Switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering

doi:10.1007/s13238-011-1057-7

Prot Cell

2011, Vol. 2

Issue (6) : 497-506 https://doi.org/10.1007/s13238-011-1057-7 PMID: 21748600

RESEARCH ARTICLE

Switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering

Chang Liu^2,3, Guangyu Yang¹, Lie Wu², Guohe Tian², Zuoming Zhang², Yan Feng^1,2(

)

1. State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; 2. Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, China; 3. Changchun Institute of Biological Products, China National Biotec Group, Changchun 130061, China

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Abstract

The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/T560W was generated, which had the highest catalytic efficiency (k_cat/K_m) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.

Keywords acylaminoacyl peptidase esterase substrate specificity protein engineering solvent engineering

Corresponding Author(s): Feng Yan,Email:yfeng2009@sjtu.edu.cn

Issue Date: 01 June 2011

Cite this article:

Chang Liu,Guangyu Yang,Lie Wu, et al. Switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering[J]. Prot Cell, 2011, 2(6): 497-506.

URL:

https://academic.hep.com.cn/pac/EN/10.1007/s13238-011-1057-7
https://academic.hep.com.cn/pac/EN/Y2011/V2/I6/497

Tab.1 Specific activities of selected variants of apAAP with esterase, peptidase and thioesterase substrates

Fig.1 Specific activities of the variants of apAAP in different concentrations of acetonitrile (A), DMSO (B), and DMF (C) at 60°C, with NP-C12 as the substrate.

Fig.2 Substrate specificities of the variants of apAAP towards -nitrophenyl alkanoate esters with various acyl chain lengths.
(A) Specificities in the absence of organic solvents. (B) Specificities in the presence of 10% acetonitrile. (C) Specificities in the presence of 30% DMSO. (D) Specificities in the presence of 20% DMF. (E) Evolution pathway of the substrate selectivity between NP-C12 and NP-C4. Values for wild type apAAP were assigned as one.

Tab.2 Kinetic parameters of wild type apAAP and its mutants for the hydrolysis of NP-C12 at 60°C

Fig.3 Modeled structures of selected variants of apAAP.
(A) The overall structure of mutant C01 identified in this study. The main chain of the enzyme is shown as a ribbon, while the mutation sites are shown as spheres. The catalytic triad of the enzyme is shown in red, and the substrate NP-C12 is shown in dark grey. (B) Molecular geometry of the substrate binding pocket of the parental enzyme P01. (C) Molecular geometry of the substrate binding pocket of the best mutant C01.

Fig.4 Results of molecular dynamics simulations of the variants of apAAP.
(A) RMSD of the backbone atoms of P01 (black), S01 (red), and S02 (blue) as a function of time. (B) The percentage of preserved secondary structure of the mutants after the MD simulation reached equilibrium. (C) Superimposition of the active sites of the mutants P01 (yellow) S01 (red), and S02 (blue) after the MD simulation reached equilibrium. The backbone and hydrogen bonding networks represented in the figure are belonging to P01. (D) A snapshot of the penetration of acetonitrile 3097 into the S01 mutant.

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