Electronic Thesis and Dissertation Repository


Master of Science




Dr. Frédéric Marsolais


Asparagine aminotransferase transforms asparagine into α-ketosuccinamate, which is further deamidated by an ω-amidase. Serine:glyoxylate aminotransferase, encoded by AGT1 in Arabidopsis, was identified as asparagine aminotransferase. In the roots of 10-day-old Arabidopsis seedlings treated with 20 mM asparagine, AGT1 transcript levels increased by 2-fold while ω-amidase transcripts were decreased by 30%. Recombinant AGT1 had a substrate preference for asparagine when compared with alanine and serine as amino group donors. An ω-amidase candidate gene, AT5G12040, was identified based on amino acid sequence identity with mammalian gene Nitrilase 2. RT-PCR of a T-DNA insertion mutant line showed that ω-amidase expression was abolished compared with wild-type. In the roots of 10-day-old seedlings grown without asparagine or with 2 mM asparagine, the amount of ω-amidase substrates, α-ketosuccinamate and α-hydroxysuccinamate, was higher in mutant than in wild-type under both conditions. Kinetic analysis indicated that recombinant ω-amidase has a preference for α-hydroxysuccinamate over α-ketosuccinamate and α-hydroxysuccinamate.