Structure of Oxalacetate Acetylhydrolase, a Virulence Factor of the Chestnut Blight Fungus

Journal Article · Mon Nov 15 04:00:00 EST 2010 · J. Biol. Chem.

DOI:https://doi.org/10.1074/jbc.M110.117804· OSTI ID:1002623

Chen, Chen; Sun, Qihong; Narayanan, Buvaneswari; Nuss, Donald L; Herzberg, Osnat ^[1]

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1. UMBI

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 {approx}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 {angstrom}, 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 {center_dot} 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.

• Research Organization: Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)
• Sponsoring Organization: USDOE
• OSTI ID: 1002623
• Journal Information: J. Biol. Chem., Journal Name: J. Biol. Chem. Journal Issue: (34) ; 08, 2010 Vol. 285; ISSN JBCHA3; ISSN 0021-9258
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

36 MATERIALS SCIENCE
CHESTNUT TREES
CRYSTAL STRUCTURE
DIMERS
ENZYMES
ESCHERICHIA COLI
GENES
HYDROLYSIS
LYASES
OXALATES
OXALIC ACID
PHOSPHOENOLPYRUVATE
RESOLUTION
SUBSTRATES
VIRULENCE