TY - JOUR
T1 - The active site of the Mycobacterium tuberculosis branched-chain amino acid biosynthesis enzyme dihydroxyacid dehydratase contains a 2Fe-2S cluster
AU - Bashiri, Ghader
AU - Grove, Tyler L.
AU - Hegde, Subray S.
AU - Lagautriere, Thomas
AU - Gerfen, Gary J.
AU - Almo, Steven C.
AU - Squire, Christopher J.
AU - Blanchard, John S.
AU - Baker, Edward N.
N1 - Funding Information:
This work was supported by the Tertiary Education Commission of New Zealand (Centre of Research Excellence funding to the Maurice Wilkins Centre); a Health Research Council of New Zealand program grant (to E. N. B.); a Hercus Health Research Fellowship (to G. B.); and National Institutes of Health Grants RO1-060899 (to J. S. B.), R21-AI133329 (to T. L. G. and S. C. A.), P01-GM118303-01 (to J. A. Gerlt and S. C. A.), U54-GM093342 (to J. A. Gerlt and S. C. A.), and U54-GM094662 (to S. C. A.). We thank the Advanced Photon Source (Argonne National Laboratory, Argonne, IL) for access to X-ray data collection facilities (Beamline 21-ID-D, LS-CAT).
Funding Information:
This work was supported by the Tertiary Education Commission of New Zea-land (Centre of Research Excellence funding to the Maurice Wilkins Cen-tre); a Health Research Council of New Zealand program grant (to E. N. B.); a Hercus Health Research Fellowship (to G. B.); and National Institutes of Health Grants RO1–060899 (to J.S.B.), R21-AI133329 (to T.L.G. and S. C. A.), P01-GM118303-01 (to J. A. Gerlt and S. C. A.), U54-GM093342 (to J. A. Gerlt and S. C. A.), and U54-GM094662 (to S. C. A.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2019 Bashiri et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2019/8/30
Y1 - 2019/8/30
N2 - 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. Here 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.
AB - 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. Here 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.
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U2 - 10.1074/jbc.RA119.009498
DO - 10.1074/jbc.RA119.009498
M3 - Article
C2 - 31315931
AN - SCOPUS:85071696134
SN - 0021-9258
VL - 294
SP - 13158
EP - 13170
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 35
ER -
