Oxaloacetate hydrolase, the C-C bond lyase of oxalate secreting fungi
Oxaloacetate hydrolase, the C-C bond lyase of oxalate secreting fungi
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DOI：
10.1074/jbc.m608961200
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发表时间：
2007-03-30
影响因子：
4.8
通讯作者：
Dunaway-Mariano, Debra
中科院分区：
文献类型：
--
作者：
Han, Ying;Joosten, Henk-Jan;Dunaway-Mariano, Debra
已知真菌分泌的草酸盐与真菌发病机制有关。此外，草酸盐毒性是真菌在食品和药品工业中商业应用的一个问题。尽管草酸盐是通过几种不同的生化途径产生的，但草酰乙酸乙酰水解酶 (OAH) 催化的草酰乙酸水解似乎是一个特别重要的途径。下面，我们报道了灰葡萄孢 oahA 基因的克隆，并证明该基因的破坏会导致草酸盐形成的丧失。此外，通过互补，我们发现完整的灰霉病菌 oahA 基因可以恢复缺乏功能性 oahA 基因的黑曲霉突变株中的草酸盐产生。这些观察结果清楚地表明，黑曲霉和灰霉病菌中草酸盐的产生完全依赖于 OAH 催化的草酰乙酸的水解裂解。此外，B. cinera oahA 基因在大肠杆菌中过表达，纯化的 OAH 用于确定催化效率、底物特异性和金属离子活化。这些结果与基于机制的紧密结合 OAH 抑制剂 3,3-二氟草酰乙酸 (K-i = 68 nm) 的发现一起报告。最后，我们提出细胞摄取这种抑制剂可以减少草酸盐的产生。
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.