Gene cloning and characterization of arylamine N-acetyltransferase from Bacillus cereus strain 10-L-2.
Bacillus cereus strain 10-L-2 synthesizes two arylamine N-acetyltransferases (Nat-a and Nat-b) with broad substrate specificities toward aniline and its derivatives. In southern blot analysis using probes encoding the NH2-terminus of Nat-b and a conserved region of N-acetyltransferases, digested total DNA of strain 10-L-2 showed one positive band. We cloned and sequenced the gene encoding Nat-b. The NH2-terminal amino acid sequence predicted from the open reading frame (768 base pairs) corresponded to that of purified Nat-b. The cloned Nat-b gene was expressed in Escherichia coli. The expressed enzyme (BcNAT) from the recombinant strain was partially purified and characterized. Nat-b from strain 10-L-2 and BcNAT from the recombinant strain were slightly different from each others in substrate specificity and thermo-stability. We examined the biotransformations of 2-aminophenols and phenylenediamines by the whole cells of the recombinant strain. The cells converted these compounds into their corresponding acetanilides. Only one amino group of phenylenediamines was acetylated. The cells utilized 4-nitroacetanilide as an acetyl donor instead of acetyl-CoA. 4-Aminoacetanilide was produced and 4-nitroaniline was released almost stoichiometrically.
Takenaka
Shinji
S
Department of Applied Biological Chemistry, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan.
Cheng
Minyi
M
Mulyono
Koshiya
Atsushi
A
Murakami
Shuichiro
S
Aoki
Kenji
K
D000083:Acetanilides / Q000737:chemistry; D000595:Amino Acid Sequence; D001191:Arylamine N-Acetyltransferase / Q000737:chemistry* / Q000235:genetics*; D001409:Bacillus cereus / Q000201:enzymology*; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D019800:Phenol / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000083:Acetanilides; D019800:Phenol; D001191:Arylamine N-Acetyltransferase; C033553:4-nitroacetanilide