Molecular cloning, expression, and functional analysis of a predicted sulfotransferase STF9 from Mycobacterium avium

• Published in: Molecular and cellular biochemistry 2011-04, Vol.350 (1-2), p.155-162
• Main Authors: Hossain, Md. Murad, Moriizumi, Yuuji, Tanaka, Shotaro, Kimura, Makoto, Kakuta, Yoshimitsu
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Analysis; Bacterial proteins; Biochemistry; Biomedical and Life Sciences; Biosynthesis; Cardiology; Catalysis; Catalytic Domain - genetics; Cloning; Cloning, Molecular; Computational Biology; E coli; Enzymes; Escherichia coli; Esters; Forecasting; Gene expression; Gene Expression Profiling; Life Sciences; Medical Biochemistry; Molecular biology; Molecular Sequence Data; Mutagenesis, Site-Directed; Mycobacterium avium; Mycobacterium avium - enzymology; Mycobacterium avium - genetics; Mycobacterium avium - metabolism; Oncology; Sulfates; Sulfotransferases - chemistry; Sulfotransferases - genetics; Sulfotransferases - isolation & purification; Sulfotransferases - physiology
• ISSN: 0300-8177; 1573-4919
• DOI: 10.1007/s11010-010-0693-1

Sulfotransferases catalyze the transfer of sulfate group from para-nitrophenyl sulfate (pNPS) or 3′-phosphoadenosine-5′-phosphosulfate (PAPS) onto acceptor molecules in the biosynthesis of sulfate esters. Human pathogenic mycobacteria are known to produce numerous sulfated molecules on their cell surface which have been implicated as important mediators in host-pathogen interactions. The open reading frame stf9 , a predicted homologue of sulfotransferase in the Mycobacterium avium genomic data, was cloned and over expressed in Escherichia coli . The recombinant STF9 conserved the characteristic PAPS binding motif of sulfotransferase and was purified as a 44 kDa soluble protein which exhibited transfer of sulfate group from pNPS ( K m 1.34 mM, V max 7.56 nmol/min/mg) onto 3′-phosphoadenosine-5′-phosphate ( K m 0.24 mM, V max 10.36 nmol/min/mg). The recombinant STF9 protein was also capable of transferring sulfate group from PAPS onto certain acceptor substrates in E. coli , and showed binding affinity to the PAP-agarose resin, supporting the sulfotransferase activity of the recombinant STF9 protein. This is the first report of molecular evidence for sulfotransferase activity of a protein from M. avium . Mutation of Arg96 to Ala and Glu170 to Ala abolishes sulfotransferase activity, indicating the importance of Arg96 and Glu170 in STF9 activity catalysis.