title: Identification and biochemical characterization of plant acylamino acid-releasing enzyme.
abstract: Plant acylamino acid-releasing enzyme (AARE) catalyzing the N-terminal hydrolysis of N(alpha)-acylpeptides to release N(alpha)-acylated amino acids, was biochemically characterized using recombinant and native AAREs. A cDNA encoding a deduced Arabidopsis thaliana AARE (AtAARE) was cloned and sequenced. The deduced amino acid sequence encoded a 764 amino acid protein of 83.9 kDa, which was 31.8% identical with that of rat AARE. In particular, the proposed catalytic residues (Ser, Asp, and His) of AARE, called the "catalytic triad residues, " were completely conserved. Recombinant AtAARE was expressed in Escherichia coli and confirmed to be a functional AARE. Native AAREs were prepared from A. thaliana and cucumber (Cucumis sativus, L.) plants. Both native AAREs were tetrameric proteins of 350 kDa comprising four subunits of 82 kDa, and showed typical enzymological properties of other AAREs, i.e. sensitivity to diisopropyl fluorophosphate, an optimum pH of around 7.0, and an optimum temperature of 37 degrees C. Both the native and recombinant AAREs were immunochemically homologous. Intracelluar fractionation analysis showed that the AARE was mainly present in the stroma of chloroplasts. Native AARE degraded the glycated ribulose-1,5-bisphoshate carboxylase/oxygenase protein but not the native protein. Thus, plant AARE might be involved in not only catalysis of the N-terminal hydrolysis of N(alpha)-acylpeptides but also the elimination of glycated proteins.
authors.affiliation: Laboratory of Plant Biotechnology, Faculty of Agriculture, Tottori University, Koyama, Tottori 680-8553. yamauchi@muses.tottori-u.ac.jp
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yamauchi
authors.forename: Yukinori
authors.initials: Y
authors.lastname: Ejiri
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Toyoda
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018553:Cucumis sativus / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D009842:Oligopeptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; C038220:acylaminoacyl-peptidase; D012273:Ribulose-Bisphosphate Carboxylase