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Abstract
Brevundimonas diminuta TPU 5720 produces an amidase acting l-stereoselectively on phenylalaninamide. The enzyme (LaaA^sub Bd^) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The final preparation gave a single band on SDS-PAGE with a molecular weight of [asymptotically =]53,000. The native molecular weight of the enzyme was about 288,000 based on gel filtration chromatography, suggesting that the enzyme is active as a homohexamer. It had maximal activity at 50°C and pH 7.5. LaaA^sub Bd^ lost its activity almost completely on dialysis against potassium phosphate buffer (pH 7.0), and the amidase activity was largely restored by the addition of Co^sup 2+^ ions. The enzyme was, however, inactivated in the presence of ethylenediaminetetraacetic acid even in the presence of Co^sup 2+^, suggesting that LaaA^sub Bd^ is a Co^sup 2+^-dependent enzyme. LaaA^sub Bd^ had hydrolyzing activity toward a broad range of l-amino acid amides including l-phenylalaninamide, l-glutaminamide, l-leucinamide, l-methioninamide, l-argininamide, and l-2-aminobutyric acid amide. Using information on the N-terminal amino acid sequence of the enzyme, the gene encoding LaaA^sub Bd^ was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 4,446 bp of the cloned DNA revealed the presence of seven open-reading frames (ORFs), one of which (laaA ^sub Bd^) encodes the amidase. LaaA^sub Bd^ is composed of 491 amino acid residues (calculated molecular weight 51,127), and the deduced amino acid sequence exhibits significant similarity to that of ORFs encoding hypothetical cytosol aminopeptidases found in the genomes of Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Mesorhizobium loti, and Agrobacterium tumefaciens, and leucine aminopeptidases, PepA, from Rickettsia prowazekii, Pseudomonas putida ATCC 12633, and Escherichia coli K-12. The laaA ^sub Bd^ gene modified in the nucleotide sequence upstream from its start codon was overexpressed in an E. coli transformant. The activity of the recombinant LaaA^sub Bd^ in cell-free extracts of the E. coli transformant was 25.9 units mg^sup -1^ with l-phenylalaninamide as substrate, which was 50 times higher than that of B. diminuta TPU 5720.[PUBLICATION ABSTRACT]