Insight into subtilisin E-S7 cleavage pattern based on crystal structure and hydrolysates peptide analysis

Heng Tang, Juan Zhang, Ke Shi, Hideki Aihara, Guocheng Du

Research output: Contribution to journalArticle

Abstract

The X-ray crystallographic structure of the mature form of subtilisin E-S7 (SES7) at 1.90 Å resolution is reported here. Structural comparisons between the previously reported propeptide-subtilisin E complex (1SCJ) and our mature form subtilisin E-S7 (6O44) provide insight into active site adjustments involved in catalysis and specificity. To further investigate the protease substrate selectivity mechanism, we used SES7 to hydrolyze skim milk and analyzed the hydrolysates by LC-MS for peptide identification. The cleavage pattern suggests a high preference for proline at substrate P2 position. The results based on the peptide analysis are consistent with our structural observations, which is instrumental in future protein engineering by rational design. Furthermore, the ACE-inhibitor and NLN-inhibitor activity of the hydrolysates were determined to assess the utility of SES7 for further industrial applications; IC 50 -ACE = 67 ± 0.92 μg/mL and IC 50 -NLN = 263 ± 13 μg/mL.

Original languageEnglish (US)
Pages (from-to)623-628
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume512
Issue number3
DOIs
StatePublished - May 7 2019

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ethyl-2-methylthio-4-methyl-5-pyrimidine carboxylate
Subtilisins
Crystal structure
Peptides
Protein Engineering
Substrates
Catalysis
Angiotensin-Converting Enzyme Inhibitors
Proline
Industrial applications
Catalytic Domain
Milk
Peptide Hydrolases
X-Rays
X rays
Proteins

Keywords

  • ACE-inhibitor
  • Crystal structure
  • NLN-inhibitor
  • Structural comparisons
  • Subtilisin E-S7

PubMed: MeSH publication types

  • Journal Article

Cite this

Insight into subtilisin E-S7 cleavage pattern based on crystal structure and hydrolysates peptide analysis. / Tang, Heng; Zhang, Juan; Shi, Ke; Aihara, Hideki; Du, Guocheng.

In: Biochemical and Biophysical Research Communications, Vol. 512, No. 3, 07.05.2019, p. 623-628.

Research output: Contribution to journalArticle

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abstract = "The X-ray crystallographic structure of the mature form of subtilisin E-S7 (SES7) at 1.90 {\AA} resolution is reported here. Structural comparisons between the previously reported propeptide-subtilisin E complex (1SCJ) and our mature form subtilisin E-S7 (6O44) provide insight into active site adjustments involved in catalysis and specificity. To further investigate the protease substrate selectivity mechanism, we used SES7 to hydrolyze skim milk and analyzed the hydrolysates by LC-MS for peptide identification. The cleavage pattern suggests a high preference for proline at substrate P2 position. The results based on the peptide analysis are consistent with our structural observations, which is instrumental in future protein engineering by rational design. Furthermore, the ACE-inhibitor and NLN-inhibitor activity of the hydrolysates were determined to assess the utility of SES7 for further industrial applications; IC 50 -ACE = 67 ± 0.92 μg/mL and IC 50 -NLN = 263 ± 13 μg/mL.",
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AU - Zhang, Juan

AU - Shi, Ke

AU - Aihara, Hideki

AU - Du, Guocheng

PY - 2019/5/7

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N2 - The X-ray crystallographic structure of the mature form of subtilisin E-S7 (SES7) at 1.90 Å resolution is reported here. Structural comparisons between the previously reported propeptide-subtilisin E complex (1SCJ) and our mature form subtilisin E-S7 (6O44) provide insight into active site adjustments involved in catalysis and specificity. To further investigate the protease substrate selectivity mechanism, we used SES7 to hydrolyze skim milk and analyzed the hydrolysates by LC-MS for peptide identification. The cleavage pattern suggests a high preference for proline at substrate P2 position. The results based on the peptide analysis are consistent with our structural observations, which is instrumental in future protein engineering by rational design. Furthermore, the ACE-inhibitor and NLN-inhibitor activity of the hydrolysates were determined to assess the utility of SES7 for further industrial applications; IC 50 -ACE = 67 ± 0.92 μg/mL and IC 50 -NLN = 263 ± 13 μg/mL.

AB - The X-ray crystallographic structure of the mature form of subtilisin E-S7 (SES7) at 1.90 Å resolution is reported here. Structural comparisons between the previously reported propeptide-subtilisin E complex (1SCJ) and our mature form subtilisin E-S7 (6O44) provide insight into active site adjustments involved in catalysis and specificity. To further investigate the protease substrate selectivity mechanism, we used SES7 to hydrolyze skim milk and analyzed the hydrolysates by LC-MS for peptide identification. The cleavage pattern suggests a high preference for proline at substrate P2 position. The results based on the peptide analysis are consistent with our structural observations, which is instrumental in future protein engineering by rational design. Furthermore, the ACE-inhibitor and NLN-inhibitor activity of the hydrolysates were determined to assess the utility of SES7 for further industrial applications; IC 50 -ACE = 67 ± 0.92 μg/mL and IC 50 -NLN = 263 ± 13 μg/mL.

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