Molecular characterization of a novel bacterial aryl acylamidase belonging to the amidase signature enzyme family.
In seeking aryl acylamidase (EC 3.5.1.13) acting on an amide bond in p-acetaminophenol (Tylenol), we identified a novel gene encoding 496 residues of a protein. The gene revealed a conserved amidase signature region with a canonical catalytic triad. The gene was expressed in E. coli and characterized for its biochemical properties. The optimum pH and temperature for the activity on p-acetaminophenol were 10 and 37 degrees C, respectively. The half-life of enzyme activity at 37 degrees C was 192 h and 90% of its activity remained after 3 h incubation at 40 degrees C. Divalent metals was found to inhibit the activity of enzyme. The K (m) values for various aryl acylamides such as 4-nitroacetanilide, p-acetaminophenol, phenacetin, 4-chloroacetanilide and acetanilide were 0.10, 0.32, 0.83, 1.9 and 19 mM, respectively. The reverse reaction activity (amide synthesis) was also examined using various chain lengths (C(1) approximately C(4) and C(10)) of carboxylic donors and aniline as substrates. These kinetic parameters and substrate specificity in forward and reverse reaction indicated that the aryl acylamidase in this study has a preference for aryl substrate having polar functional groups and hydrophobic carboxylic donors.
Ko
Hyeok-Jin
HJ
Division of Biotechnology, Korea University, Seoul, 136-713, Korea.
Lee
Eun Woo
EW
Bang
Won-Gi
WG
Lee
Cheol-Koo
CK
Kim
Kyoung Heon
KH
Choi
In-Geol
IG
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001709:Biotechnology; D003001:Cloning, Molecular; D004789:Enzyme Activation; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D010802:Phylogeny; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000581:Amidohydrolases; C019732:aryl acylamidase