Carbon monoxide adducts of KatG and KatG(S315T) as probes of the heme site and isoniazid binding.
KatG, the catalase peroxidase from Mycobacterium tuberculosis, is important in the activation of the antitubercular drug, isoniazid. About 50% of isoniazid-resistant clinical isolates contain a mutation in KatG wherein the serine at position 315 is substituted with threonine, KatG(S315T). The heme pockets of KatG and KatG(S315T) and their interactions with isoniazid are probed using resonance Raman (rR) spectroscopy to characterize their ferrous CO complexes. Three vibrational modes, C-O and Fe-C stretching and Fe-CO bending, are assigned using 12CO and 13CO isotope shifts. Two conformers are observed for KatG-CO and KatG(S315T)-CO. Resonance Raman features assigned to form I are consistent with it having a neutral proximal histidine ligand and the Fe-C-O moiety hydrogen bonded to a distal residue. The nu(C-O) band for form I is sharp, consistent with a conformationally homogeneous Fe-CO unit. Form II also has a neutral proximal histidine ligand but is not hydrogen bonded. This appears to result in a conformationally disordered Fe-CO unit, as evidenced by a comparatively broad C-O stretching band. The 13CO-sensitive bands assigned to form II are predominant in the KatG(S315T)-CO rR spectrum. Isoniazid binding is apparent from the resonance Raman signatures of both WT KatG-CO and KatG(S315T)-CO. Moreover, isoniazid binding elicits an increase in the form I population of wild-type KatG-CO while having little, if any, effect on the already low population of form I of KatG(S315T)-CO. Since oxyKatG (compound III) also contains a low-spin diatomic ligand-heme adduct (heme-O2), it is reasonable to suggest that it too would exist as a mixture of conformers. Because the small form I population of KatG(S315T)-CO correlates with its inability to activate INH, we hypothesize that form I plays a role in INH activation.
Lukat-Rodgers
G S
GS
Department of Chemistry, North Dakota State University, Fargo, North Dakota 58105, USA.
Wengenack
N L
NL
Rusnak
F
F
Rodgers
K R
KR
D019943:Amino Acid Substitution / Q000235:genetics; D000995:Antitubercular Agents / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites / Q000235:genetics; D002248:Carbon Monoxide / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D007538:Isoniazid / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D013059:Spectrum Analysis, Raman; D013912:Threonine / Q000235:genetics
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D005290:Ferric Compounds; D013912:Threonine; D006418:Heme; D012694:Serine; D002248:Carbon Monoxide; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria; C076136:catalase HPI; D007538:Isoniazid