title: Purification and characterization of enzyme responsible for N-myristoylation of octapeptide in aqueous solution without ATP and coenzyme a from Pseudomonas aeruginosa.
abstract: The enzyme that catalyzes N-acyl linkage between myristic acid and the NH(2)-terminal glycine residue of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-NH(2) in aqueous solution without ATP and coenzyme A was found in Pseudomonas aeruginosa. The enzyme was purified from cell-free crude extract using DEAE-Cellulose, Sephadex G-200, CM-Sephadex C-50, and hydroxyapatite column chromatographies, and then purified approximately 1900-fold with about 1.5% recovery of enzyme activity from the crude extract. Finally, the purified enzyme showed a main band on SDS polyacrylamide gel electrophoresis after staining with Coomassie Brilliant Blue. The band corresponded to a molecular mass of approximately 60 kDa. The K(m)s of the purified enzyme for the substrate myristic acid and the octapeptide were 0.36 and 2.6 mM, respectively. When myristoyl-CoA instead of myristic acid was used as the substrate for the enzyme reaction, myristoyl octapeptide could be synthesized as observed in the case of myristic acid. The K(m) of myristoyl-CoA was 0.17 mM.
authors.affiliation: Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, Sugimoto, Osaka, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Jyomoto
authors.forename: Masayuki
authors.initials: M
authors.lastname: Azuma
authors.forename: Nazneen Naher
authors.initials: NN
authors.lastname: Islam
authors.forename: Ryo
authors.initials: R
authors.lastname: Fujinami
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Takao
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ogawa
authors.forename: Masaki
authors.initials: M
authors.lastname: Tabuchi
authors.forename: Koichi
authors.initials: K
authors.lastname: Igarashi
authors.forename: Jyoji
authors.initials: J
authors.lastname: Kato
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ooshima
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism; D019814:Myristic Acid / Q000737:chemistry* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D009842:Oligopeptides; D019814:Myristic Acid; D000255:Adenosine Triphosphate; D019883:Alkyl and Aryl Transferases; D003065:Coenzyme A