title: Purification and characterization of a puromycin-hydrolyzing enzyme from blasticidin S-producing Streptomyces morookaensis.
abstract: Blasticidin S-producing Streptomyces morookaensis JCM4673 produces an enzyme which inactivates puromycin (PM) by hydrolyzing an amide linkage between its aminonucleoside and O-methyl-L-tyrosine moieties [Nishimura et al. (1995) FEMS Microbiol. Lett. 132, 95-100]. In this study, we purified to homogeneity the enzyme from the cell-free extracts of S. morookaensis. The molecular weight of PM-hydrolyzing enzyme, estimated by SDS-PAGE and gel filtration, was 68 and 66 kDa, respectively, suggesting that this protein is monomeric. The PM-hydrolyzing activity was strongly inhibited by Zn2+, Fe2+, Cu2+, Hg2+, and N-bromosuccinimide, but was stimulated by DTT. The optimum pH and temperature for PM-hydrolyzing activity were 8.0 and 45 degrees C, respectively. Several L-aminoacyl-beta-naphthylamides were good substrates for the enzyme, suggesting that the PM-inactivating enzyme has an aminopeptidase activity. The N-terminal sequence of the first 14 amino acids (Val-Ser-Thr-Ala-Pro-Tyr-Gly-Ala-Trp-Gln-Ser-Pro-Ile-Asp) of the enzyme showed no significant homology with any published hydrolase sequences.
authors.affiliation: Department of Chemical and Biological Engineering, Ube National College of Technology, Tokiwadai, Yamaguchi. nisimura@ube-k.ac.jp
authors.forename: M
authors.initials: M
authors.lastname: Nishimura
authors.forename: H
authors.initials: H
authors.lastname: Matsuo
authors.forename: A
authors.initials: A
authors.lastname: Nakamura
authors.forename: M
authors.initials: M
authors.lastname: Sugiyama
mesh_terms: D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006868:Hydrolysis; D019216:Metals, Heavy / Q000494:pharmacology; D008969:Molecular Sequence Data; D009705:Nucleosides / Q000096:biosynthesis; D011691:Puromycin / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D019216:Metals, Heavy; D009705:Nucleosides; D011691:Puromycin; C004500:blasticidin S; D000626:Aminopeptidases; C022450:prolyl aminopeptidase