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J. Biol. Chem., Vol. 283, Issue 25, 17531-17541, June 20, 2008

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Crystal Structures of F₄₂₀-dependent Glucose-6-phosphate Dehydrogenase FGD1 Involved in the Activation of the Anti-tuberculosis Drug Candidate PA-824 Reveal the Basis of Coenzyme and Substrate Binding^*

From the School of Biological Sciences and Centre of Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland, New Zealand

The modified flavin coenzyme F₄₂₀ is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F₄₂₀-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F₄₂₀ for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F₄₂₀ and citrate at resolutions of 1.90 and 1.95Å_, respectively. The structure reveals a highly specific F₄₂₀ binding mode, which is shared with several other F₄₂₀-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F₄₂₀ and is shown to be a competitive inhibitor (IC₅₀ 43 µM). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F₄₂₀ and helps promote a butterfly bend conformation that facilitates F₄₂₀ reduction and catalysis.

Received for publication, March 7, 2008 , and in revised form, April 8, 2008.

The atomic coordinates and structure factors (codes 3C8N and 3B4Y) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the New Economy Research Fund of New Zealand, the Health Research Council of New Zealand, and Centres of Research Excellence funding to the Maurice Wilkins Centre of Molecular Biodiscovery. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a Doctoral Scholarship from the Iranian Ministry of Science, Research, and Technology.

² To whom correspondence should be addressed: School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Tel.: 64-9-373-7599; Fax: 64-9-373-7619; E-mail: ted.baker{at}auckland.ac.nz.

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