Enzyme promiscuity: mechanism and applications

Hult, Karl; Berglund, Per

doi:10.1016/j.tibtech.2007.03.002

Cited by 578 publications

(352 citation statements)

References 46 publications

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“…It is widely accepted that promiscuous enzymes can evolve rapidly, allowing organisms to survive environmental changes. Recently, numerous reports have shown that promiscuous enzymes can evolve novel or altered functions, which provides a useful approach for generating new enzymes with specific properties (Aharoni et al, 2005;Hult and Berglund, 2007).…”

Section: Introductionmentioning

confidence: 99%

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

Liu

Yang

et al. 2011

Protein Cell

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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.

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Section: Introductionmentioning

confidence: 99%

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

Liu

Yang

et al. 2011

Protein Cell

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“…[11,12,13,14] Many lipases have been found to be stable under a broad range of temperature and pH, and active in organic solvents, [15,16] which greatly expands their utility in synthetic applications. More recently, evidence has emerged of catalytic promiscuity [17,18] (i.e. the ability of an enzyme to exhibit catalysis of other than its native reaction) towards e.g.…”

Section: Introductionmentioning

confidence: 99%

Computational design of a lipase for catalysis of the Diels-Alder reaction

et al. 2010

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Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the Diels-Alder reaction by a modified lipase. Six variants of the versatile enzyme {\em Candida Antarctica} lipase B (CALB) have been rationally engineered {\em in silico} based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity shape and size, and can be controlled by a few rational mutations. The well-documented S105A mutation is essential to enable sufficient space in the vicinity of the oxyanion hole. Moreover, bulky residues on the edge of the active site hinders the formation of a sandwich-like near-attack conformer (NAC), and the I189A mutation is needed to obtain enough space above the face of the $\alpha$,$\beta$-double bond on the dienophile. The double mutant S105A/I189A performs quite well for two of three dienophiles. Based on binding constants and NAC energies obtained from MD simulations combined with activation energies from DFT computations, relative catalytic rates ($v_{cat}/v_{uncat}$) of up to $10^3$ are predicted.Response to Reviewers: We are happy with the favorable comments by the reviewers. We have have corrected the manuscript as suggested by reviewer 1 1. A sentence explaining catalytic promiscuity has been added. 2. A paragraph discussing the relationship between the NAC concept and the Reaction Force has been added.We have shortened the manuscript as suggested by reviewer 2. In particular, we have moved information regarding the structural relaxation of protein structure in MD simulations to the supplementary material. Abstract Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the DielsAlder reaction by a modified lipase. Six variants of the versatile enzyme Candida Antarctica lipase B (CALB) have been rationally engineered in silico based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity s...

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“…Nevertheless, these aggregates had higher activity in both aqueous and non-aqueous conditions. Hult and Berglund (2007) [69] classified enzyme promiscuity into three classes: condition promiscuity, substrate promiscuity and catalytic promiscuity. Enzymes carrying out different catalytic activities (from those in aqueous buffers) in nearly anhydrous organic solvents can be said to exhibit condition promiscuity (Figure 2).…”

Section: Three Phase Partitioning (Tpp) Of Enzymesmentioning

confidence: 99%

“…These chemical reactions are distinct enough to involve different transition states. While doi: 10.7243/2053-7670-1-1 considerable amount of work has been already reported on "induced catalytic promiscuity" which involves enzymes engineered by protein engineering and directed evolution methods [69,70] we will again restrict ourselves to "accidental catalytic promiscuity" shown by wild enzymes in low water containing organic solvents. As Busto et al, (2010) [70] have recently reviewed this as well extensively, only brief treatment will be provided here with a focus on the current status of catalytic efficiency in such cases.…”

Section: Three Phase Partitioning (Tpp) Of Enzymesmentioning

confidence: 99%

Use of high activity enzyme preparations in neat organic solvents for organic synthesis

Gupta¹,

Mukherjee²,

Malhotra³

2013

Univers Org Chem

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The use of enzymes in nearly anhydrous organic solvents generally results in low initial rates/percentage conversions. The current review focuses on some biocatalyst designs like immobilization on nanomaterials, enzyme precipitated and rinsed with organic solvents (EPROS), crosslinked enzyme crystals (CLEC), crosslinked enzyme aggregates (CLEA), protein coated microcrystals (PCMC) and crosslinked protein coated microcrystals (CLPCMC) which show much better catalytic efficiency in such media as compared to other kinds of biocatalyst preparations. The basic methodology and principle behind these efficient designs are described. The relatively recent results on catalytic promiscuity as seen in low water media have also been covered. It is hoped that this will further encourage wider applications of enzymes in neat organic solvents in organic synthesis.

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Enzyme promiscuity: mechanism and applications

Cited by 578 publications

References 46 publications

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

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

Computational design of a lipase for catalysis of the Diels-Alder reaction

Use of high activity enzyme preparations in neat organic solvents for organic synthesis

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