Oxaloacetate Hydrolase, the C–C Bond Lyase of Oxalate Secreting Fungi

Abstract

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.Numerous filamentous fungi, including the food biotechnology fungus Aspergillus niger, the opportunistic human pathogen Aspergillus fumigatus, the phytopathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum, as well as many brown-rot and white-rot basidiomycetes, are able to efficiently produce large quantities of oxalate (1, 2). It is known that oxalate secretion is associated with fungal pathogenesis (1, 3-6). In the wood-rotting fungus Fomitopsis palustris oxalate is formed as the product of glucose metabolism (7). We recently initiated investigations of the oxalate biosynthetic pathway to develop a genomic-based method for distinguishing between oxalate producing and non-producing fungi. An additional goal of this effort was to identify enzyme inhibitors that could be used to arrest oxalate formation in targeted fungi.To attenuate oxalate production in fungi, it is necessary to first identify the major pathway responsible for oxalate formation. There are three potential routes for production of oxalate in fungi: oxidation of glyoxylate (8, 9), oxidation of glycolaldehyde (10), and hydrolysis of oxaloacetate (11). The results of studies of [ 14 C]CO 2 incorporation into the metabolite pools of A. niger indicate that oxalate is derived from oxaloacetate (12). This finding parallels the results of earlier work on the purification of an enzyme "oxalacetalase" (now known as oxaloacetate acetylhydrolase or OAH) 4 that catalyzes the hydrolytic cleavage of oxaloacetate to form acetate and oxalate (11). In a subsequent study, a mutant A. niger strain, NW228 (13), was found to be deficient in both oxalate production and in the synthesis of active OAH (14). These observations suggest that oxalate is ...

Structure and Function of PA4872 from Pseudomonas aeruginosa, a Novel Class of Oxaloacetate Decarboxylase from the PEP Mutase/Isocitrate Lyase Superfamily,

“…Nor is it a substrate for oxaloacetate hydrolase catalyzed hydrolytic cleavage (31). Whereas oxaloacetate exists predominantly in the C(2) keto form (35), the 3,3-difluoroxaloacetate exists predominantly in the C(2) gem diol (hydrate) form (31). Oxaloacetate hydrolase catalyzes C(2)-C(3) bond cleavage in the oxaloacetate gem diol.…”

“…Oxaloacetate hydrolase catalyzes C(2)-C(3) bond cleavage in the oxaloacetate gem diol. The tight binding of 3,3-difluoroxaloacetate (K i = 68 nM, (31)) suggests that the enzyme active site is specialized in recruiting or generating the gem diol form of oxaloacetate. It is not clear why the fluorine atoms at C(3) impair the C(2)-C(3) cleavage step.…”

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

“…The virulence mechanism is believed to be due to acidification that triggers lignocellulose degradation, reduces viability of host tissue in favor of pathogen proliferation, and induces crystallization of calcium oxalate, which blocks vessels or bronchioles (4,6,7). Studies of the human pathogen Aspergillus fumigatus and the plant pathogen Botrytis cinerea showed that oxalic acid secretion is solely due to hydrolysis of oxalacetate catalyzed by OAH (1).…”

Organic acid production in Aspergillus niger and other filamentous fungi

“…To remove gluconate and oxalate as confounding factors, but without having to exert control over the external pH and thereby influencing iron availability, we worked with A. niger strain NW186. This strain bears two mutations, one leading to a frameshift in the gene encoding glucose oxidase (goxC17), and a nonsense mutation in the gene encoding oxaloacetate hydrolase (oahA), making this strain a gluconate and oxalate non-producer (Han et al, 2007;Ruijter et al, 1999).…”

“…Both of these strains are gluconate non-producers, and NW186 differs from its oxalate producing equivalent NW305 only by dysfunctional OahA (Han et al, 2007;Ruijter et al, 1999). This mutation deprives NW186 of the possibility to produce oxalate via the cytosolic route in which oxaloacetate is hydrolysed to oxalate and acetate ( Figure 3.1); the established route of oxalate biosynthesis on glucose as carbon source (Kubicek et al, 1988).…”