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Gene cloning, nucleotide sequencing, and purification and characterization of the D‐stereospecific amino‐acid amidase from Ochrobactrum anthropi SV3

Komeda, Hidenobu, Asano, Yasuhisa
European journal of biochemistry 2000 v.267 no.7 pp. 2028-2035
Bacillus cereus, DNA, Escherichia coli, Ochrobactrum anthropi, Streptomyces, active sites, amidase, amides, amino acid sequences, ammonium sulfate, beta-lactamase, electrophoresis, fractionation, gene overexpression, genes, hydrophobicity, liquid chromatography, molecular cloning, molecular weight, pH, proteins, sequence homology, start codon
The gene encoding the d‐stereospecific amino‐acid amidase from Ochrobactrum anthropi SV3 was cloned and sequenced. Analysis of 7.3 kb of genomic DNA revealed the presence of six ORFs, one of which (daaA) encodes the d‐amino‐acid amidase. This enzyme, DaaA, is composed of 363 amino‐acid residues (molecular mass 40 082 Da), and the deduced amino‐acid sequence exhibits homology to alkaline d‐peptidase from Bacillus cereus DF4‐B (32% identity), dd‐peptidase from Streptomyces R61 (29% identity), and other penicillin‐recognizing proteins. The DaaA protein contains the typical SXXK, YXN, and H(K)XG active‐site motifs identified in the penicillin‐binding proteins and β‐lactamases. The daaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant DaaA enzyme in cell‐free extracts of E. coli was 33.6 U·mg−1 with d‐phenylalaninamide as substrate, which is about 350‐fold higher than in extracts of O. anthropi SV3. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and three column chromatography steps. On gel‐filtration chromatography, DaaA appeared to be a monomer with a molecular mass of 40 kDa. It had maximal activity at 45 °C and pH 9.0, and was completely inactivated in the presence of phenylmethanesulfonyl fluoride or Zn2+. DaaA had hydrolyzing activity toward d‐amino‐acid amides with aromatic or hydrophobic side chains, but did not act on the substrates for the dd‐peptidase and β‐lactamase, despite their sequence similarity to DaaA. The characteristics of the recombinant DaaA are similar to those found for the native enzyme partially purified from O. anthropi SV3.