title: Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1.
abstract: A novel staphylolytic enzyme, ALE-1, acting on Staphylococcus aureus, was purified from a Staphylococcus capitis EPK1 culture supernatant. The optimal pH range for staphylolytic activity was 7 to 9. ALE-1 contains one Zn2+ atom per molecule. Analysis of peptidoglycan fragments released by ALE-1 indicated that the enzyme is a glycylglycine endopeptidase. The effects of various modulators were determined, and we found that o-phenanthroline, iodoacetic acid, diethylpyrocarbonate, and Cu2+ reduced the staphylolytic activity of ALE-1. beta-Casein, elastin, and pentaglycine were poor substrates for ALE-1. Molecular cloning data revealed that ALE-1 is composed of 362 amino acid residues and is synthesized as a precursor protein which is cleaved after Ala at position 35, thus producing a mature ALE-1 of 35.6 kDa. The primary structure of mature ALE-1 is very similar to the proenzyme form of lysostaphin. It has the modular design of an N-terminal domain of tandem repeats of a 13-amino-acid sequence fused to the active site containing C-terminal domain. Unlike lysostaphin, ALE-1 does not undergo processing of the N-terminal repeat domain in broth culture. ale-1 is encoded on the plasmid. Protein homology search suggested that ALE-1 and lysostaphin are members of the novel Zn2+ protease family with a homologous 38-amino-acid-long motif, Tyr-X-His-X(11)-Val-X(12/20)-Gly-X(5-6)-His.
authors.affiliation: Department of Microbiology, Hiroshima University School of Dentistry, Japan. sugai@ipc.hiroshima-u.ac.jp
authors.forename: M
authors.initials: M
authors.lastname: Sugai
authors.forename: T
authors.initials: T
authors.lastname: Fujiwara
authors.forename: T
authors.initials: T
authors.lastname: Akiyama
authors.forename: M
authors.initials: M
authors.lastname: Ohara
authors.forename: H
authors.initials: H
authors.lastname: Komatsuzawa
authors.forename: S
authors.initials: S
authors.lastname: Inoue
authors.forename: H
authors.initials: H
authors.lastname: Suginaka
mesh_terms: D000595:Amino Acid Sequence; D001433:Bacteriolysis; D001482:Base Composition; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008248:Lysostaphin / Q000737:chemistry / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000032:analysis
chemical_list: D004792:Enzyme Precursors; D008666:Metalloendopeptidases; C104239:glycylglycyl endopeptidase ALE-1; D008248:Lysostaphin; D015032:Zinc