Mapping multi-substrate specificity of Arabidopsis aminotransferases

Nitrogen is an essential element in all organisms, and its availability and use efficiency directly impact organismal growth and performance, especially in plants. Aminotransferases are core enzymes of the nitrogen metabolic network for synthesizing various organonitrogen compounds. Although each aminotransferase can potentially catalyse hundreds of transamination reactions with different combinations of amino and keto acid substrates, the full functionality of many aminotransferases remains elusive. Here we employed high-throughput gene synthesis and enzyme assay platforms to determine the substrate specificities of 38 aminotransferases of Arabidopsis thaliana and unveiled many previously unrecognized activities among a total of 4,104 reactions tested. The integration of these biochemical data in an enzyme-constrained metabolic model of Arabidopsis and in silico simulation further revealed that the promiscuity of aminotransferases may alter nitrogen distribution profiles and contribute to the robustness of the nitrogen metabolic network. This study provides foundational knowledge for deciphering the plant nitrogen metabolic network and improving nitrogen use efficiency in crops.