Identification and characterization of omega-amidase as an enzyme metabolically linked to asparagine transamination in Arabidopsis.

In higher plants, asparagine (Asn) is a major form of organic nitrogen used for transport and storage. There are two pathways of Asn metabolism, involving asparaginase and Asn aminotransferase. The enzyme serine:glyoxylate aminotransferase encoded by AGT1 has been identified as an asparagine aminotransferase in Arabidopsis. The product of asparagine transamination, alpha-ketosuccinamate, can be hydrolyzed by the enzyme omega-amidase to form oxaloacetate and ammonia. A candidate gene was identified in Arabidopsis based on its sequence similarity with mouse omega-amidase. Recombinant omega-amidase exhibited comparable catalytic activities with alpha-hydroxysuccinamate, alpha-ketosuccinamate and alpha-ketoglutaramate, the product of glutamine transamination. A mutant with a T-DNA inserted in the first exon accumulated alpha-ketosuccinamate and alpha-hydroxysuccinamate as compared with wild-type, both under control conditions and after treatment with Asn. Treatment with Asn led to decreased transcript levels of omega-amidase in root, while transcript levels of AGT1 are increased under these conditions, suggesting that excess Asn may lead to the accumulation of alpha-ketosuccinamate and alpha-hydroxysuccinamate. Crown