Intermolecular interactions in the AhpC/AhpD antioxidant defense system of Mycobacterium tuberculosis.

The AhpC/AhpD system of Mycobacterium tuberculosis provides important antioxidant protection, particularly when the KatG catalase-peroxidase activity is depressed, as it is in many isoniazid resistant strains. In the absence of lipoamide or bovine dihydrolipoamide dehydrogenase (DHLDH), components of the normal catalytic system, covalent dimers, tetramers, and hexamers are formed when a mixture of AhpC and AhpD is exposed to peroxide. Each of the oligomers contains equimolar amounts of AhpC and AhpD. This oligomerization is reversible because the oligomers can be fully reduced to the monomeric species by dithiothreitol. Using mutagenesis, we confirm here that Cys61 and Cys174 of AhpC as well as Cys133 and Cys130 of AhpD are critical for activity in the fully reconstituted system consisting of AhpC, AhpD, lipoamide, DHLDH, and NADH. A key step in the reduction of oxidized AhpC by reduced AhpD is formation of a disulfide cross-link between Cys61 of AhpC and Cys133 of AhpD. This cross-link can be reduced by intramolecular reaction with either Cys174 of AhpC or Cys130 of AhpD. Cys176 can also, to some extent, substitute for Cys174, providing a measure of redundancy that helps to maintain the efficiency of this antioxidant protective system.

[1]  J. Sacchettini,et al.  Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis. , 1998, Science.

[2]  P. Ortiz de Montellano,et al.  The AhpC and AhpD Antioxidant Defense System of Mycobacterium tuberculosis * , 2000, The Journal of Biological Chemistry.

[3]  G. Church,et al.  Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Cole,et al.  The catalase—peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis , 1992, Nature.

[5]  S. Rhee,et al.  Thioredoxin-dependent peroxide reductase from yeast. , 1994, The Journal of biological chemistry.

[6]  P. Ortiz de Montellano,et al.  The Crystal Structure of Mycobacterium tuberculosisAlkylhydroperoxidase AhpD, a Potential Target for Antitubercular Drug Design* , 2002, The Journal of Biological Chemistry.

[7]  L. Poole,et al.  Requirement for the two AhpF cystine disulfide centers in catalysis of peroxide reduction by alkyl hydroperoxide reductase. , 1997, Biochemistry.

[8]  P. Schultz,et al.  Mechanistic Studies of the Oxidation of Isoniazid by the Catalase Peroxidase from Mycobacterium tuberculosis , 1994 .

[9]  C. E. Barry,et al.  The genetics and biochemistry of isoniazid resistance in mycobacterium tuberculosis. , 2000, Microbes and infection.

[10]  S. Dhandayuthapani,et al.  The extreme sensitivity of Mycobacterium tuberculosis to the front-line antituberculosis drug isoniazid , 1996, Nature Biotechnology.

[11]  P. Brown,et al.  Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[12]  B. Barrell,et al.  Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence , 1998, Nature.

[13]  P. Karplus,et al.  Dimers to doughnuts: redox-sensitive oligomerization of 2-cysteine peroxiredoxins. , 2002, Biochemistry.

[14]  Shekhar C Mande,et al.  Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C. , 2002, The Biochemical journal.

[15]  P. Ortiz de Montellano,et al.  The Mechanism of Mycobacterium tuberculosis Alkylhydroperoxidase AhpD as Defined by Mutagenesis, Crystallography, and Kinetics* , 2003, Journal of Biological Chemistry.

[16]  S. Ryu,et al.  Crystal structure of a novel human peroxidase enzyme at 2.0 Å resolution , 1998, Nature Structural Biology.

[17]  Clifton E. Barry,et al.  Compensatory ahpC Gene Expression in Isoniazid-Resistant Mycobacterium tuberculosis , 1996, Science.

[18]  S. Dhandayuthapani,et al.  Molecular basis for the exquisite sensitivity of Mycobacterium tuberculosis to isoniazid. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[19]  G. Mahairas,et al.  Disparate responses to oxidative stress in saprophytic and pathogenic mycobacteria. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[20]  N. Wengenack,et al.  Evidence for isoniazid-dependent free radical generation catalyzed by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG(S315T). , 2001, Biochemistry.

[21]  V. Deretic,et al.  Silencing of Oxidative Stress Response in Mycobacterium tuberculosis: Expression Patterns of ahpC in Virulent and Avirulent Strains and Effect ofahpC Inactivation , 2001, Infection and Immunity.

[22]  C. Pace,et al.  How to measure and predict the molar absorption coefficient of a protein , 1995, Protein science : a publication of the Protein Society.

[23]  C D Lima,et al.  Metabolic Enzymes of Mycobacteria Linked to Antioxidant Defense by a Thioredoxin-Like Protein , 2002, Science.

[24]  A. Vagin,et al.  Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A resolution. , 2000, Structure.

[25]  Z. A. Wood,et al.  Structure, mechanism and regulation of peroxiredoxins. , 2003, Trends in biochemical sciences.