Characterization of Enzymes in the L-Asparagine Metabolism Pathway in Legumes
Abstract
Improving the quality of economically and nutritionally valuable legume crops, such as soybean, requires a comprehensive understanding of nitrogen metabolism. L-asparagine (Asn) is an important source of nitrogen stored and transported in higher plants, favoured in legumes due to its advantageous 2N:4C ratio. The catabolism of Asn in tissues occurs through two major pathways: deamidation and transamination. The transamination pathway employs an unidentified α-ketosuccinamate (α-KSM ) reducing dehydrogenase (α-KSDH), as well as an ω-amidase which has not been fully characterized. Additionally, α-ketosuccinamate and α-hydroxysuccinamate are potentially toxic metabolites generated in this pathway. The goal of this research is to identify α-KSDH in soybean and characterize the role of ω-amidase in the transamination pathway in Arabidopsis. Fractionation, ammonium sulfate precipitation, size exclusion chromatography, and LC-MS/MS were employed to sequentially purify and identify an α-KSDH that showed activity with α-KSM. Altogether, these techniques revealed that soybean hydroxyphenylpyruvate reductase (GmHPPR) and Arabidopsis hydroxypyruvate reductase 2 (AtHPR2) catalyze the reduction of α-KSM into α-HSM. Based on this activity, these are likely the enzymes responsible for α-KSDH function in vivo of their respective species, however in vivo studies are required to confirm this function. The broad substrate specificity of HPPR and HPR2 and its activity with 2-hydroxyacids indicate that its main role in vivo is to convert intermediary metabolites into metabolites that can be directly used for other important pathways within the plant. An ω-amidase T-DNA insertion loss of function mutant was found to be embryo lethal in Arabidopsis, cause shortened siliques in heterozygotes, and overall have a detrimental effect on reproduction. The mechanism of these effects is likely a shortage of methionine and polyamines, toxic metabolite accumulation, or a combination of the two. Like ω-amidase, HPPR is known as a “clean-up” enzyme, and it can be assumed that it also plays a role in mitigating the levels of toxic intermediary metabolites as a “repair” enzyme. This research has advanced our understanding of Asn metabolism in higher plants and may ultimately contribute to advancements in crop nitrogen use efficiency.