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 63000 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 Escherichia 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.
Trott
S
S
Institut für Mikrobiologie, Universität Stuttgart, 70569 Stuttgart, Germany.
Bauer
R
R
Knackmuss
H J
HJ
Stolz
A
A
D000081:Acetamides / Q000737:chemistry* / Q000378:metabolism*; D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism
D000081:Acetamides; D000581:Amidohydrolases; C019665:amidase