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 journalArticlepeer-review

1 Scopus citations

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

Bibliographical note

Funding Information:
We thank Surajit Banerjee for his assistance during diffraction data collection at the Advanced Photon Source. This work was supported by the National Natural Science Foundation of China ( 31470160 ), Program for Changjiang Scholars and Innovative Research Team in University (No. IRT_15R26 ), and a grant from the US National Institutes of Health ( R35 GM118047 ). And the China Scholarship Council fund for financial support.

Keywords

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

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