Oxaloacetate hydrolase, the C-C bond lyase of oxalate secreting fungi.
Oxalate secretion by fungi is known to be associated with fungal pathogenesis. In addition, oxalate toxicity is a concern for the commercial application of fungi in the food and drug industries. Although oxalate is generated through several different biochemical pathways, oxaloacetate acetylhydrolase (OAH)-catalyzed hydrolytic cleavage of oxaloacetate appears to be an especially important route. Below, we report the cloning of the Botrytis cinerea oahA gene and the demonstration that the disruption of this gene results in the loss of oxalate formation. In addition, through complementation we have shown that the intact B. cinerea oahA gene restores oxalate production in an Aspergillus niger mutant strain, lacking a functional oahA gene. These observations clearly indicate that oxalate production in A. niger and B. cinerea is solely dependent on the hydrolytic cleavage of oxaloacetate catalyzed by OAH. In addition, the B. cinera oahA gene was overexpressed in Escherichia coli and the purified OAH was used to define catalytic efficiency, substrate specificity, and metal ion activation. These results are reported along with the discovery of the mechanism-based, tight binding OAH inhibitor 3,3-difluorooxaloacetate (K(i) = 68 nM). Finally, we propose that cellular uptake of this inhibitor could reduce oxalate production.
Han
Ying
Y
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131.
Joosten
Henk-Jan
HJ
Laboratory of Microbiology Section Fungal Genomics, Wageningen University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.
Niu
Weiling
W
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131.
Zhao
Zhiming
Z
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131.
Mariano
Patrick S
PS
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131.
McCalman
Melisa
M
Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands.
van Kan
Jan
J
Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands.
Schaap
Peter J
PJ
Laboratory of Microbiology Section Fungal Genomics, Wageningen University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands. Electronic address: peter.schaap@wur.nl.
Dunaway-Mariano
Debra
D
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131. Electronic address: dd39@unm.edu.
D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics; D020171:Botrytis / Q000201:enzymology* / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D010070:Oxalates / Q000378:metabolism*
D005656:Fungal Proteins; D010070:Oxalates; D006867:Hydrolases; C054172:oxaloacetase