Genetic and biochemical characterization of an enantioselective amidase from Agrobacterium tumefaciens strain d3

An enantioselective amidase was purified to homogeneity from Agrobacterium tumefaciens d3. The enzyme has a molecular mass of about 490000 Da and is composed of identical subunits with a molecular mass of about: 63 000 Da. The purified enzyme converted racemic 2-phenylpropionamide to the corresponding S-acid with an enantiomeric excess (ee) value > 95% at almost 50% conversion of the racemic amide. The purified enzyme was digested with trypsin and the amino acid sequences of the N terminus and different tryptic peptides determined. These amino acid sequences were used to clone the encoding gene. Finally, a 9330 bp DNA fragment was sequenced and the amidase gene identified. The deduced amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. No indications of a structural coupling of the amidase gene with the genes for a nitrile hydratase could be found on the cloned DNA fragment. The amidase gene was encoded by an approximately 500 kb circular plasmid in A; tumefaciens d3. The amidase was heterologously expressed in Eseherichia coli and, as well as 2-phenylpropionamide, was shown to hydrolyse alpha -chloro- and alpha -methoxyphenylacetamide and 2-methyl-3-phenylpropionamide highly enantioselectively. Some amino acids within a highly conserved region common amongst all known enantioselective amidases ('amidase signature') were changed by site-specific mutagenesis and significant changes in the relative activities with different amides observed.