Improving the pyrophosphate-inosine phosphotransferase activity of Escherichia blattae acid phosphatase by sequential site-directed mutagenesis.
Escherichia blattae acid phosphatase/phosphotransferase (EB-AP/PTase) exhibits C-5'-position selective pyrophosphate-nucleoside phosphotransferase activity in addition to its intrinsic phosphatase. Improvement of its phosphotransferase activity was investigated by sequential site-directed mutagenesis. By comparing the primary structures of higher 5'-inosinic acid (5'-IMP) productivity and lower 5'-IMP productivity acid phosphatase/phosphotransferase, candidate residues of substitution were selected. Then a total of 11 amino acid substitutions were made with sequential substitutions. As the number of substituted amino acid residues increased, the 5'-IMP productivity of the mutant enzyme increased, and the activity of the 11 mutant phosphotransferases of EB-AP/PTase reached the same level as that of Morganella morganii AP/PTase. This result shows that Leu63, Ala65, Glu66, Asn69, Ser71, Asp116, Thr135, and Glu136, whose relevance was not directly established by structural analysis alone, also plays an important role in the phosphotransferase activity of EB-AP/PTase.
Mihara
Yasuhiro
Y
Applied Microbiology Laboratory, Fermentation and Biotechnology Laboratories, Ajinomoto Co, Inc, Kaeasaki-shi, Japan. yasuhiro_mihara@ajinomoto.com
Ishikawa
Kohki
K
Suzuki
Ei-Ichiro
E
Asano
Yasuhisa
Y
D000135:Acid Phosphatase / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D011756:Diphosphates / Q000378:metabolism; D004925:Escherichia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D007288:Inosine / Q000378:metabolism*; D007291:Inosine Monophosphate / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009711:Nucleotides / Q000096:biosynthesis; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D011756:Diphosphates; D009711:Nucleotides; D007291:Inosine Monophosphate; D007288:Inosine; D010770:Phosphotransferases; C021892:nucleoside phosphotransferase; D000135:Acid Phosphatase