Reduced Affinity for Isoniazid in the S315T Mutant ofMycobacterium tuberculosis KatG Is a Key Factor in Antibiotic Resistance

Abstract

Catalase-peroxidase (KatG) fromMycobacterium tuberculosis is responsible for the activation of the antitubercular drug isonicotinic acid hydrazide (INH) and is important for survival of M. tuberculosis in macrophages. Characterization of the structure and catalytic mechanism of KatG is being pursued to provide insights into drug (INH) resistance in M. tuberculosis. Site-directed mutagenesis was used to prepare the INH-resistant mutant KatG[S315T], and the overexpressed enzyme was characterized and compared with wild-type KatG. KatG[S315T] exhibits a reduced tendency to form six-coordinate heme, because of coordination of water to iron during purification and storage, and also forms a highly unstable Compound III (oxyferrous enzyme). Catalase activity and peroxidase activity measured usingt-butylhydroperoxide and o-dianisidine were moderately reduced in the mutant compared with wild-type KatG. Stopped-flow spectrophotometric experiments revealed a rate of Compound I formation similar to wild-type KatG using peroxyacetic acid to initiate the catalytic cycle, but no Compound I was detected when bulkier peroxides (chloroperoxybenzoic acid,t-butylhydroperoxide) were used. The affinity of resting (ferric) KatG[S315T] for INH, measured using isothermal titration calorimetry, was greatly reduced compared with wild-type KatG, as were rates of reaction of Compound I with the drug. These observations reveal that although KatG[S315T] maintains reasonably good steady state catalytic rates, poor binding of the drug to the enzyme limits drug activation and brings about INH resistance. Catalase-peroxidase (KatG) fromMycobacterium tuberculosis is responsible for the activation of the antitubercular drug isonicotinic acid hydrazide (INH) and is important for survival of M. tuberculosis in macrophages. Characterization of the structure and catalytic mechanism of KatG is being pursued to provide insights into drug (INH) resistance in M. tuberculosis. Site-directed mutagenesis was used to prepare the INH-resistant mutant KatG[S315T], and the overexpressed enzyme was characterized and compared with wild-type KatG. KatG[S315T] exhibits a reduced tendency to form six-coordinate heme, because of coordination of water to iron during purification and storage, and also forms a highly unstable Compound III (oxyferrous enzyme). Catalase activity and peroxidase activity measured usingt-butylhydroperoxide and o-dianisidine were moderately reduced in the mutant compared with wild-type KatG. Stopped-flow spectrophotometric experiments revealed a rate of Compound I formation similar to wild-type KatG using peroxyacetic acid to initiate the catalytic cycle, but no Compound I was detected when bulkier peroxides (chloroperoxybenzoic acid,t-butylhydroperoxide) were used. The affinity of resting (ferric) KatG[S315T] for INH, measured using isothermal titration calorimetry, was greatly reduced compared with wild-type KatG, as were rates of reaction of Compound I with the drug. These observations reveal that although KatG[S315T] maintains reasonably good steady state catalytic rates, poor binding of the drug to the enzyme limits drug activation and brings about INH resistance. tuberculosis catalase-peroxidase wild-type Isoniazid (isonicotinic acid hydrazide) compound I compound II compound III peroxyacetic acid 3-chloroperoxybenzoic acid tert-butylhydroperoxide five-coordinate six-coordinate electron paramagnetic resonance isothermal titration calorimetry δ-aminolevulinic acid horseradish peroxidase minimal inhibitory concentration Tuberculosis (TB)1infection is the leading cause of death due to a single infectious agent, causing over 2 million deaths annually (1World Health Organization (2002) Tuberculosis Fact Sheet (August) No. 104.Google Scholar). Mycobacterium tuberculosis is the causative agent of this infection, and antibiotics have been used to treat the infection since the 1940s. Although Isoniazid (isonicotinic acid hydrazide, INH) is still one of the most effective antibiotics against tuberculosis (2Robitzek E.H. Selikoff I.J. Am. Rev. Tuberc. 1952; 65: 402-410PubMed Google Scholar), the number of INH and other drug-resistant strains has increased dramatically. Outbreaks of multidrug-resistant M. tuberculosis make the solution of this worldwide health problem even more urgent. The mechanism of action of INH is not fully understood, but it has been clearly demonstrated that the catalase-peroxidase of M. tuberculosis, encoded by the katG gene, is required for bacterial sensitivity to INH (3Zhang Y. Heym B. Allen B. Young D. Cole S. Nature. 1992; 358: 591-593Crossref PubMed Scopus (1078) Google Scholar, 4Zhang Y. Garbe T. Young D. Mol. Microbiol. 1993; 8: 521-524Crossref PubMed Scopus (157) Google Scholar). The katG gene alone can restore INH susceptibility to resistant mutants (katG−/−) of Mycobacterium smegmatis and M. tuberculosis (4Zhang Y. Garbe T. Young D. Mol. Microbiol. 1993; 8: 521-524Crossref PubMed Scopus (157) Google Scholar, 5Rouse D.A. DeVito J.A. Li Z. Byer H. Morris S.L. Mol. Microbiol. 1996; 22: 583-592Crossref PubMed Scopus (149) Google Scholar), and its expression can make certain strains of Escherichia colisensitive to INH (3Zhang Y. Heym B. Allen B. Young D. Cole S. Nature. 1992; 358: 591-593Crossref PubMed Scopus (1078) Google Scholar, 4Zhang Y. Garbe T. Young D. Mol. Microbiol. 1993; 8: 521-524Crossref PubMed Scopus (157) Google Scholar). INH is a pro-drug that requires activation by KatG leading to its ultimate bactericidal effects that target mycolic acid biosynthesis (6Johnsson K. Schultz P.G. J. Am. Chem. Soc. 1994; 116: 7425-7426Crossref Scopus (268) Google Scholar, 7Johnsson K. King D.S. Schultz P.G. J. Am. Chem. 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M. tuberculosis KatG, a catalase-peroxidase containing 80-kDa subunits with one heme per subunit in the dimer, is homologous to yeast cytochrome c peroxidase in its N-terminal region, especially in the distal and proximal heme regions (12Hochman A. Goldberg I. Biochim. Biophys. Acta. 1991; 1077: 299-307Crossref PubMed Scopus (74) Google Scholar, 13Hicks D.B. Biochim. Biophys. Acta. 1995; 1229: 347-355Crossref PubMed Scopus (25) Google Scholar, 14Fraaije M.W. Roubroeks H.P. Hagen W.R. Van Berkel W.J. Eur. J. Biochem. 1996; 235: 192-198Crossref PubMed Scopus (70) Google Scholar, 15Yumoto I. Fukumori Y. Yamanaka T. J. Biochem. (Tokyo). 1990; 108: 583-587Crossref PubMed Scopus (43) Google Scholar, 16Cendrin F. Jouve H.M. Gaillard J. Thibault P. Zaccai G. Biochim. Biophys. Acta. 1994; 1209: 1-9Crossref PubMed Scopus (29) Google Scholar, 17Marcinkeviciene J.A. Magliozzo R.S. Blanchard J.S. J. Biol. 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Full Text Full Text PDF PubMed Scopus Google Scholar). INH activation by KatG can in a mechanism the formation of I (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar, S. S. S. Magliozzo R.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. Magliozzo R.S. PubMed Scopus Google Scholar). radical is formed in of the resting enzyme with though its is not S. S. S. Magliozzo R.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of acid in KatG is one of the most in clinical INH-resistant strains (5Rouse D.A. DeVito J.A. Li Z. Byer H. Morris S.L. Mol. Microbiol. 1996; 22: 583-592Crossref PubMed Scopus (149) Google Scholar, Z. Y. J. Infect. Dis. 1995; PubMed Scopus Google Scholar, B. Cole Mol. Microbiol. 1995; PubMed Scopus Google Scholar, S. P. M. D. J. Infect. Dis. 1995; PubMed Scopus Google Scholar, D.A. Li Z. Morris S.L. Antimicrob. Agents Chemother. 1995; PubMed Scopus Google Scholar, D.A. Morris S.L. Infect. 1995; PubMed Google Scholar, J.M. D. J. Infect. Dis. 1996; PubMed Scopus Google Scholar, Antimicrob. Agents Chemother. 1996; 40: PubMed Google Scholar, S. J.M. Dis. 1998; Full Text PDF PubMed Scopus Google Scholar, D. E. H. M.W. J. Infect. Dis. PubMed Scopus Google Scholar). For example, more of INH-resistant clinical isolates the in a in minimal inhibitory concentration for INH in The KatG[S315T] mutant is in the that this not in a of bacterial this to a major of activity peroxidase activity measured with in B. H. M. K. Cole Biochem. J. PubMed Scopus Google Scholar, B. Cole Infect. PubMed Scopus Google Scholar, S. F. J. Infect. Dis. 1997; PubMed Scopus Google Scholar). the of KatG[S315T] in INH S. F. J. Infect. Dis. 1997; PubMed Scopus Google Scholar) that it was moderately KatG in reaction to no other of peroxidase activity was and this mutant was more in INH compared with KatG in a reaction by in the of enzyme but not with H.M. F. J. Am. Chem. Soc. Scopus Google Scholar). The a reaction KatG[S315T] and as the of INH resistance. a in INH binding to KatG[S315T] was binding for INH in KatG[S315T] and KatG were from S. F. Biochemistry. 1998; PubMed Scopus (70) Google Scholar, S. S. F. J. Am. Chem. Soc. Scopus Google Scholar). to the problem of INH resistance of the the resting KatG[S315T] enzyme and the drug and the reaction of the drug with catalytically competent enzyme as I. this the of the mutant enzyme KatG[S315T], its and and spectroscopic using electron paramagnetic resonance and as as isothermal titration calorimetry were used to the of the and the of in enzyme that a in INH binding in KatG[S315T] is a major in the of to of this for INH was from was to in and was with for to by of the enzyme by and the of were from and were from II from was used for and The II from was used as an for KatG K. Froland W.A. Schultz P.G. J. Biol. Chem. 1997; 272: 2834-2840Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) and as the of the katG gene that was into to used for E. was used as a for the and and was used in the mutagenesis E. J. M. Biochem. Biol. 1990; PubMed Scopus (29) Google Scholar) was the for was using the mutagenesis from The of the was into the in to in mutagenesis was The mutagenesis was the was was with a the the enzyme of by the F. S. S. A. PubMed Scopus Google Scholar) was used to the The katG was from the using containing the was to the to the wild-type the The and mutant used in this were and from an in E. II were in in expression was by the of acid when the of acid was to to the of (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar). were a using of J.A. Magliozzo R.S. Blanchard J.S. J. Biol. Chem. 1995; 270: 22290-22295Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). were for KatG and for concentration was using the heme Catalase activity was measured by the in concentration of activity is as the of enzyme that of in in a solution B. H. M. K. Cole Biochem. J. PubMed Scopus Google Scholar). activity was also by the for in in the of for the J.A. Magliozzo R.S. Blanchard J.S. J. Biol. Chem. 1995; 270: 22290-22295Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). of peroxidase activity is as the of enzyme that the formation of of were using an was and using an was used to during I formation from resting (ferric) enzyme and for the reaction of I with and and were in as by (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar). titration calorimetry was using a and were using the with the KatG and KatG[S315T] were in were in the to minimal from titration were using enzyme in the and an INH concentration in the that an of KatG[S315T] the of the per were and the of reaction per per was by of the of of INH was by of INH into and was from the of binding to The the of of the of binding per of heme, and the from of per of the were with a with a and for K. was used for (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar). was using an to a S. S. S. Magliozzo R.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The KatG[S315T] used in was using an the tuberculosis katG The mutant enzyme a reduced compared with KatG for this the of (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar) that KatG and in E. in in the of the heme δ-aminolevulinic acid a of enzyme and in the of the enzyme is For KatG[S315T] in the of was in of was in the of though KatG[S315T] still a compared with KatG. to this is the of heme in the mutant in to enzyme is in KatG The enzyme formed by coordination of a of water to heme iron has a is in the for the wild-type from in containing heme, to in containing a of heme S. S. S. S. Magliozzo R.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The for KatG[S315T] from to in that the for heme in and mutant enzymes are the and that the for the mutant also of the mutant enzyme enzyme and are heme concentration the is not for in experiments The not to peroxidase in the heme to by and J.A. Magliozzo R.S. Blanchard J.S. J. Biol. Chem. 1995; 270: 22290-22295Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). to the in a of the and a of the were in the mutant compared with KatG and These differences are also with a of heme in and and rate for I formation in KatG and in are the of for KatG for usingt-butylhydroperoxide and o-dianisidine are the of for KatG and rate for I formation using in were from of rates as a of in B. H. M. K. Cole Biochem. J. PubMed Scopus Google Scholar). are the of for KatG for usingt-butylhydroperoxide and o-dianisidine J.A. Magliozzo R.S. Blanchard J.S. J. Biol. Chem. 1995; 270: 22290-22295Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). are the of for KatG and using in (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar) were from of rates as a of in a Catalase and peroxidase of KatG[S315T] are reduced by and compared with the wild-type enzyme using the (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar). in catalytic have been by (5Rouse D.A. DeVito J.A. Li Z. Byer H. Morris S.L. Mol. Microbiol. 1996; 22: 583-592Crossref PubMed Scopus (149) Google Scholar, B. H. M. K. Cole Biochem. J. PubMed Scopus Google Scholar, S. F. J. Infect. Dis. 1997; PubMed Scopus Google Scholar). steady state not provide into the of the in the catalytic experiments were KatG[S315T] maintains of catalytic formation of I other peroxidase was important to especially in the of INH resistance (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar, S. S. Magliozzo R.S. PubMed Scopus Google Scholar) rate for formation of I in KatG and mutant using peroxides and also that INH with this intermediate in KatG and of the formation of I in the mutant KatG[S315T] revealed differences from the wild-type For example, to a in of the resting mutant enzyme to I clearly to this intermediate in KatG. for I formation was demonstrated in of KatG[S315T] with by a and in the and with a for KatG[S315T] with but with of I were using The of I not using of The for the mutant is that for KatG similar because of differences in for resting enzyme and I in enzymes the used to KatG KatG[S315T] I to the resting enzyme in the of a that the was to of during of the enzyme and its to the resting (ferric) state when (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar, S. S. Magliozzo R.S. PubMed Scopus Google Scholar). the the of this in the mutant is that for KatG with are not this and KatG[S315T] I formation and resting KatG KatG[S315T] with are of the rate of I formation (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar) of rate to and similar to KatG of were from of rates as a of is important in of the poor reaction of the peroxides with resting mutant For KatG, III (oxyferrous is formed by of a of to the resting though this intermediate is not the of KatG[S315T], to of not in as for KatG (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar). even of not the of III in the the in was a in of the a with a of the These not formation of III and and but are with coordination of as a of the for this was in experiments KatG I was (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar) to catalytically competent in INH used to this reaction in KatG and the single using KatG KatG[S315T] with formation of I was 2 and and I for the KatG I formed during an reaction was with INH in a of and and were INH the of I to the resting state in in a and The a of that of I to II and II to the resting though II is not that (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar) demonstrated that in the of INH II from the as a in the peroxidase that II is unstable in KatG, its are in the in for is the the to a in as a of binding of INH to I. in the of the were during this to the I and for J. F. M. A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). this a reaction the of the resting enzyme in the in the and in the is the formation of an intermediate a the is the of INH the in and is with formation of enzyme of the peroxidase cycle, by its to enzyme as INH to the The during this is also the concentration of For the similar but to a in KatG. the affinity for the drug and the for heme to this from of to heme because of the of water INH binding to KatG has been in of the enzyme with the drug S. F. Biochemistry. 1998; PubMed Scopus (70) Google Scholar, S. S. S. S. Magliozzo R.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to its with the of I in the of INH as the required for the to its the was and INH) for KatG KatG[S315T] I more with INH, and even the drug concentration of this was compared with the rate with alone also the rate of of I in the mutant not as for KatG (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar). no as II detected in the The and the concentration of the for formation of the in KatG. was not in the reaction of mutant I with INH drug These to poor the drug and I of the mutant The concentration of the rates for the and the are and rates were not was used by and in other S. S. Magliozzo R.S. PubMed Scopus Google Scholar, S. F. Biochemistry. 1998; PubMed Scopus (70) Google Scholar) to affinity for the binding of INH to KatG. the that titration of KatG with drug in the resting enzyme with of heme into the concentration of enzyme in the enzyme is no can detected of INH, as for KatG in heme S. S. S. S. Magliozzo R.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of heme in KatG[S315T] that a more for of INH affinity a as this a is into a solution of the and the during in the titration is binding is from this and for the number of binding and and of binding are for KatG titration of the enzyme with a of INH of INH to heme, of and to and were The with the drug concentration to the for wild-type M. tuberculosis similar a binding titration was for of KatG[S315T] with of INH of a of of that for KatG. the binding of INH to the mutant is demonstrated to in the of the for the INH-resistant M. in (5Rouse D.A. DeVito J.A. Li Z. Byer H. Morris S.L. Mol. Microbiol. 1996; 22: 583-592Crossref PubMed Scopus (149) Google Scholar, B. Cole Mol. Microbiol. 1995; PubMed Scopus Google Scholar). is the of and the of KatG enzymes for the drug in in more to the of iron coordination number in KatG have S. S. S. S. Magliozzo R.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that the of and heme is in KatG and of used for purification and The most for wild-type enzyme was that the heme in the enzyme the of heme as a of over a of the of KatG, KatG for and KatG[S315T] also for The of KatG[S315T] has and similar to the wild-type enzyme purification These were to and heme can also is to a For the wild-type enzyme storage, the is no is because of to heme coordination of a of water to These were using and J. P. M. that the mutant is more resistant to to the observations with heme in KatG[S315T] F. Biochemistry. PubMed Scopus Google Scholar). to the catalytic of KatG[S315T] in the of is about this drug-resistant mutant from and studies. The to to the strains this are and B. Cole Infect. PubMed Scopus Google Scholar, B. Cole Microbiol. 1992; PubMed Scopus Google Scholar), a binding for the drug is in the mutant a from the heme iron to that in KatG S. F. Biochemistry. 1998; PubMed Scopus (70) Google Scholar, S. S. F. J. Am. Chem. Soc. Scopus Google Scholar), to experiments F. Biochemistry. 40: PubMed Scopus Google Scholar) KatG[S315T] in the of of INH and the peroxidase of as KatG over a of the mutant INH more KatG in the of other peroxidase S. F. J. Infect. Dis. 1997; PubMed Scopus Google Scholar). in drug by KatG[S315T] compared with KatG were also in the of though was when was to enzymes H.M. F. J. Am. Chem. Soc. Scopus Google Scholar). for reduced catalytic of the mutant from in other but mechanistic in the peroxidase cycle, is the most for drug activation in have not been Although it been that KatG catalytically competent in drug R.S. J.A. J. Am. Chem. Soc. 1996; Scopus (70) Google Scholar), the peroxidase is to and for INH activation that KatG I is competent and is reduced by INH and that INH as a single electron in a peroxidase in (20Chouchane S. Lippai I. Magliozzo R.S. Biochemistry. PubMed Scopus Google Scholar). The spectroscopic of KatG[S315T] demonstrated a reduced affinity for heme in this with a of compared with heme, the as the form of KatG. the wild-type demonstrated the of heme from heme because of of water during purification and a of S. S. S. S. Magliozzo R.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of KatG and this mutant to for the of heme in this are also with this of the These observations an iron coordination number and the structure of the enzyme in a from the distal coordination number is to by the distal as in M. tuberculosis KatG, by to the in H. is a heme the a of and the in heme binding of to reasonably to a poor for this the heme in the mutant also with the in the heme that formation of with the reduced tendency for water coordination in the mutant is its affinity for S. F. Biochemistry. 1998; PubMed Scopus (70) Google Scholar). These differences KatG and the mutant are not important in INH because the rate of formation of KatG I is not for and S. S. S. S. Magliozzo R.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). KatG[S315T] has activity KatG and also peroxidase activity compared with KatG when measured that not formation of I in the mutant using in peroxidase activity can using that I is in its no of this intermediate was detected a of in These observations are with and of I with is to to the of the a in an from the of the enzyme Y. T. T. PubMed Scopus Google Scholar), in the mutant is by a bulkier increased heme to the peroxidase activity in the mutant because of poor to the other rates of of resting enzyme with to I are not reduced compared with KatG, with of this about this being to heme been in KatG[S315T] B. H. M. K. Cole Biochem. J. PubMed Scopus Google Scholar). not the in the but for For example, III not in the mutant when it was with of that to III in KatG. the mutant I formed in the of form with KatG peroxidase reaction I. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of III in the mutant that a rate for reaction of I with of II in other reaction a highly unstable III is formed in the against the is the of activity in the compared with KatG. of the are the observations of H.M. F. J. Am. Chem. Soc. Scopus Google Scholar), that by the mutant enzyme KatG[S315T] are compared with similar in KatG. by a reduced affinity of the mutant enzyme for that for KatG, III can as heme to resting with of The increased of heme in the mutant also in of an unstable III in The poor reaction of KatG[S315T] with effects similar to responsible for of to the heme The of experiments the affinity of the resting mutant enzyme for the drug. alone an for drug resistance because of this it was that the catalytically intermediate I the poor binding in the resting enzyme drug binding to I a this a more binding reaction an increased rate of KatG[S315T] I with the drug. of observations that INH affinity for the resting enzyme is with of the drug with KatG I and a drug binding in enzyme KatG[S315T] in the resting state and in its I intermediate affinity for INH, is the of susceptibility to INH in strains this that of the INH to drug binding a to resistance in into a in KatG[S315T] not survival drug resistance in clinical to cause a worldwide problem in