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

A. Argyrou, J. S. Blanchard

Research output: Contribution to journalArticle

36 Scopus citations

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
Publication statusPublished - Sep 25 2001

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry

Cite this