Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase

Hiroomi Tamura, K. Tameishi, A. Yamada, M. Tomita, Y. Matsuo, K. Nishikawa, H. Ikezawa

Research output: Contribution to journalArticle

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Abstract

Four aspartic acid residues (Asp126, Asp156, Asp233 and Asp295) of Bacillus cereus sphingomyelinase (SMase) in the conservative regions were changed to glycine by in vitro mutagenesis, and the mutant SMases [D126G (Asp126 → Gly etc.), D156G, D233G and D295G] were produced in Bacillus brevis 47, a protein-producing strain. The sphingomyelin (SM)-hydrolysing activity of D295G was completely abolished and those of D126G and D156G were reduced by more than 80%, whereas that of D233G was not so profoundly affected, Two mutant enzymes (D126G and D156G) were purified and characterized further. The hydrolytic activities of D126G and D156G toward four phosphocholine-containing substrates with different hydrophobicities, SM, 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP), lysophosphatidylcholine (lysoPC) and p-nitrophenylphosphocholine (p-NPPC), were compared with those of the wild-type. The activity of D126G toward water-soluble p-NPPC was comparable with that of the wild-type. On the other hand, D156G catalysed the hydrolysis of hydrophilic substrates such as HNP and p-NPPC more efficiently (> 4-fold) than the wild-type. These results suggested that Asp126 and Asp156, located in the highly conserved region, may well be involved in a substrate recognition process rather than catalytic action. Haemolytic activities of the mutant enzymes were found to be parallel with their SM-hydrolysing activities. Two regions, including the C-terminal region containing Asp295, were found to show considerable sequence identity with the corresponding regions of bovine pancreatic DNase I. Structural predictions indicated structural similarity between SMase and DNase I. An evolutionary relationship based on the catalytic function was suggested between the structures of these two phosphodiesterases.

Original languageEnglish
Pages (from-to)757-764
Number of pages8
JournalBiochemical Journal
Volume309
Issue number3
Publication statusPublished - 1995
Externally publishedYes

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Bacillus cereus
Sphingomyelin Phosphodiesterase
Sphingomyelins
Deoxyribonuclease I
Aspartic Acid
Mutation
Substrates
Mutagenesis
Lysophosphatidylcholines
Phosphorylcholine
Phosphoric Diester Hydrolases
Bacilli
Enzymes
Hydrophobicity
Hydrophobic and Hydrophilic Interactions
Glycine
Bacillus
Hydrolysis
Water
4-nitrophenylphosphocholine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Tamura, H., Tameishi, K., Yamada, A., Tomita, M., Matsuo, Y., Nishikawa, K., & Ikezawa, H. (1995). Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase. Biochemical Journal, 309(3), 757-764.

Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase. / Tamura, Hiroomi; Tameishi, K.; Yamada, A.; Tomita, M.; Matsuo, Y.; Nishikawa, K.; Ikezawa, H.

In: Biochemical Journal, Vol. 309, No. 3, 1995, p. 757-764.

Research output: Contribution to journalArticle

Tamura, H, Tameishi, K, Yamada, A, Tomita, M, Matsuo, Y, Nishikawa, K & Ikezawa, H 1995, 'Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase', Biochemical Journal, vol. 309, no. 3, pp. 757-764.
Tamura, Hiroomi ; Tameishi, K. ; Yamada, A. ; Tomita, M. ; Matsuo, Y. ; Nishikawa, K. ; Ikezawa, H. / Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase. In: Biochemical Journal. 1995 ; Vol. 309, No. 3. pp. 757-764.
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abstract = "Four aspartic acid residues (Asp126, Asp156, Asp233 and Asp295) of Bacillus cereus sphingomyelinase (SMase) in the conservative regions were changed to glycine by in vitro mutagenesis, and the mutant SMases [D126G (Asp126 → Gly etc.), D156G, D233G and D295G] were produced in Bacillus brevis 47, a protein-producing strain. The sphingomyelin (SM)-hydrolysing activity of D295G was completely abolished and those of D126G and D156G were reduced by more than 80{\%}, whereas that of D233G was not so profoundly affected, Two mutant enzymes (D126G and D156G) were purified and characterized further. The hydrolytic activities of D126G and D156G toward four phosphocholine-containing substrates with different hydrophobicities, SM, 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP), lysophosphatidylcholine (lysoPC) and p-nitrophenylphosphocholine (p-NPPC), were compared with those of the wild-type. The activity of D126G toward water-soluble p-NPPC was comparable with that of the wild-type. On the other hand, D156G catalysed the hydrolysis of hydrophilic substrates such as HNP and p-NPPC more efficiently (> 4-fold) than the wild-type. These results suggested that Asp126 and Asp156, located in the highly conserved region, may well be involved in a substrate recognition process rather than catalytic action. Haemolytic activities of the mutant enzymes were found to be parallel with their SM-hydrolysing activities. Two regions, including the C-terminal region containing Asp295, were found to show considerable sequence identity with the corresponding regions of bovine pancreatic DNase I. Structural predictions indicated structural similarity between SMase and DNase I. An evolutionary relationship based on the catalytic function was suggested between the structures of these two phosphodiesterases.",
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T1 - Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase

AU - Tamura, Hiroomi

AU - Tameishi, K.

AU - Yamada, A.

AU - Tomita, M.

AU - Matsuo, Y.

AU - Nishikawa, K.

AU - Ikezawa, H.

PY - 1995

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