Structural, kinetic and chemical mechanism of isocitrate dehydrogenase-1 from mycobacterium tuberculosis

Christine E. Quartararo, Saugata Hazra, Timin Hadi, John S. Blanchard

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Mycobacterium tuberculosis (Mtb) is the leading cause of death due to a bacterial infection. The success of the Mtb pathogen has largely been attributed to the nonreplicating, persistence phase of the life cycle, for which the glyoxylate shunt is required. In Escherichia coli, flux through the shunt is controlled by regulation of isocitrate dehydrogenase (ICDH). In Mtb, the mechanism of regulation is unknown, and currently, there is no mechanistic or structural information about ICDH. We optimized expression and purification to a yield sufficiently high to perform the first detailed kinetic and structural studies of Mtb ICDH-1. A large solvent kinetic isotope effect [D2OV = 3.0 ± 0.2, and D2O(V/Kisocitrate) = 1.5 ± 0.3] and a smaller primary kinetic isotope effect [DV = 1.3 ± 0.1, and D(V/K[2R-2H]isocitrate) = 1.5 ± 0.2] allowed us to perform the first multiple kinetic isotope effect studies on any ICDH and suggest a chemical mechanism. In this mechanism, protonation of the enolate to form product α-ketoglutarate is the rate-limiting step. We report the first structure of Mtb ICDH-1 to 2.18 Å by X-ray crystallography with NADPH and Mn2+ bound. It is a homodimer in which each subunit has a Rossmann fold, and a common top domain of interlocking β sheets. Mtb ICDH-1 is most structurally similar to the R132H mutant human ICDH found in glioblastomas. Similar to human R132H ICDH, Mtb ICDH-1 also catalyzes the formation of α-hydroxyglutarate. Our data suggest that regulation of Mtb ICDH-1 is novel.

Original languageEnglish (US)
Pages (from-to)1765-1775
Number of pages11
JournalBiochemistry
Volume52
Issue number10
DOIs
StatePublished - Mar 12 2013

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Isocitrate Dehydrogenase
Mycobacterium tuberculosis
Kinetics
Isotopes
Protonation
X ray crystallography
X Ray Crystallography
Pathogens
Glioblastoma
Life Cycle Stages
NADP
Bacterial Infections
Escherichia coli
Purification
Life cycle
Cause of Death
Fluxes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural, kinetic and chemical mechanism of isocitrate dehydrogenase-1 from mycobacterium tuberculosis. / Quartararo, Christine E.; Hazra, Saugata; Hadi, Timin; Blanchard, John S.

In: Biochemistry, Vol. 52, No. 10, 12.03.2013, p. 1765-1775.

Research output: Contribution to journalArticle

Quartararo, Christine E. ; Hazra, Saugata ; Hadi, Timin ; Blanchard, John S. / Structural, kinetic and chemical mechanism of isocitrate dehydrogenase-1 from mycobacterium tuberculosis. In: Biochemistry. 2013 ; Vol. 52, No. 10. pp. 1765-1775.
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