The active site of the Mycobacterium tuberculosis branched-chain amino acid biosynthesis enzyme dihydroxyacid dehydratase contains a 2Fe–2S cluster
Iron–sulfur clusters are protein cofactors with an ancient evolutionary origin. These clusters are best known for their roles in redox proteins such as ferredoxins, but some iron–sulfur clusters have nonredox roles in the active sites of enzymes. Such clusters are often prone to oxidative degradation, making the enzymes difficult to characterize. In this study we report a structural and functional characterization of dihydroxyacid dehydratase (DHAD) from Mycobacterium tuberculosis (Mtb), an essential enzyme in the biosynthesis of branched-chain amino acids. Conducting this analysis under fully anaerobic conditions, we solved the DHAD crystal structure, at 1.88 Å resolution, revealing a 2Fe–2S cluster in which one iron ligand is a potentially exchangeable water molecule or hydroxide. UV and EPR spectroscopy both suggested that the substrate binds directly to the cluster or very close to it. Kinetic analysis implicated two ionizable groups in the catalytic mechanism, which we postulate to be Ser-491 and the iron-bound water/hydroxide. Site-directed mutagenesis showed that Ser-491 is essential for activity, and substrate docking indicated that this residue is perfectly placed for proton abstraction. We found that a bound Mg2+ ion 6.5 Å from the 2Fe–2S cluster plays a key role in substrate binding. We also identified a putative entry channel that enables access to the cluster and show that Mtb-DHAD is inhibited by a recently discovered herbicide, aspterric acid, that, given the essentiality of DHAD for Mtb survival, is a potential lead compound for the design of novel anti-TB drugs.
- Research Organization:
- Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Organization:
- USDOE; National Institutes of Health (NIH); Tertiary Education Commission of New Zealand; Health Research Council of New Zealand
- Grant/Contract Number:
- RO1-06089; R21-AI133329; P01-GM118303-01; U54-GM093342; U54-GM094662
- OSTI ID:
- 1569878
- Journal Information:
- Journal of Biological Chemistry, Vol. 294, Issue 35; ISSN 0021-9258
- Publisher:
- American Society for Biochemistry and Molecular BiologyCopyright Statement
- Country of Publication:
- United States
- Language:
- ENGLISH
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