Characterization and rational design for substrate specificity of a prolyl endopeptidase from Stenotrophomonas maltophilia

https://doi.org/10.1016/j.enzmictec.2020.109548Get rights and content

Highlights

  • A novel prolyl endopeptidase, SmPEP, was cloned and expressed in E. coli.

  • The specific activity of recombinant SmPEP was 68.3 U/mg at pH 8.0 and 37 °C.

  • An inter-domain open configuration was rational-designed for substrate entrance.

  • F263 V achieved catalysis efficiency on 13-mer peptide 10.2 times of wild type.

  • F263 V significantly improved the ACE inhibitory activity of the casein hydrolysate.

Abstract

A novel prolyl endopeptidase from Stenotrophomonas maltophilia, SmPEP, was discovered and characterized. The specific activity of the recombinant SmPEP expressed by Escherichia coli BL21 (DE3), was 68.3 U/mg at pH 8.0 and 37 °C. In order to improve the substrate specificity for long-chain peptide, rational design was applied based on the structure constructed by homology modeling. Inter-domain sites within the β-propeller domain were chosen for the mutation to weaken the inter-domain interaction and form an open conformation for long-chain substrate entering into the active site. The substrate specificity on a designed long-chain substrate, PQPQLPYPQPQLP, of the mutants F263A and E184 G increased 8.77 and 5.75 times respectively versus wild-type. After the saturated mutation of the both sites, the reactive rate of mutant F263 V on 13-mer peptide was 10.2 times higher than that of the wild-type. Then the mutant F263 V was used in the hydrolysis of casein, and the ACE inhibitory activity of the hydrolysate was significantly improved compared with wild type enzyme, which verified the efficiency of the design strategy.

Introduction

Hypertension is a major health concern afflicting approximately a quarter of adults worldwide. It is estimated that the global hypertensive population will increase to 1.5 billion by 2025 [1]. The angiotensin converting enzyme (EC 3.4.15.1) in renin-angiotensin system, plays a significant role in regulating blood pressure [2,3]. Therefore, ACE is a key target for blood pressure controlling [4,5]. Drugs such as captopril, enalapril and lisinopril have pril-medicine inhibitory model in common, with proline structure in C-terminus. The proline part can bind with the S2’ pocket of ACE and result in ACE inactivation [6,7].

Food-derived ACE inhibitory peptides become attractive because of their controlling blood pressure in a safer way than traditional medicine. And peptides with proline in C-terminus were proved to have higher inhibition, owing to the pyrrole ring binding with ACE active pocket specifically [[8], [9], [10]]. However, such type of peptides cannot be generated through gastrointestinal hydrolysis. Since pepsin, trypsin and chymotrypsin fail to recognize the proline at P1 site [11,12]. A protease which enable to cut food protein targeted at Pro-Xaa site is essential for the production of ACE inhibitory peptides with C-terminal proline.

Prolyl endopeptidase (PEP, EC 3.4.21.26) preferentially hydrolyzes the peptide bond on the carboxyl side of proline residues [13]. The enzyme was used in the degrading of gluten to avoid allergic reaction in gluten intolerant people [14,15]. PEP has been initially identified in several microbes, such as Flavobacterium meningosepticum [16], Xanthomonas sp. [17], Aeromonas hydrophilic [18], Sphingomonas capsulate [19], Halobacterium halobium S9 [20], Lactobacillus helveticus [21], Myxococcus xanthus [22], Aspergillus niger [23] and Aspergillus oryzae [24]. These have all shown ability to break down toxic gluten peptides under in vitro conditions. However, as peptidases, most of them are restricted to cleave peptides shorter than 30 residues but not intact proteins. Most of PEPs consist of a characteristic β-propeller domain and an α/β hydrolase catalytic domain. The β-propeller domain of the enzyme operated as a gating filter and excludes large structured peptides or protein from the hydrolase catalytic domain [25], which hindered the application prospects of PEPs in purposes for treating gluten allergy and processing ACE inhibitors.

There were two pathways for substrate entrance of PEP reported. One was the entrance in 4 Å diameter within the β-propeller domain [26]. The other was the cleft between the catalytic domain and the β-propeller domain [27]. In the former case, the narrow entrance could be enlarged by substrate induction. However, it was found that PEP could only hydrolyze peptides up to 30 amino acids due to such small entrance [28]. In the latter case, the substrate channel of inter-domain cleft was discovered by comparison between an open configuration of the active site of S. capsulate PEP and a close form of inhibitor-bound M. xanthus PEP [25]. Then the up-and-down movement of the catalytic domain was predicted to twist between the two domains, and facilitate inter-domain entrance for substrate through molecular dynamics simulations [29]. The open configuration of PEP from A. punctate displayed a 30 Å entrance between the two domains, which allowed substrate HP35 with 35 amino acids to bind with the active pocket [30]. Accordingly, the engineering on the inter-domain may be an effective solution to develop the substrate specificity of long-chain peptide or intact protein.

The aim of present work is the characterization of a novel PEP from Stenotrophomonas maltophilia (SmPEP) and improvement of substrate specificity based on rational design. SmPEP was cloned and expressed. The recombinant enzyme then was purified for the enzymatic properties determination. Rational design was applied to enlarge the inter-domain of the enzyme. Saturated mutation of the chosen site was afterwards carried out to further improve the substrate specificity and illustrate the structure-activity relationship of SmPEP. Finally, the mutant was applied to hydrolyze casein to verify its substrate specificity on intact protein and capability in ACE inhibitory peptides production.

Section snippets

Strains, vectors and chemicals

The strain S. maltophilia was isolated from contaminated culture of Pichia pastoris GS115, which was used to express a recombinant PEP from Aspergillus oryzae (the GenBank accession No. of the encoding sequence, XM_001825944.2) in the Lab. The GenBank accession No. of the encoding sequence of SmPEP is KIS37168.1. Escherichia coli BL21 (DE3) and pET-28a (+) (Invitrogen, Calsbad, CA, USA) was used as host cells and expression vector, respectively. Chromogenic substrate, Z-Gly-Pro-pNA, was

Sequence analysis of SmPEP

The strain S. maltophilia was firstly sequenced in 2015 [34]. The gene encoding a PEP was discovred among the sequence. However, PEP from such strain was not reported before. The gene (GenBank Accession No. KIS37168.1) contained an open reading frame of 2097 bps, which encoded a protein of 698 amino acids. The calculated molecular weight of this protein was 77.0 kDa, and its calculated pI was 6.78. The amino acid sequence of SmPEP displayed 24.1%, 23.7%, 23.7%, 27.1% and 20.4% identity to the

Conclusions

In summary, a novel prolyl endopeptidase, SmPEP, was cloned and expressed in E. coli. The enzyme properties were then investigated. In order to efficiently apply the enzyme to produce ACE inhibitory peptide, the substrate specificity on long-chain peptides or intact proteins of the enzyme was improved through rational design. The strategy is to weaken the inter-domain interaction and induce an open configuration of inter-domain, which enable the long-chain substrate to enter the active site

Declaration of Competing Interest

There are no conflicts of interest to declare.

CRediT authorship contribution statement

Junjie Yu: Investigation, Writing - original draft. Junjie Wu: Methodology, Writing - original draft. Dewei Xie: Data curation. Lei Du: Formal analysis. Ya-Jie Tang: Funding acquisition. Jingli Xie: Conceptualization, Writing - review & editing. Dongzhi Wei: Supervision.

Acknowledgements

This work was supported by the Open Funding Project of the Key Laboratory of Fermentation Engineering (Ministry of Education) of China, the Opening Project of Shanghai Key Laboratory of New Drug Design (Grant No. 17DZ2271000), China and the National Natural Science Foundation of China (31801489).

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