The mechanism of Mycobacterium tuberculosis alkylhydroperoxidase AhpD as defined by mutagenesis, crystallography, and kinetics.
AhpD, a protein with two cysteine residues, is required for physiological reduction of the Mycobacterium tuberculosis alkylhydroperoxidase AhpC. AhpD also has an alkylhydroperoxidase activity of its own. The AhpC/AhpD system provides critical antioxidant protection, particularly in the absence of the catalase-peroxidase KatG, which is suppressed in most isoniazid-resistant strains. Based on the crystal structure, we proposed recently a catalytic mechanism for AhpD involving a proton relay in which the Glu118 carboxylate group, via His137 and a water molecule, deprotonates the catalytic residue Cys133 (Nunn, C. M., Djordjevic, S., Hillas, P. J., Nishida, C., and Ortiz de Montellano, P. R. (2002) J. Biol. Chem. 277, 20033-20040). A possible role for His132 in subsequent formation of the Cys133-Cys130 disulfide bond was also noted. To test this proposed mechanism, we have expressed the H137F, H137Q, H132F, H132Q, E118F, E118Q, C133S, and C130S mutants of AhpD, determined the crystal structures of the H137F and H132Q mutants, estimated the pKa values of the cysteine residues, and defined the kinetic properties of the mutant proteins. The collective results strongly support the proposed catalytic mechanism for AhpD.
Koshkin
Aleksey
A
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-2280, USA.
Nunn
Christine M
CM
Djordjevic
Snezana
S
Ortiz de Montellano
Paul R
PR
D000975:Antioxidants / Q000494:pharmacology; D001426:Bacterial Proteins; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002455:Cell Division; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010100:Oxygen / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics*; D054464:Peroxiredoxins; D011487:Protein Conformation; D011522:Protons; D013879:Thioredoxins / Q000737:chemistry; D014466:Ultraviolet Rays
D000975:Antioxidants; D001426:Bacterial Proteins; D004220:Disulfides; D011522:Protons; D018698:Glutamic Acid; D006639:Histidine; D013879:Thioredoxins; D010544:Peroxidases; D054464:Peroxiredoxins; C076136:catalase HPI; D003545:Cysteine; D010100:Oxygen