Computational Tools Applied to Enzyme Design − a review

García-Guevara, Fernando; Avelar, Mayra; Ayala, Marcela; Segovia, Lorenzo

doi:10.1515/boca-2015-0009

Cited by 13 publications

(10 citation statements)

References 23 publications

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“…Understanding the molecular factors that modulate the oxidative capacity of these enzymes is fundamental for the design of improved biocatalysts with biotechnological potential [ 20 , 21 ]. Computational tools are increasingly used in protein engineering, combined with experimental approaches such as directed evolution and/or site-directed mutagenesis [ 22 ]. Protein simulation allows to predict the effect of site-directed mutagenesis as well as to explain experimental results.…”

Section: Introductionmentioning

confidence: 99%

Exploring the Role of Phenylalanine Residues in Modulating the Flexibility and Topography of the Active Site in the Peroxygenase Variant PaDa-I

Ramirez-Ramirez

Martin-Diaz

Pastor

et al. 2020

IJMS

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Unspecific peroxygenases (UPOs) are fungal heme-thiolate enzymes able to catalyze a wide range of oxidation reactions, such as peroxidase-like, catalase-like, haloperoxidase-like, and, most interestingly, cytochrome P450-like. One of the most outstanding properties of these enzymes is the ability to catalyze the oxidation a wide range of organic substrates (both aromatic and aliphatic) through cytochrome P450-like reactions (the so-called peroxygenase activity), which involves the insertion of an oxygen atom from hydrogen peroxide. To catalyze this reaction, the substrate must access a channel connecting the bulk solution to the heme group. The composition, shape, and flexibility of this channel surely modulate the catalytic ability of the enzymes in this family. In order to gain an understanding of the role of the residues comprising the channel, mutants derived from PaDa-I, a laboratory-evolved UPO variant from Agrocybe aegerita, were obtained. The two phenylalanine residues at the surface of the channel, which regulate the traffic towards the heme active site, were mutated by less bulky residues (alanine and leucine). The mutants were experimentally characterized, and computational studies (i.e., molecular dynamics (MD)) were performed. The results suggest that these residues are necessary to reduce the flexibility of the region and maintain the topography of the channel.

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Section: Introductionmentioning

confidence: 99%

Exploring the Role of Phenylalanine Residues in Modulating the Flexibility and Topography of the Active Site in the Peroxygenase Variant PaDa-I

Ramirez-Ramirez

Martin-Diaz

Pastor

et al. 2020

IJMS

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“…Finally, by optimizing for substrate binding only, enzyme turnover rate ( k cat ) has not been considered, which will contribute to the overall efficiency of the enzyme. To this end, each transition state of the multistep reaction would need to be optimized using QM/MM hybrid methods to estimate the activation barriers for each mutant (good reviews in refs − ). To our knowledge, these have not been evaluated in high throughput with thousands of variants because of the computational cost of these calculations.…”

Section: Results and Discussionmentioning

confidence: 99%

“Site and Mutation”-Specific Predictions Enable Minimal Directed Evolution Libraries

Moore¹,

Rodriguez-Granillo²,

Crespo³

et al. 2018

ACS Synth. Biol.

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Directed evolution experiments designed to improve the activity of a biocatalyst have increased in sophistication from the early days of completely random mutagenesis. Sequence-based and structure-based methods have been developed to identify "hotspot" positions that when randomized provide a higher frequency of beneficial mutations that improve activity. These focused mutagenesis methods reduce library sizes and therefore reduce screening burden, accelerating the rate of finding improved enzymes. Looking for further acceleration in finding improved enzymes, we investigated whether two existing methods, one sequence-based (Protein GPS) and one structure-based (using Bioluminate and MOE), were sufficiently predictive to provide not just the hotspot position, but also the amino acid substitution that improved activity at that position. By limiting the libraries to variants that contained only specific amino acid substitutions, library sizes were kept to less than 100 variants. For an initial round of ATA-117 R-selective transaminase evolution, we found that the methods used produced libraries where 9% and 18% of the amino acid substitutions chosen were amino acids that improved reaction performance in lysates. The ability to create combinations of mutations as part of the initial design was confounded by the relatively large number of predicted mutations that were inactivating (30% and 45% for the sequence-based and structure-based methods, respectively). Despite this, combining several mutations identified within a given method produced variant lysates 7- and 9-fold more active than the wild-type lysate, highlighting the capability of mutations chosen this way to generate large advances in activity in addition to the reductions in screening.

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“…THz: terahertz spectroscopy, EPR: electron paramagnetic resonance spectroscopy, FTIR: Fourier-transform infrared spectroscopy, smFRET: single molecule fluorescence resonance energy transfer, SMX: serial millisecond crystallography, SSX: serial synchrotron crystallography, SFX: serial femtosecond crystallography, hNOE: heteronuclear nuclear Overhauser effect, CPMG: Carr–Purcell–Meiboom–Gill, AFM: atomic force microscopy, ED: electron diffraction, SAXS: small angle X-ray scattering, SANS: small angle neutron scattering, MD: molecular dynamics, QM: quantum mechanics, MM: molecular mechanics. The contents of this figure were excerpted from the contents of the study mentioned in [ 15 , 16 , 17 , 18 , 19 ].…”

Section: Figurementioning

confidence: 99%

Molecular Dynamics—From Small Molecules to Macromolecules

Nam

2021

IJMS

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All molecular systems, from small molecules to macromolecules, exhibit specific characteristics for a specific environment and time. In order to gain an accurate understanding of the functions of all types of molecules, studies of their structure and dynamics are essential. Through dynamic studies, using techniques such as spectroscopy, structure determination, and computer analysis, it is possible to collect functional information on molecules at specific times and in specific environments. Such information not only reveals the properties and mechanisms of action of molecules but also provides insights that can be applied to various industries, such as the development of new materials and drugs. Herein, I discuss the importance of molecular dynamics studies, present the time scale of molecular motion, and review techniques for analyzing molecular dynamics.

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Computational Tools Applied to Enzyme Design − a review

Cited by 13 publications

References 23 publications

Exploring the Role of Phenylalanine Residues in Modulating the Flexibility and Topography of the Active Site in the Peroxygenase Variant PaDa-I

Exploring the Role of Phenylalanine Residues in Modulating the Flexibility and Topography of the Active Site in the Peroxygenase Variant PaDa-I

“Site and Mutation”-Specific Predictions Enable Minimal Directed Evolution Libraries

Molecular Dynamics—From Small Molecules to Macromolecules

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