Molecular cloning of the arylsulfate sulfotransferase gene and characterization of its product from Enterobacter amnigenus AR-37

The gene encoding the Enterobacter amnigenus AR-37 arylsulfate sulfotransfe rase (ASST) was cloned, sequenced, and expressed in Escherichia coli NM522. Sequencing led to the identification of three contiguous open reading fram es (ORFs) on the same strand. Based on amino acid sequence homology, ORF1, ORF2, and ORF3 are designated astA, dsbA, and dsbB, respectively. A multipl e sequence alignment revealed conserved regions in ASST. An N-terminal amin o acid sequence analysis of the purified ASST from E. coli NM522 (pEAST72) showed that it is subject to N-terminal processing. The specific activity o f purified ASST is 436.5 U/mg of protein. The enzyme is a monomeric protein with a molecular mass of 64 kDa. Using phenol as an acceptor substrate, 4- methylumbelliferyl sulfate is the best donor substrate, followed by beta-na phthyl sulfate, p-nitrophenyl sulfate (PNS), and alpha-naphthyl sulfate. Fo r PNS, alpha-naphthol is the best acceptor substrate, followed by phenol, r esorcinol, p-acetaminophen, tyramine, and tyrosine. The enzyme has a differ ent acceptor specificity than the enzyme purified from Eubacterium A-44. It is similar to Klebsiella K-36 and Haemophilus K-12. The apparent K-m value s for PNS using phenol as an acceptor and for phenol using PNS as a donor a re 0.163 and 0.314 mM, respectively. The pr and optimum pH are 6.1 and 9.0, respectively. (C) 1999 Academic Press.