Structure of Oxalacetate Acetylhydrolase, a Virulence Factor of the Chestnut Blight Fungus
| Autor: | Donald L. Nuss, Chen Chen, Osnat Herzberg, Buvaneswari Narayanan, Qihong Sun |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Hydrolases
Protein Conformation Virulence Factors Molecular Sequence Data Crystallography X-Ray Ligands Biochemistry Substrate Specificity chemistry.chemical_compound Phosphoenolpyruvate mutase Protein structure Catalytic Domain Oxaloacetic acid Hydrolase Cloning Molecular Molecular Biology Plant Diseases biology Fungi Active site Cell Biology Isocitrate lyase Lyase Enzyme structure chemistry Protein Structure and Folding biology.protein Protein Multimerization |
| Zdroj: | Journal of Biological Chemistry. 285:26685-26696 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m110.117804 |
| Popis: | Oxalacetate acetylhydrolase (OAH), a member of the phosphoenolpyruvate mutase/isocitrate lyase superfamily, catalyzes the hydrolysis of oxalacetate to oxalic acid and acetate. This study shows that knock-out of the oah gene in Cryphonectria parasitica, the chestnut blight fungus, reduces the ability of the fungus to form cankers on chestnut trees, suggesting that OAH plays a key role in virulence. OAH was produced in Escherichia coli and purified, and its catalytic rates were determined. Oxalacetate is the main OAH substrate, but the enzyme also acts as a lyase of (2R,3S)-dimethyl malate with approximately 1000-fold lower efficacy. The crystal structure of OAH was determined alone, in complex with a mechanism-based inhibitor, 3,3-difluorooxalacetate (DFOA), and in complex with the reaction product, oxalate, to a resolution limit of 1.30, 1.55, and 1.65 A, respectively. OAH assembles into a dimer of dimers with each subunit exhibiting an (alpha/beta)(8) barrel fold and each pair swapping the 8th alpha-helix. An active site "gating loop" exhibits conformational disorder in the ligand-free structure. To obtain the structures of the OAH.ligand complexes, the ligand-free OAH crystals were soaked briefly with DFOA or oxalacetate. DFOA binding leads to ordering of the gating loop in a conformation that sequesters the ligand from the solvent. DFOA binds in a gem-diol form analogous to the oxalacetate intermediate/transition state. Oxalate binds in a planar conformation, but the gating loop is largely disordered. Comparison between the OAH structure and that of the closely related enzyme, 2,3-dimethylmalate lyase, suggests potential determinants of substrate preference. |
| Databáze: | OpenAIRE |
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