4.7 Review

Tools for computational design and high-throughput screening of therapeutic enzymes

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Summary: This study presents the crystal structure of a (S)-6-hydroxy-nicotine oxidase enzyme with high catalytic efficiency, which has potential applications in nicotine detection, therapeutic intervention, and waste remediation in the tobacco industry. By employing a rational design approach, the researchers were able to improve the enzyme's oxidative turnover rate and reduce its affinity for nicotine. These findings are a step forward in engineering a nicotine oxidase enzyme with desired kinetic parameters for various functional applications.

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Summary: The transplantation of loops between structurally related proteins is a method to improve enzyme activity, specificity, and stability. However, there are limited methods for loop-based rational protein design. The unique dynamism and identification of successful excision points are practical challenges. LoopGrafter is a web server that guides users through the loop grafting process, providing step-by-step procedures for loop identification, calculation of geometry, assessment of similarities and dynamics, and selection of loops for transplantation.

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SoluProtMut(DB): A manually curated database of protein solubility changes upon mutations

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Summary: This review summarizes the computational methods developed over the past 30 years for predicting the change in thermodynamic stability of proteins due to mutations, as well as their current applications. It discusses the limitations of existing methods and provides guidance on selecting the most suitable tools for different needs.

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Summary: Advancements in technology and algorithms have revolutionized the field of protein design and engineering, with computational approaches playing a key role in tailoring proteins for various biotechnological applications. New tools and methods are continuously developed each year to meet the growing demands and challenges in protein engineering.

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DynaMut2: Assessing changes in stability and flexibility upon single and multiple point missense mutations

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Summary: DynaMut2 is a web server that combines NMA methods and graph-based signatures to investigate the effects of missense mutations on protein stability and dynamics. It accurately predicts the effects of missense mutations, achieving good performance on single-point and multiple-point missense mutations.

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SoluProt: prediction of soluble protein expression in Escherichia coli

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Computational design of enzymes for biotechnological applications

Joan Planas-Iglesias et al.

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FireProtASR: A Web Server for Fully Automated Ancestral Sequence Reconstruction

Milos Musil et al.

Summary: Researchers are increasingly interested in improving the stability of proteins, and ancestral sequence reconstruction has been proven to be an effective method for designing stable proteins. FireProt(ASR) offers a fully automated workflow to help users obtain ancestral sequences based on just a sequence query.

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Yanan Wang et al.

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D. A. Mokhtari et al.

Summary: Accurate predictions from models based on physical principles are crucial in our biophysical understanding. Despite progress in structure prediction, quantitative prediction of enzyme function remains challenging. Ground truth data and technological advances are necessary to guide the development and testing of these quantitative models, leading us towards a predictive understanding of enzyme structure and function.

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James R. Marshall et al.

Summary: Over 300 new imine reductases have been discovered in this study, and a large and diverse panel of 384 enzymes has been produced for screening. A high-throughput screening method has also been developed. Through these methods, imine reductase biocatalysts capable of accepting structurally demanding ketones and amines for excellent synthesis have been identified.

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Therapeutic enzymes: Discoveries, production and applications

Siddhi Tandon et al.

Summary: Enzymes are increasingly being used in pharmaceutical industries for their ability to efficiently catalyze biochemical reactions in living systems. Therapeutic enzymes are produced using various fermentation techniques and advanced technologies to improve their stability and catalytic activity, providing new opportunities for the treatment of various diseases.

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Cell-free Directed Evolution of a Protease in Microdroplets a Ultrahigh Throughput

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Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering

Kristin J. Adolfsen et al.

Summary: In this study, a more potent EcN-based PKU strain was developed through the optimization of whole cell PAL activity, showcasing approximately two-fold increase in in vivo PAL activity compared to the previous strain. The biosensor-based ultra-high-throughput screening approach utilized in this study could potentially lead to improved in vivo performance for PKU treatment in the future.

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Computational Enzyme Stabilization Can Affect Folding Energy Landscapes and Lead to Catalytically Enhanced Domain-Swapped Dimers

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Summary: Enzyme functionality depends on its unique three-dimensional structure, with computational algorithms being used to stabilize proteins for research and biotech applications. In a unique case, introducing eleven stabilizing mutations affected the protein folding energy landscape and resulted in advantageous catalytic domain-swapped dimers.

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Exploring mechanism of enzyme catalysis by on-chip transient kinetics coupled with global data analysis and molecular modeling

David Hess et al.

Summary: The development of a droplet-based microfluidic platform has enabled high-throughput acquisition of transient kinetic data for enzymes under various substrate concentrations and temperatures. This platform significantly reduces assay volumes and increases throughput when compared to conventional methods. Through studying model enzymes and engineered variants, new insights into enzyme design and applications have been gained, complemented by molecular dynamics simulations.
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Ancestral lysosomal enzymes with increased activity harbor therapeutic potential for treatment of Hunter syndrome

Natalie M. Hendrikse et al.

Summary: Ancestral sequence reconstruction can enhance the activity of iduronate-2-sulfatase, potentially improving treatment outcomes for Hunter syndrome. Ancestral variants showed up to 2-fold higher activity than human IDS in vitro and could offer a more effective therapeutic effect, reducing treatment burden.

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D3DistalMutation: a Database to Explore the Effect of Distal Mutations on Enzyme Activity

Xiaoyu Wang et al.

Summary: Enzyme activity can be significantly affected by distal mutations, with the majority of mutations decreasing or abolishing enzyme activity. Only a small percentage of distal mutations may increase enzyme activity, particularly Y to F, S to D, and T to D mutations. Understanding the impact of distal mutations on enzyme activity is crucial for both industrial catalysis and allosteric drug design.

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Kai Blin et al.

Summary: antiSMASH, a tool for detecting biosynthetic gene clusters in microorganisms, has been updated to version 6, which increases the supported cluster types, displays the structure of multi-modular BGCs, adds a new comparison algorithm, allows integration of results from other prediction tools, and improves detection of tailoring enzymes in RiPP clusters.

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Computationally designed pyocyanin demethylase acts synergistically with tobramycin to kill recalcitrant Pseudomonas aeruginosa biofilms

Chelsey M. VanDrisse et al.

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Lorea Alejaldre et al.

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Yang Yang et al.

Summary: Genetic variations have diverse effects on proteins, particularly affecting protein-solvent interactions, aggregation, or solubility. The developed prediction method, PON-Sol2, utilizes a machine learning tool with gradient boosting algorithm to accurately identify amino acid substitutions that impact protein solubility. The method shows superior performance in comparison to previous methods and is freely available for predicting solubility effects of variants in any organism, even in large-scale projects.

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