Mycobacterium tuberculosis KatG(S315T) catalase-peroxidase retains all active site properties for proper catalytic function

Sofia M. Kapetanaki, Salem Chouchane, Shengwei Yu, Xiangbo Zhao, Richard S. Magliozzo, Johannes Schelvis

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Abstract

Mycobacterium tuberculosis (Mtb) KatG is a catalase-peroxidase that is thought to activate the antituberculosis drug isoniazid (INH). The local environment of Mtb KatG and its most prevalent INH-resistant mutant, KatG(S315T), is investigated with the exogenous ligands CO and NO in the absence and presence of INH by using resonance Raman, FTIR, and transient absorption spectroscopy. The Fe-His stretching vibration is detected at 244 cm-1 in the ferrous forms of both the wild-type enzyme and KatG(S315T). The ferrous-CO complex of both enzymes exhibits v(CO), v(Fe-CO), and δ(Fe-C-O) vibrations at 1925, 525, and 586 cm-1, respectively, indicating a positive electrostatic environment for the CO complex, which is probably weakly hydrogen-bonded to a distal residue. The CO geometry is nonlinear as indicated by the unusually high intensity of the Fe-C-O bending vibration. The v(Fe III-NO) and δ(FeIII-N-O) vibrations are detected at 596 and 571 cm-1, respectively, in the ferric forms of wild-type and mutant enzyme and are indicative of a nonlinear binding geometry in support of the CO data. Although the presence of INH does not affect the vibrational frequencies of the CO- and NO-bound forms of either enzyme, it seems to perturb slightly their Raman intensities. Our results suggest a minimal, if any, perturbation of the distal heme pocket in the S315T mutant. Instead, the S315T mutation seems to induce small changes in the KatG conformation/dynamics of the ligand access channel as indicated by CO rebinding kinetics in flash photolysis experiments. The implications of these findings for the catalytic mechanism and mechanism of INH resistance in KatG(S315T) are discussed.

Original languageEnglish
Pages (from-to)243-252
Number of pages10
JournalBiochemistry
Volume44
Issue number1
DOIs
StatePublished - 11 Jan 2005

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Carbon Monoxide
Mycobacterium tuberculosis
Catalase
Peroxidase
Catalytic Domain
Vibration
Enzymes
Ligands
Geometry
Photolysis
Isoniazid
Vibrational spectra
Fourier Transform Infrared Spectroscopy
Static Electricity
Heme
Absorption spectroscopy
Stretching
Conformations
Hydrogen
Electrostatics

Cite this

Kapetanaki, Sofia M. ; Chouchane, Salem ; Yu, Shengwei ; Zhao, Xiangbo ; Magliozzo, Richard S. ; Schelvis, Johannes. / Mycobacterium tuberculosis KatG(S315T) catalase-peroxidase retains all active site properties for proper catalytic function. In: Biochemistry. 2005 ; Vol. 44, No. 1. pp. 243-252.
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title = "Mycobacterium tuberculosis KatG(S315T) catalase-peroxidase retains all active site properties for proper catalytic function",
abstract = "Mycobacterium tuberculosis (Mtb) KatG is a catalase-peroxidase that is thought to activate the antituberculosis drug isoniazid (INH). The local environment of Mtb KatG and its most prevalent INH-resistant mutant, KatG(S315T), is investigated with the exogenous ligands CO and NO in the absence and presence of INH by using resonance Raman, FTIR, and transient absorption spectroscopy. The Fe-His stretching vibration is detected at 244 cm-1 in the ferrous forms of both the wild-type enzyme and KatG(S315T). The ferrous-CO complex of both enzymes exhibits v(CO), v(Fe-CO), and δ(Fe-C-O) vibrations at 1925, 525, and 586 cm-1, respectively, indicating a positive electrostatic environment for the CO complex, which is probably weakly hydrogen-bonded to a distal residue. The CO geometry is nonlinear as indicated by the unusually high intensity of the Fe-C-O bending vibration. The v(Fe III-NO) and δ(FeIII-N-O) vibrations are detected at 596 and 571 cm-1, respectively, in the ferric forms of wild-type and mutant enzyme and are indicative of a nonlinear binding geometry in support of the CO data. Although the presence of INH does not affect the vibrational frequencies of the CO- and NO-bound forms of either enzyme, it seems to perturb slightly their Raman intensities. Our results suggest a minimal, if any, perturbation of the distal heme pocket in the S315T mutant. Instead, the S315T mutation seems to induce small changes in the KatG conformation/dynamics of the ligand access channel as indicated by CO rebinding kinetics in flash photolysis experiments. The implications of these findings for the catalytic mechanism and mechanism of INH resistance in KatG(S315T) are discussed.",
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Mycobacterium tuberculosis KatG(S315T) catalase-peroxidase retains all active site properties for proper catalytic function. / Kapetanaki, Sofia M.; Chouchane, Salem; Yu, Shengwei; Zhao, Xiangbo; Magliozzo, Richard S.; Schelvis, Johannes.

In: Biochemistry, Vol. 44, No. 1, 11.01.2005, p. 243-252.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Magliozzo, Richard S.

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N2 - Mycobacterium tuberculosis (Mtb) KatG is a catalase-peroxidase that is thought to activate the antituberculosis drug isoniazid (INH). The local environment of Mtb KatG and its most prevalent INH-resistant mutant, KatG(S315T), is investigated with the exogenous ligands CO and NO in the absence and presence of INH by using resonance Raman, FTIR, and transient absorption spectroscopy. The Fe-His stretching vibration is detected at 244 cm-1 in the ferrous forms of both the wild-type enzyme and KatG(S315T). The ferrous-CO complex of both enzymes exhibits v(CO), v(Fe-CO), and δ(Fe-C-O) vibrations at 1925, 525, and 586 cm-1, respectively, indicating a positive electrostatic environment for the CO complex, which is probably weakly hydrogen-bonded to a distal residue. The CO geometry is nonlinear as indicated by the unusually high intensity of the Fe-C-O bending vibration. The v(Fe III-NO) and δ(FeIII-N-O) vibrations are detected at 596 and 571 cm-1, respectively, in the ferric forms of wild-type and mutant enzyme and are indicative of a nonlinear binding geometry in support of the CO data. Although the presence of INH does not affect the vibrational frequencies of the CO- and NO-bound forms of either enzyme, it seems to perturb slightly their Raman intensities. Our results suggest a minimal, if any, perturbation of the distal heme pocket in the S315T mutant. Instead, the S315T mutation seems to induce small changes in the KatG conformation/dynamics of the ligand access channel as indicated by CO rebinding kinetics in flash photolysis experiments. The implications of these findings for the catalytic mechanism and mechanism of INH resistance in KatG(S315T) are discussed.

AB - Mycobacterium tuberculosis (Mtb) KatG is a catalase-peroxidase that is thought to activate the antituberculosis drug isoniazid (INH). The local environment of Mtb KatG and its most prevalent INH-resistant mutant, KatG(S315T), is investigated with the exogenous ligands CO and NO in the absence and presence of INH by using resonance Raman, FTIR, and transient absorption spectroscopy. The Fe-His stretching vibration is detected at 244 cm-1 in the ferrous forms of both the wild-type enzyme and KatG(S315T). The ferrous-CO complex of both enzymes exhibits v(CO), v(Fe-CO), and δ(Fe-C-O) vibrations at 1925, 525, and 586 cm-1, respectively, indicating a positive electrostatic environment for the CO complex, which is probably weakly hydrogen-bonded to a distal residue. The CO geometry is nonlinear as indicated by the unusually high intensity of the Fe-C-O bending vibration. The v(Fe III-NO) and δ(FeIII-N-O) vibrations are detected at 596 and 571 cm-1, respectively, in the ferric forms of wild-type and mutant enzyme and are indicative of a nonlinear binding geometry in support of the CO data. Although the presence of INH does not affect the vibrational frequencies of the CO- and NO-bound forms of either enzyme, it seems to perturb slightly their Raman intensities. Our results suggest a minimal, if any, perturbation of the distal heme pocket in the S315T mutant. Instead, the S315T mutation seems to induce small changes in the KatG conformation/dynamics of the ligand access channel as indicated by CO rebinding kinetics in flash photolysis experiments. The implications of these findings for the catalytic mechanism and mechanism of INH resistance in KatG(S315T) are discussed.

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