Enhanced transaminase activity of a bifunctional l-aspartate 4-decarboxylase

l-Aspartate 4-decarboxylase (Asd) catalyzes mainly the β-decarboxylation of aspartate and also transamination with α-keto acids. To investigate residues that are critical in directing the reaction pathway, seven point mutations were designed based on the differences between Asd and amiontransferases in conservative amino acid residues. All mutant Asds were purified and characterized. The F204W mutant enhanced aminotransferase activity, and its ratio to β-decarboxylase activity was 3.8-fold. Its Km values for aspartate and α-ketoglutarate were 1.3 and 0.17 mM, respectively, representing a large increase in the binding affinity with substrates. The K347R mutation did not increase transaminase activity. The D360P mutation decreased transaminase activity and was more specific in catalyzing β-decarboxylation reaction. This is the first study that successfully increased transaminase activity in Asd via site-directed mutagenesis. The modeled protein structure reveals how the residue may involve in reaction specificity, providing insights into comprehending the molecular evolution of this bifunctional enzyme.