Purification and characterization of enzyme responsible for N-myristoylation of octapeptide in aqueous solution without ATP and coenzyme a from Pseudomonas aeruginosa.
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.
Jyomoto
Takahiro
T
Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, Sugimoto, Osaka, Japan.
Azuma
Masayuki
M
Islam
Nazneen Naher
NN
Fujinami
Ryo
R
Takao
Toshifumi
T
Ogawa
Takayuki
T
Tabuchi
Masaki
M
Igarashi
Koichi
K
Kato
Jyoji
J
Ooshima
Hiroshi
H
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*
D001426:Bacterial Proteins; D009842:Oligopeptides; D019814:Myristic Acid; D000255:Adenosine Triphosphate; D019883:Alkyl and Aryl Transferases; D003065:Coenzyme A