Can constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo-β-glucanase

Contreras, Francisca; Nutschel, Christina; Beust, Laura; Davari, Mehdi D.; Gohlke, Holger; Schwaneberg, Ulrich

doi:10.1016/j.csbj.2020.12.034

Cited by 9 publications

(4 citation statements)

References 70 publications

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“…The Hotspot Wizard 3.0 server also provides other selection methods, such as correlated mutations and structural flexibility [22]. In addition, other methods such as constraint network analysis [23,24] can be used to identify weak spots for protein engineering. Optimal amino acid substitution relies on precise modeling of the influence of mutations in a protein.…”

Section: Discussionmentioning

confidence: 99%

Glycine Substitution of Residues with Unfavored Dihedral Angles Improves Protein Thermostability

Zhang

Zhong

et al. 2022

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Single mutations that can substantially enhance stability are highly desirable for protein engineering. However, it is generally rare for this kind of mutant to emerge from directed evolution experiments. This study used computational approaches to identify hotspots in a diacylglycerol-specific lipase for mutagenesis with functional hotspot and sequence consensus strategies, followed by ∆∆G calculations for all possible mutations using the Rosetta ddg_monomer protocol. Single mutants with significant ∆∆G changes (≤−2.5 kcal/mol) were selected for expression and characterization. Three out of seven tested mutants showed a significantly enhanced thermostability, with Q282W and A292G in the catalytic pocket and D245G located on the opposite surface of the protein. Remarkably, A292G increased the T5015 (the temperature at which 50% of the enzyme activity was lost after a 15 min of incubation) by ~7 °C, concomitant with a twofold increase in enzymatic activity at the optimal reaction temperature. Structural analysis showed that both A292 and D245 adopted unfavored dihedral angles in the wild-type (WT) enzyme. Substitution of them by glycine might release a steric strain to increase the stability. In sum, substitution by glycine might be a promising strategy to improve protein thermostability.

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Section: Discussionmentioning

confidence: 99%

Glycine Substitution of Residues with Unfavored Dihedral Angles Improves Protein Thermostability

Zhang

Zhong

et al. 2022

Catalysts

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“…E̅ region,CNA was calculated for each MD trajectory separately and then averaged over all replicas. The computations were done with the Constraint Network Analysis (CNA) program (version 4.0) developed by us, which has been applied in the context of protein thermostability, − allosteric signaling, , and substitution influences on the function , before.…”

Section: Materials and Methodsmentioning

confidence: 99%

F/G Region Rigidity is Inversely Correlated to Substrate Promiscuity of Human CYP Isoforms Involved in Metabolism

Becker

Bharatam

Gohlke

2021

J. Chem. Inf. Model.

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Of 57 human cytochrome P450 (CYP) enzymes, 12 metabolize 90% of xenobiotics. To our knowledge, no study has addressed the relation between enzyme dynamics and substrate promiscuity for more than three CYPs. Here, we show by constraint dilution simulations with the Constraint Network Analysis for the 12 isoforms that structural rigidity of the F/G region is significantly inversely correlated to the enzymes' substrate promiscuity. This highlights the functional importance of structural dynamics of the substrate tunnel.

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“…Four rounds of HTS were conducted, resulting in selection and sequencing of the top 12 most active variants from approximately 8000 independent transformants. Contreras et al cultured active mutants of EGLII (endo-β-glucanase gene egl II from Penicillium verruculosum ) in 96-well plates to produce enzymes and measured their absorbance at 590 nm . Finally, mutants with >80% initial activity of wild-type EGLII were screened out.…”

Section: Hts Techniques In Improving Enzyme Thermostabilitymentioning

confidence: 99%

“…Contreras et al cultured active mutants of EGLII (endo-β-glucanase gene eglII from Penicillium verruculosum) in 96-well plates to produce enzymes and measured their absorbance at 590 nm. 33 Finally, mutants with >80% initial activity of wild-type EGLII were screened out. Garci ́a-Ruiz et al investigated the thermostability mutant libraries of lignin-degrading oxidoreductases expressed in yeast by measuring residual activity across a temperature gradient from 30 to 90 °C compared to the initial activity at room temperature.…”

mentioning

confidence: 99%

High-Throughput Screening Techniques for the Selection of Thermostable Enzymes

Li,

Liu,

Bai

et al. 2024

J. Agric. Food Chem.

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The acquisition of a thermostable enzyme is an indispensable prerequisite for its successful implementation in industrial applications and the development of novel functionalities. Various protein engineering approaches, including rational design, semirational design, and directed evolution, have been employed to enhance thermostability. However, all of these approaches require sensitive and reliable high-throughput screening (HTS) technologies to efficiently and rapidly identify variants with improved properties. While numerous reviews focus on modification strategies for enhancing enzyme thermostability, there is a dearth of literature reviewing HTS methods specifically aimed at this objective. Herein, we present a comprehensive overview of various HTS methods utilized for modifying enzyme thermostability across different screening platforms. Additionally, we highlight significant recent examples that demonstrate the successful application of these methods. Furthermore, we address the technical challenges associated with HTS technologies used for screening thermostable enzyme variants and discuss valuable perspectives to promote further advancements in this field. This review serves as an authoritative reference source offering theoretical support for selecting appropriate screening strategies tailored to specific enzymes with the aim of improving their thermostability.

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Can constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo-β-glucanase

Abstract: Graphical abstract

Cited by 9 publications

References 70 publications

Glycine Substitution of Residues with Unfavored Dihedral Angles Improves Protein Thermostability

Glycine Substitution of Residues with Unfavored Dihedral Angles Improves Protein Thermostability

F/G Region Rigidity is Inversely Correlated to Substrate Promiscuity of Human CYP Isoforms Involved in Metabolism

High-Throughput Screening Techniques for the Selection of Thermostable Enzymes

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