Design of salt-bridge cyclization peptide tags for stability and activity enhancement of enzymes
Graphical abstract
Introduction
For an industrial enzyme with a defined substrate, activity and stability are the two most important properties [[1], [2], [3]]. However, the trade-off between activity and stability generally limits the biotechnological applications of enzymes [4]. Industrial bio-catalytic processes, which are increasingly important in the production of diverse bulk or fine chemicals and medicine intermediates, have seen considerable growth and are in need of enzymes with both high efficiency and stability. Although several methodologies have been developed, such as directed evolution [[5], [6], [7]], rational design [8,9], and immobilization [3,10], there remains a need for a novel strategy of both simplicity and effectiveness to enhance the activity and stability of target enzymes.
The salt-bridge is a strong interaction connecting positively and negatively charged amino acids. Its intensity generally falls between that of covalent bonds and hydrogen bonds. It participates in the folding and formation of the tertiary structure of enzymes and it is insensitive to temperature change or a polar environment, thus it plays a key role in stabilizing the enzyme structures [11,12]. Under stress conditions such as high temperature, inappropriate pH and organic solvents, the structure of well-folded enzymes will be deformed, which will consequently cause activity loss of the enzymes [13]. Comparative studies on the structures of mesophilic and thermophilic enzymes showed that some special strong interactions, including the disulfide bond, salt bridge and hydrogen bond, are of great significance for the outstanding stability of the thermophilic enzymes [14]. Particularly, extremely thermophilic enzymes generally have more salt bridges compared to mesophilic enzymes [[15], [16], [17]].
Several studies have investigated the function of salt-bridges in different parts of enzymes, including the surface, the vicinity of active site, and the terminus of the enzymes (Table S1). Strop et al. observed the contribution of the salt bridge to the stability of the super thermophilic enzyme PFRD-XC4 [18]. Kumara et al. showed that more salt bridges are found near the active site in the thermophilic glutamate dehydrogenase [19]. Cobucci-Ponzano et al. reported that the salt bridge network at the C-terminus of the β-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus plays an important role in the thermal stabilization of the quaternary structure [20]. Foglia et al. also reported on the importance of N-terminal salt bridges to the stability of thermophilic esterase 2 [21]. A short peptide loop was applied to cyclize the polypeptide backbone of enzymes to enhance the stability [22,23].
Nitrilase hydratase (NHase) and nitrilase are two remarkable nitrile-converting enzymes that are widely used to hydrolyze the available nitrile to corresponding amides and acids, respectively. Previous studies regarding the modification of NHase and nitrilase for stability improvement usually caused activity loss. For example, Shi et al. increased the thermal stability of NHase by stabilizing the rigid α-helix structure, but this resulted in a 30–50% activity loss [24]. Cui et al. presented a site targeted amino recombination method that enhanced both the thermal stability and acrylamide tolerance of NHase, but its activity was decreased by 20% [25]. Liu et al. fused NHase with two self-assembling peptides (EAK16-AEAEAKAKAEAEAKAK and ELK16-LELELKLKLELELKLK) and obtained stability-enhanced enzymes, but the activity was not simultaneously enhanced either [26]. Yang et al. introduced an amphipathic short peptide 18 A (EWLKAFYEKVLEKLKELF) to the C-terminus of a nitrilase, which resulted in higher stability but lower activity [27].
Our previous study investigated the thermal sensitive regions of NHase through molecular dynamics (MD) simulations and found that the N- and C-terminus are the most unstable regions that are responsible for the initial heat denaturation of NHase [28]. For the NHase from the Rhodococcus ruber TH (NHaseM-TH) [29], the global stability was significantly enhanced when it was introduced by a salt-bridge module into the C-terminus of its β-subunit [30].
Enlightened by these studies, this work describes a novel idea to design a salt-bridge cyclization peptide tag (SbCPT) for enzyme stability enhancement. The SbCPT, similar to the widely used His-tag for fast protein purification, can be conveniently attached to the subunit terminus of an enzyme. We proposed that the SbCPT would prevent large fluctuations of the sensitive subunit terminus and stabilize the target enzyme under elevated thermal or other environmental stress. Meanwhile, the SbCPT would also enhance, or at least would not be detrimental to, the activity of the enzyme. Using Co-type NHaseM–TH from R. ruber TH [31,32] and aliphatic nitrilase from R. rhodochrous tg1-A6 (Nit-tg1 A6) [33,34] as model enzymes, a series of SbCPTs were designed and investigated both computationally and experimentally.
Section snippets
Models and systems
The structures of SbCPTs of different lengths (GRPEG, GRGPGEG, and GRGGPGGEG) and different types (GRPEG, GRPDG, GKPEG, and GKPDG) were constructed. Their N-termini and C-termini were capped with neutral acetyl group (CH3CO-) and a methylamino group (-NHCH3), respectively, to avoid the electrostatic interactions between the charged residues and the termini of the SbCPTs. For example, GRPEG SbCPT was constructed as CH3CO-Gly-Arg-Pro-Glu-Gly-NHCH3. All the peptide bonds were in the trans
Design and evaluation of SbCPTs with different lengths
SbCPTs of different lengths were investigated to find the optimal size and structure for enzyme terminal stabilization. An arginine (Arg, R) and a glutamic acid (Glu, E) were employed in the SbCPT as the basic and acidic amino acids, respectively, to form a salt bridge. The turn structure was introduced by a proline (Pro, P) residue. SbCPTs of 5, 7, and 9 amino acid lengths were chosen and named as follows: SB1 (GRPEG), SB2 (GRGPGEG), and SB3 (GRGGPGGEG). The MD simulations of the three SbCPTs
Discussion
Several studies have shown that a salt bridge in the terminus of an enzyme would enhance the enzyme stability [20,21,30,47]. Here, we proposed an enzyme stabilization strategy by terminal insertion of a SbCPT. The core idea is to enable an oligo-peptide to form a cyclization structure via salt-bridge interaction and apply it to reduce the extensional freedom of the enzyme terminus, and thereby universally enhance stability and activity of diverse enzymes.
Design of SbCPTs with different length
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 21476126 and 21776157), National Key Basic Research Project 973 (2013CB733600) and the China Postdoctoral Science Foundation (No. 2015M581110).
References (49)
- et al.
Industrial applications of enzyme biocatalysis: Current status and future aspects
Biotechnol. Adv.
(2015) - et al.
Strategies for design of improved biocatalysts for industrial applications
Bioresour. Technol.
(2017) - et al.
Improving and repurposing biocatalysts via directed evolution
Curr. Opin. Chem. Biol.
(2015) - et al.
Directed evolution of carbon-hydrogen bond activating enzymes
Curr. Opin. Biothenol.
(2019) - et al.
Trends on enzyme immobilization researched based on bibliometric analysis
Process Biochem.
(2019) - et al.
Physical and molecular bases of protein thermal stability and cold adaption
Curr. Opin. Struct. Biol.
(2017) - et al.
Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey
Structure
(2000) - et al.
The role of salt bridges on the temperature adaptation of aqualysin I, a thermostable subtilisin-like proteinase
Biochim. Biophys. Acta
(2014) - et al.
Role of the N-terminal region for the conformational stability of esterase 2 from Alicyclobacillus acidocaldarius
Biophys. Chem.
(2007) - et al.
Circular -lactamase: stability enhancement by cyclizing the backbone
FEBS Lett.
(1999)
An over expression and high efficient mutation system of a cobalt-containing nitrile hydratase
J. Mol. Catal., B Enzym.
Improvement of stability of nitrile hydratase via protein fragment swapping
Biochem. Biophys. Res. Commun.
Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide
J. Biosci. Bioeng.
Insights into thermal stability of thermophilic nitrile hydratases by molecular dynamics simulation
J. Mol. Graph. Model.
Identification of nitrile hydratase-producing Rhodococcus ruber TH and characterization of an amiE-negative mutant
Bioresour. Technol.
Cloning of the nitrile hydratase gene from Nocardia sp. in Escherichia coli and Pichia pastoris and its functional expression using site-directed mutagenesis
Enzyme Microb. Technol.
Crystal structural of cobalt-containing nitrile hydratase
Biochem. Biophys. Res. Commun.
VMD: visual molecular dynamics
J. Mol. Graph. Model.
Atomic-level protein structure refinement using fragment-guided molecular dynamics conformation sampling
Structure
Cloning of the nitrile hydratase gene from Nocardia sp. in Escherichia coli and Pichia pastoris and its functional expression using site-directed mutagenesis
Enzyme Microb. Technol.
Ubiquitin-independent proteasomal degradation
Biochim. Biophys. Acta - Mol. Cell Res.
Integrating enzyme immobilization and protein engineering: an alternative path for the development of novel and improved industrial biocatalysts
Biotechnol. Adv.
How protein stability and new functions trade off
PLoS Comput. Biol.
Methods for the directed evolution of proteins
Nat. Rev. Genet.
Cited by (7)
The stability improvement of dextransucrase by artificial extension modification of the V domain of the enzyme
2021, Enzyme and Microbial TechnologyCitation Excerpt :In addition, there are other methods that improve the stability by minor modifications of the protein structure. The modification of residues [13,14], substitution domain, fusing functional domains or peptide tag [15–17], removing the sensitive domain [18], adding molecular chaperones [19], synthetic enzymes (implanting of enzyme in molecular Brush) [20], truncation, and cyclization [21] have been reported in the past studies. Enzymes are proteins formed by the winding of polypeptide chains consisting of amino acids.
Functional expression of an echinocandin B deacylase from Actinoplanes utahensis in Escherichia coli
2021, International Journal of Biological MacromoleculesCitation Excerpt :GE and 6K had a better solubilizing effect on ECBD when compared with other peptide tags. The enhanced activity after introducing peptide tags may be due to the repulsive electrostatic interaction of peptide tags to prevent the aggregation of nascent proteins [27–29,40]. Meanwhile, the main reason behind the not evident increase in activity could be ECBD is composed of two subunits, and the introduction of a single peptide tag cannot significantly increase the activity.
Effect and mechanism analysis of different linkers on efficient catalysis of subunit-fused nitrile hydratase
2021, International Journal of Biological MacromoleculesCitation Excerpt :However, as the nitrile hydration being an exothermic process, numerous energy cost is wasted to maintain low reaction temperature, and the high concentration of both nitrile substrate and amide product lead to huge loss of NHase activity. Although numerous efforts have been made to improve the stability of NHase [16–22], industry still calls for robust NHase with high thermostability and strong tolerance to either nitriles or amides. Here, to further clarify the regular pattern of suitable type of linker for fusion proteins, NHase from Pseudomonas putida was used as a target protein and a subunit fusion strategy was carried out to improve its thermostability and tolerance to either nitriles or amides.
Nitrile Hydratases: From Industrial Application to Acetamiprid and Thiacloprid Degradation
2021, Journal of Agricultural and Food Chemistry
- 1
These two authors contributed equally to this work.