A kinetic comparison of asparagine synthetase isozymes from higher plants

Four previously identified maize asparagine synthetase (AsnS) genes and a soy AsnS gene have been cloned and expressed in Escherichia coli. The enzymes have been purified and kinetically characterized. The plant AsnS proteins were expressed mainly in the inclusion bodies although small amounts of one form (ZmAsnS2) were recovered in the soluble fraction. In order to measure the kinetic properties of these enzymes a sensitive assay based on the detection of Asn by HPLC has been developed. In addition a method to refold the recombinant plant AsnS to produce active enzyme has been developed. The plant AsnS enzymes are kinetically distinct with substantial differences in Km (Gln) and Vmax values when compared to each other. These differences may be important factors for transgenic studies using AsnS genes for crop improvement.

Research highlights
► Four maize and one soybean, asparagine synthetase (AsnS) genes have been cloned and expressed in E. coli.
► A sensitive assay based on the detection of Asn by HPLC has been developed.
► A method to refold the recombinant plant AsnS to produce active enzyme has been developed.
► The plant AsnS enzymes are kinetically distinct with substantial differences in Km (Gln) and Vmax values when compared to each other. These differences may be important factors for transgenic studies using AsnS genes for crop improvement.