Arylamine N-acetyltransferase responsible for acetylation of 2-aminophenols in Streptomyces griseus

Abstract

An arylamine N-acetyltransferase (NAT) responsible for the N acetylation of exogenous 3-amino-4-hydroxybenzoic acid in Streptomyces griseus was identified and characterized. This enzyme was distinct from other eukaryotic and bacterial NATs in that it acetylated various 2-aminophenol derivatives more effectively than it acetylated 5-aminosalicylic acid, and thus it may be involved in the metabolism of xenobiotic compounds.

FIG. 1.

Disruption of the chromosomal natA gene and N acetylation of exogenous 3,4-AHBA by SgNAT. (A) Gene organization in the neighborhood of natA on the S. griseus chromosome and schematic diagram of construction of a ΔnatA mutant. Most of the natA coding sequence was replaced by the kanamycin resistance gene (aphII) as a result of a double crossover. Probes used for Southern hybridization are also indicated. HP, hypothetical protein. (B) Southern hybridization to check for correct disruption. Digoxigenin-labeled probes 1 and 2 were hybridized with BglII-digested chromosomal DNA from the wild-type strain and ΔnatA mutant. Hybridized probes were detected using an anti-digoxigenin Fab fragment conjugated to alkaline phosphatase with 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate. (C) N acetylation of exogenous 3,4-AHBA by the wild-type strain and ΔnatA mutant. S. griseus cells were incubated at 30°C for 2 days in YPD medium supplemented with 1 mM 3,4-AHBA, and 10 μl of the culture broth was analyzed by HPLC.

FIG. 2.

Purification of His-tagged SgNAT from E. coli. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of molecular size markers (lane 1) and SgNAT purified from E. coli BL21(DE3)/pLysS harboring pET-natA (lane 2). Proteins were stained with Coomassie brilliant blue R-250. (B) Reaction catalyzed by SgNAT.