Leveraging intrinsic flexibility to engineer enhanced enzyme catalytic activity

Karamitros, Christos S.; Murray, Kyle; Winemiller, Brent; Lamb, Candice; Stone, Everett; D’Arcy, Sheena; Johnson, Kenneth A.; Georgiou, George

doi:10.1073/pnas.2118979119

Cited by 25 publications

(11 citation statements)

References 78 publications

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“…84 On the other hand, mutations further from the binding site can have ever smaller e ects, 85,86 enabling ne-tuning of mechanics (Fig. 3), [87][88][89][90][91][92] and structure. 93,94 For example, in a separate recent study of ours, we studied the e ect of single mutations on structure (using proteins in the protein data bank, and AlphaFold), nding that structural perturbations are felt up to 2 nm away from the mutated residue.…”

Section: Discussionmentioning

confidence: 99%

General theory of specific binding: insights from a genetic-mechano-chemical protein model

McBride

Eckmann

Tlusty³

2022

Preprint

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Proteins need to selectively interact with specific targets among a multitude of similar molecules in the cell. Despite a firm physical understanding of binding interactions, we lack a general theory of how proteins evolve high specificity. Here, we present a model that combines chemistry, mechanics and genetics, and explains how their interplay governs the evolution of specific protein-ligand interactions. The model shows that there are many routes to achieving discrimination -- by varying degrees of flexibility and shape/chemistry complementarity -- but the key ingredient is precision. Harder discrimination tasks require more collective and precise coaction of structure, forces and movements. Proteins can achieve this through correlated mutations extending far from a binding site, which fine tune the localized interaction with the ligand. Thus, the solution of more complicated tasks is aided by increasing the protein, and proteins become more evolvable and robust when they are larger than the bare minimum required for discrimination. Our model makes testable, specific predictions about the role of flexibility in discrimination, and how to independently tune affinity and specificity. Thus, the proposed theory of molecular discrimination addresses the natural question ``why are proteins so big?'' A possible answer is that molecular discrimination is often a hard task best performed by adding more layers to the protein.

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Section: Discussionmentioning

confidence: 99%

General theory of specific binding: insights from a genetic-mechano-chemical protein model

McBride

Eckmann

Tlusty³

2022

Preprint

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“…Interestingly, it has been observed that the mutagenesis of residues located at flexible regions remote from the active center could also significantly improve the enzyme catalytic efficiency. Recently, such a method was applied to engineer Homo sapiens kynureninase (HsKYNase) for increasing its catalytic activity towards a non-preferred substrate, L-kynurenine (KYN) ( Karamitros et al, 2022 ). B-factor analysis, hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) and MD simulations were first applied to predict the highly flexible regions distal to the active center ( Figures 5A–C – C ).…”

Section: Rational Design Strategy Based On Dynamics Modificationmentioning

confidence: 99%

“…Further analysis by HDX-MS and MD simulations revealed that the distal mutations in BF-HsKYNase allosterically influenced the flexibility of the cofactor pyridoxal-5′-phosphate (PLP) binding pocket. This probably altered the conformational ensemble and led to sampling states more favorable to the catalyzed reaction, thereby affecting the reaction rate ( Karamitros et al, 2022 ). Additionally, a conformational dynamics-guided loop engineering strategy was employed to improve activity of an alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) towards a non-native substrate ( Liu et al, 2019 ).…”

Section: Rational Design Strategy Based On Dynamics Modificationmentioning

confidence: 99%

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Rational design of enzyme activity and enantioselectivity

Song

Zhang

Wu³

et al. 2023

Front. Bioeng. Biotechnol.

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The strategy of rational design to engineer enzymes is to predict the potential mutants based on the understanding of the relationships between protein structure and function, and subsequently introduce the mutations using the site-directed mutagenesis. Rational design methods are universal, relatively fast and have the potential to be developed into algorithms that can quantitatively predict the performance of the designed sequences. Compared to the protein stability, it was more challenging to design an enzyme with improved activity or selectivity, due to the complexity of enzyme molecular structure and inadequate understanding of the relationships between enzyme structures and functions. However, with the development of computational force, advanced algorithm and a deeper understanding of enzyme catalytic mechanisms, rational design could significantly simplify the process of engineering enzyme functions and the number of studies applying rational design strategy has been increasing. Here, we reviewed the recent advances of applying the rational design strategy to engineer enzyme functions including activity and enantioselectivity. Five strategies including multiple sequence alignment, strategy based on steric hindrance, strategy based on remodeling interaction network, strategy based on dynamics modification and computational protein design are discussed and the successful cases using these strategies are introduced.

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“…Georgiou and colleagues have increased the activity of Homo sapiens KYNase 45-fold by mutagenesis of residues in flexible regions distal to the active site. 199 Scientists at Codexis have also made breakthroughs in the development of an engineered phenylalanine lyase (CDX-6114) that is tolerant to the low pH conditions of the stomach and resistant to intestinal proteases, making it an oral enzyme candidate for the potential treatment of phenylketonuria (NCT04085666). In addition, enzymes with novel activities have been created through protein engineering.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

The Biocatalysis in Cancer Therapy

et al. 2023

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Biocatalysis can be defined as the use of isolated enzymes or enzymes inside cells to convert substrates into products. The term “biocatalysis” is often used in modern organic synthesis in connection with the environmentally friendly manufacture of chemicals and pharmaceuticals, where biocatalysis has achieved great success in recent years. Here, we review the potential use of biocatalysis in cancer therapy. We summarize the common biocatalytic reactions for activation of anticancer prodrugs, generation of excessive reactive oxygen species (ROS), and deletion of essential/immunosuppressive components in cancer therapy. We also describe the biocatalytic release of small molecule drugs from antibody–drug conjugates (ADCs) and the application of biocatalysis in living cells for cancer treatment. The diverse application of biocatalysis is an exciting and ongoing strategy that holds great promise for cancer therapy.

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Leveraging intrinsic flexibility to engineer enhanced enzyme catalytic activity

Cited by 25 publications

References 78 publications

General theory of specific binding: insights from a genetic-mechano-chemical protein model

General theory of specific binding: insights from a genetic-mechano-chemical protein model

Rational design of enzyme activity and enantioselectivity

The Biocatalysis in Cancer Therapy

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