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 c omposed of identical subunits with a molecular mass of about: 63 000 Da. Th e purified enzyme converted racemic 2-phenylpropionamide to the correspondi ng S-acid with an enantiomeric excess (ee) value > 95% at almost 50% conver sion of the racemic amide. The purified enzyme was digested with trypsin an d the amino acid sequences of the N terminus and different tryptic peptides determined. These amino acid sequences were used to clone the encoding gen e. Finally, a 9330 bp DNA fragment was sequenced and the amidase gene ident ified. The deduced amino acid sequence showed homology to other enantiosele ctive amidases from different bacterial genera. No indications of a structu ral coupling of the amidase gene with the genes for a nitrile hydratase cou ld 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-phenylpropi onamide, was shown to hydrolyse alpha -chloro- and alpha -methoxyphenylacet amide and 2-methyl-3-phenylpropionamide highly enantioselectively. Some ami no acids within a highly conserved region common amongst all known enantios elective amidases ('amidase signature') were changed by site-specific mutag enesis and significant changes in the relative activities with different am ides observed.