title: Crystal structure of Mycobacterium tuberculosis ketol-acid reductoisomerase at 1.0 Å resolution - a potential target for anti-tuberculosis drug discovery.
abstract: The biosynthetic pathway for the branched-chain amino acids is present in plants, fungi and bacteria, but not in animals, making it an attractive target for herbicidal and antimicrobial drug discovery. Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) is the second enzyme in this pathway, converting in a Mg(2+) - and NADPH-dependent reaction either 2-acetolactate or 2-aceto-2-hydroxybutyrate to their corresponding 2,3-dihydroxy-3-alkylbutyrate products. Here, we have determined the crystal structure of Mycobacterium tuberculosis (Mt) KARI, a class I KARI, with two magnesium ions bound in the active site. X-ray data were obtained to 1.0 Å resolution and the final model has an Rfree of 0.163. The structure shows that the active site is solvent-accessible with the two metal ions separated by 4.7 Å. A comparison of this structure with that of Mg(2+) -free Pseudomonas aeruginosa KARI suggests that upon magnesium binding no movement of the N domain relative to the C domain occurs. However, upon formation of the Michaelis complex, as illustrated in the structure of Slackia exigua KARI in complex with NADH.Mg(2+) . N-hydroxy-N-isopropyloxamate (IpOHA, a transition state analog), domain movements and reduction of the metal-metal distance to 3.5 Å are observed. This inherent flexibility therefore appears to be critical for initiation of the KARI-catalyzed reaction. This study provides new insights into the complex structural rearrangements required for activity of KARIs, particularly those belonging to class I, and provides the framework for the rational design of Mt KARI inhibitors that can be tested as novel antituberculosis agents.
  
  
  METHODS
  Coordinates and structure factors for the Mt KARI.Mg(2+) complex are available in the Protein Data Bank under accession number 4YPO.
authors.affiliation: School of Chemistry and Molecular Biosciences and Australian Infectious Disease Research Centre, University of Queensland, Brisbane, Australia.
authors.forename: You
authors.initials: Y
authors.lastname: Lv
authors.affiliation: School of Chemistry and Molecular Biosciences and Australian Infectious Disease Research Centre, University of Queensland, Brisbane, Australia.
authors.forename: Ajit
authors.initials: A
authors.lastname: Kandale
authors.affiliation: School of Chemistry and Molecular Biosciences and Australian Infectious Disease Research Centre, University of Queensland, Brisbane, Australia.
authors.forename: Shun Jie
authors.initials: SJ
authors.lastname: Wun
authors.affiliation: School of Chemistry and Molecular Biosciences and Australian Infectious Disease Research Centre, University of Queensland, Brisbane, Australia.
authors.forename: Ross P
authors.initials: RP
authors.lastname: McGeary
authors.affiliation: School of Chemistry and Molecular Biosciences and Australian Infectious Disease Research Centre, University of Queensland, Brisbane, Australia.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Williams
authors.affiliation: School of Chemistry and Molecular Biosciences and Australian Infectious Disease Research Centre, University of Queensland, Brisbane, Australia.|Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia.
authors.forename: Bostjan
authors.initials: B
authors.lastname: Kobe
authors.affiliation: Straubing Center of Science, Technische Universität München, Straubing, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Sieber
authors.affiliation: School of Chemistry and Molecular Biosciences and Australian Infectious Disease Research Centre, University of Queensland, Brisbane, Australia.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Schembri
authors.affiliation: School of Chemistry and Molecular Biosciences and Australian Infectious Disease Research Centre, University of Queensland, Brisbane, Australia.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Schenk
authors.affiliation: School of Chemistry and Molecular Biosciences and Australian Infectious Disease Research Centre, University of Queensland, Brisbane, Australia.
authors.forename: Luke W
authors.initials: LW
authors.lastname: Guddat
mesh_terms: D000995:Antitubercular Agents / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D004791:Enzyme Inhibitors / Q000627:therapeutic use; D006801:Humans; D050540:Ketol-Acid Reductoisomerase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D058990:Molecular Targeted Therapy; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000502:physiology; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012996:Solutions; D014376:Tuberculosis / Q000382:microbiology / Q000517:prevention & control
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D012996:Solutions; D050540:Ketol-Acid Reductoisomerase; D008274:Magnesium
keywords: X-ray crystal structure; branched-chain amino acids biosynthesis; ketol-acid reductoisomerase; metal assisted catalysis; tuberculosis