l-tryptophan-histidine synthesis by Pseudomonas serine peptidase, an amino acid ester hydrolase of the peptidase family S9

Abstract

Pseudomonas sp. KM1 produces an amino acid ester hydrolase (KM1AEH) that catalyzes peptide bond formation by acting on carboxylic ester bonds. The KM1AEH gene was cloned from genomic DNA and expressed in Escherichia coli. The recombinant enzyme (rKM1AEH) was purified, and gel filtration showed that it is a 68 kDa monomeric protein. rKM1AEH can synthesize the vasoactive dipeptide tryptophan-histidine from tryptophan methyl ester and histidine as acyl donor and acceptor, respectively. The enzyme showed maximum activity at pH 9.5 and 45 °C and was specifically inhibited by silver (Ag⁺). Mutation of the catalytic Ser459 residue in the active site of rKM1AEH with Ala, Cys, or Thr eliminated all catalytic activity. The enzyme is a novel ester hydrolase that belongs to the peptidase family S9 based on the phylogenetic analysis.

Keywords: Amino acid ester hydrolase; Enzymatic synthesis; Peptidase family S9; Purification; Tryptophan-histidine.