Studies on the active sites of Bacillus cereus sphingomyelinase substitution of some amino acids by site-directed mutagenesis

H. Ikezawa, K. Tameishi, A. Yamada, H. Tamura, K. Tsukamoto, Y. Matsuo, K. Nishikawa

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5 Citations (Scopus)

Abstract

Chemical modifications suggested that acidic amino acids such as aspartic and glutamic acids are involved in the active sites of Bacillus cereus sphingomyelinase. Among aspartic acid residues in the conserved regions of this enzyme, Asp-126, Asp-156, Asp-233 and Asp-295 were converted to glycine by site-directed mutagenesis. According to prediction on structural similarity to pancreatic DNase I, His-151 and His-296 were also converted to alanine. The Asp and His mutants, D126G, D156G, D233G, D295G, H151A and H296A, were produced in Bacillus brevis 47, a protein-hyperproducing strain. The catalytic activities of D295G, H151A and H296A were completely abolished, and sphingomyelin-hydrolyzing activity of D126G or D156G was reduced by more than 50%. The activity of D126G toward p-NPPC was comparable to that of the wild-type, while D156G catalyzed the hydrolysis of HNP and p-NPPC more efficiently than the wild-type. Hemolytic activities of the mutants were parallel to their sphingomyelin-hydrolyzing activities.

Original languageEnglish
Pages (from-to)293-298
Number of pages6
JournalAmino Acids
Volume9
Issue number3
DOIs
Publication statusPublished - 1995 Sep

Keywords

  • Amino acids
  • Bacillus cereus
  • Pancreatic DNase I
  • Replacement of alanine
  • Replacement of histidine
  • Sphingomyelinase

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Organic Chemistry

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    Ikezawa, H., Tameishi, K., Yamada, A., Tamura, H., Tsukamoto, K., Matsuo, Y., & Nishikawa, K. (1995). Studies on the active sites of Bacillus cereus sphingomyelinase substitution of some amino acids by site-directed mutagenesis. Amino Acids, 9(3), 293-298. https://doi.org/10.1007/BF00805960