title: Identification of a plant aminopeptidase with preference for aromatic amino acid residues as a novel member of the prolyl oligopeptidase family of serine proteases.
abstract: Genome analysis has indicated that plants, like animals, possess a variety of protease genes. However, bulk of putative proteases has not been characterized at the enzyme level. In this article, a novel enzyme that hydrolyses phenylalanyl-4-methylcoumaryl 7-amide (phenylalanyl-MCA) was purified from cotyledons of daikon radish by ammonium sulphate fractionation and successive chromatography with DEAE-cellulose, phenyl-Sepharose, Sephacryl S-200 and Mini-Q. The molecular mass of the enzyme was estimated to be 78 kDa by SDS-PAGE under reducing conditions and 74 kDa by gel filtration, indicating that the enzyme is a monomer. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that the enzyme is an orthologue of Arabidopsis unidentified protein, acylpeptide hydrolase-like protein (AHLP; UniProt ID: Q9FG66) belonging to the prolyl oligopeptidase (POP) family of a serine-type peptidase predicted from genetic information. Good substrates identified for the enzyme include phenylalanyl-MCA, tyrosyl-MCA and enkephalin. Neither acylamino acid-releasing activity nor endopeptidase activity was detected. The enzyme cleaved enkephalin (YGGFM, YGGFL), whereas, BAM-12 P (YGGFMRRVGRPE) and dynorphin A (YGGFLRRIRPKLK) were not digested. These results suggested that the enzyme possesses strict size selectivity of substrate. We propose the name 'tyrosyl aminopeptidase' for the uncharacterized protein AHLP.
authors.affiliation: Department of Biological Science and Technology, The University of Tokushima Graduate School, Minamijosanjima, Tokushima, Japan. tsuji@bio.tokushima-u.ac.jp
authors.forename: Akihiko
authors.initials: A
authors.lastname: Tsuji
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Fujisawa
authors.forename: Takeru
authors.initials: T
authors.lastname: Mino
authors.forename: Keizo
authors.initials: K
authors.lastname: Yuasa
mesh_terms: D024322:Amino Acids, Aromatic / Q000378:metabolism*; D000626:Aminopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018548:Cotyledon / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000085822:Prolyl Oligopeptidases; D031224:Raphanus / Q000201:enzymology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D024322:Amino Acids, Aromatic; D010940:Plant Proteins; D057057:Serine Proteases; D000626:Aminopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases