Mycobacterium tuberculosis lipoamide dehydrogenase is encoded by Rv0462 and not by the lpdA or lpdB genes

A. Argyrou, John S. Blanchard

Research output: Contribution to journalArticle

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Abstract

The gene encoding dihydrolipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity. The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer. The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD+ in the absence of D,L-lipoamide, demonstrated that the enzyme uses a ping-pong kinetic mechanism. Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C4-proS position of the nicotinamide ring are small [D(V/K)NADH = 1.12 ± 0.15, DVapp = 1.05 ± 0.07] when D,L-lipoamide is the oxidant but large and equivalent [D(V/K)NADH = DV = 2.95 ± 0.03] when 5-hydroxy-1,4-naphthoquinone is the oxidant. Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with D(V/K)APADH = 0.73 ± 0.03, D(V/K)Lip(S)2 = 0.77 ± 0.03, and DVapp = 0.77 ± 0.01. Solvent deuterium kinetic isotope effects with 4,4-dithiopyridine (DTP), the 4-thiopyridone product of which requires no protonation, are also inverse with D(V/K)APADH = 0.75 ± 0.06, D(V/K)DTP = 0.71 ± 0.02, and DVapp = 0.56 ± 0.15. All proton inventories were linear, indicating that a single proton is being transferred in the solvent isotopically sensitive step. Taken together, these results suggest that (1) the reductive half-reaction (hydride transfer from NADH to FAD) is rate limiting when a quinone is the oxidant, and (2) deprotonation of enzymic thiols, most likely Cys46 and Cys41, limits the reductive and oxidative half-reactions, respectively, when D,L-lipoamide is the oxidant.

Original languageEnglish (US)
Pages (from-to)11353-11363
Number of pages11
JournalBiochemistry
Volume40
Issue number38
DOIs
StatePublished - Sep 25 2001

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Dihydrolipoamide Dehydrogenase
Mycobacterium tuberculosis
NAD
Oxidants
Genes
Deuterium
Kinetics
Isotopes
Flavin-Adenine Dinucleotide
Protons
Complement C4
Deprotonation
Gene encoding
Niacinamide
Escherichia coli Proteins
Protonation
Reducing Agents
Enzymes
Lip
Sulfhydryl Compounds

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mycobacterium tuberculosis lipoamide dehydrogenase is encoded by Rv0462 and not by the lpdA or lpdB genes. / Argyrou, A.; Blanchard, John S.

In: Biochemistry, Vol. 40, No. 38, 25.09.2001, p. 11353-11363.

Research output: Contribution to journalArticle

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abstract = "The gene encoding dihydrolipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity. The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer. The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD+ in the absence of D,L-lipoamide, demonstrated that the enzyme uses a ping-pong kinetic mechanism. Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C4-proS position of the nicotinamide ring are small [D(V/K)NADH = 1.12 ± 0.15, DVapp = 1.05 ± 0.07] when D,L-lipoamide is the oxidant but large and equivalent [D(V/K)NADH = DV = 2.95 ± 0.03] when 5-hydroxy-1,4-naphthoquinone is the oxidant. Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with D(V/K)APADH = 0.73 ± 0.03, D(V/K)Lip(S)2 = 0.77 ± 0.03, and DVapp = 0.77 ± 0.01. Solvent deuterium kinetic isotope effects with 4,4-dithiopyridine (DTP), the 4-thiopyridone product of which requires no protonation, are also inverse with D(V/K)APADH = 0.75 ± 0.06, D(V/K)DTP = 0.71 ± 0.02, and DVapp = 0.56 ± 0.15. All proton inventories were linear, indicating that a single proton is being transferred in the solvent isotopically sensitive step. Taken together, these results suggest that (1) the reductive half-reaction (hydride transfer from NADH to FAD) is rate limiting when a quinone is the oxidant, and (2) deprotonation of enzymic thiols, most likely Cys46 and Cys41, limits the reductive and oxidative half-reactions, respectively, when D,L-lipoamide is the oxidant.",
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N2 - The gene encoding dihydrolipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity. The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer. The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD+ in the absence of D,L-lipoamide, demonstrated that the enzyme uses a ping-pong kinetic mechanism. Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C4-proS position of the nicotinamide ring are small [D(V/K)NADH = 1.12 ± 0.15, DVapp = 1.05 ± 0.07] when D,L-lipoamide is the oxidant but large and equivalent [D(V/K)NADH = DV = 2.95 ± 0.03] when 5-hydroxy-1,4-naphthoquinone is the oxidant. Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with D(V/K)APADH = 0.73 ± 0.03, D(V/K)Lip(S)2 = 0.77 ± 0.03, and DVapp = 0.77 ± 0.01. Solvent deuterium kinetic isotope effects with 4,4-dithiopyridine (DTP), the 4-thiopyridone product of which requires no protonation, are also inverse with D(V/K)APADH = 0.75 ± 0.06, D(V/K)DTP = 0.71 ± 0.02, and DVapp = 0.56 ± 0.15. All proton inventories were linear, indicating that a single proton is being transferred in the solvent isotopically sensitive step. Taken together, these results suggest that (1) the reductive half-reaction (hydride transfer from NADH to FAD) is rate limiting when a quinone is the oxidant, and (2) deprotonation of enzymic thiols, most likely Cys46 and Cys41, limits the reductive and oxidative half-reactions, respectively, when D,L-lipoamide is the oxidant.

AB - The gene encoding dihydrolipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity. The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer. The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD+ in the absence of D,L-lipoamide, demonstrated that the enzyme uses a ping-pong kinetic mechanism. Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C4-proS position of the nicotinamide ring are small [D(V/K)NADH = 1.12 ± 0.15, DVapp = 1.05 ± 0.07] when D,L-lipoamide is the oxidant but large and equivalent [D(V/K)NADH = DV = 2.95 ± 0.03] when 5-hydroxy-1,4-naphthoquinone is the oxidant. Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with D(V/K)APADH = 0.73 ± 0.03, D(V/K)Lip(S)2 = 0.77 ± 0.03, and DVapp = 0.77 ± 0.01. Solvent deuterium kinetic isotope effects with 4,4-dithiopyridine (DTP), the 4-thiopyridone product of which requires no protonation, are also inverse with D(V/K)APADH = 0.75 ± 0.06, D(V/K)DTP = 0.71 ± 0.02, and DVapp = 0.56 ± 0.15. All proton inventories were linear, indicating that a single proton is being transferred in the solvent isotopically sensitive step. Taken together, these results suggest that (1) the reductive half-reaction (hydride transfer from NADH to FAD) is rate limiting when a quinone is the oxidant, and (2) deprotonation of enzymic thiols, most likely Cys46 and Cys41, limits the reductive and oxidative half-reactions, respectively, when D,L-lipoamide is the oxidant.

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