordinates (1SJ2) were obtained from the Protein Data Bank. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] Table I. KatG Mutations Investigated in this Study Location Mutation INH-resistance conferring mutations N-terminal domain D63E Active site R104L, W107R, H108E, H108Q, N138D, N138S, Y229F, W300G Proximal side T262R, T275P, W328G, Y337C, A350S Substrate access channel S315G, S315I, S315N, S315R, S315T C-terminal domain R463L, L587M, G629S, D735N Lab mutations W107F, M255C, M255I, M255Y, T275V, W321F, W328F, R418L Figure 4. Crystal structure of the Mtb KatG dimer. The heme prosthetic group (red) and mutations examined in this study (Table I) are highlighted. Coordinates (PDB ID: 1SJ2) were obtained from the Protein Data Bank. 460 PROTEINSCIENCE.ORG Isoniazid-Resistance Conferring Mutations in Mtb KatG