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 language | English |
|---|---|
| Pages (from-to) | 293-298 |
| Number of pages | 6 |
| Journal | Amino Acids |
| Volume | 9 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 1995 Sep |
| Externally published | Yes |
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Keywords
- Amino acids
- Bacillus cereus
- Pancreatic DNase I
- Replacement of alanine
- Replacement of histidine
- Sphingomyelinase
ASJC Scopus subject areas
- Endocrinology
- Clinical Biochemistry
- Biochemistry
Cite this
Studies on the active sites of Bacillus cereus sphingomyelinase substitution of some amino acids by site-directed mutagenesis. / Ikezawa, H.; Tameishi, K.; Yamada, A.; Tamura, Hiroomi; Tsukamoto, K.; Matsuo, Y.; Nishikawa, K.
In: Amino Acids, Vol. 9, No. 3, 09.1995, p. 293-298.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Studies on the active sites of Bacillus cereus sphingomyelinase substitution of some amino acids by site-directed mutagenesis
AU - Ikezawa, H.
AU - Tameishi, K.
AU - Yamada, A.
AU - Tamura, Hiroomi
AU - Tsukamoto, K.
AU - Matsuo, Y.
AU - Nishikawa, K.
PY - 1995/9
Y1 - 1995/9
N2 - 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.
AB - 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.
KW - Amino acids
KW - Bacillus cereus
KW - Pancreatic DNase I
KW - Replacement of alanine
KW - Replacement of histidine
KW - Sphingomyelinase
UR - http://www.scopus.com/inward/record.url?scp=0028801147&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028801147&partnerID=8YFLogxK
U2 - 10.1007/BF00805960
DO - 10.1007/BF00805960
M3 - Article
VL - 9
SP - 293
EP - 298
JO - Amino Acids
JF - Amino Acids
SN - 0939-4451
IS - 3
ER -