A De Novo Designed Esterase with p-Nitrophenyl Acetate Hydrolysis Activity

Li, Guanlin; Xu, Li; Zhang, Houjin; Liu, Junjun; Yan, Jinyong; Yan, Yunjun

doi:10.3390/molecules25204658

Cited by 11 publications

(22 citation statements)

References 40 publications

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“…One distance, two angles, and three dihedral restraints are necessary to completely describe the catalytic geometry between the ligand and a residue [40]. The tolerances defined in the cst file were adapted from Guanlin et al [41]. A params file of the ligand (the PC dimer shown in Figure 2b) was generated.…”

Section: Enzyme Designmentioning

confidence: 99%

De novo design of a polycarbonate hydrolase

Holst,

Madsen,

Toftgård

et al. 2023

Protein Engineering, Design and Selection

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Enzymatic degradation of plastics is currently limited to the use of engineered natural enzymes. As of yet, all engineering approaches applied to plastic degrading enzymes retain the natural $\alpha /\beta $-fold. While mutations can be used to increase thermostability, an inherent maximum likely exists for the $\alpha /\beta $-fold. It is thus of interest to introduce catalytic activity toward plastics in a different protein fold to escape the sequence space of plastic degrading enzymes. Here, a method for designing highly thermostable enzymes that can degrade plastics is described. With the help of Rosetta an active site catalysing the hydrolysis of polycarbonate is introduced into a set of thermostable scaffolds. Through computational evaluation, a potential PCase was selected and produced recombinantly in Escherichia coli. Thermal analysis suggests that the design has a melting temperature of >95$^{\circ }$C. Activity toward polycarbonate was confirmed using atomic force spectroscopy (AFM), proving the successful design of a PCase.

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Section: Enzyme Designmentioning

confidence: 99%

De novo design of a polycarbonate hydrolase

Holst,

Madsen,

Toftgård

et al. 2023

Protein Engineering, Design and Selection

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“…[9] A more recently designed esterase showed a catalytic activity 1000-times lower than commercially available esterases, but saturation kinetics were not reported. [8] A Kemp eliminase also required eight substitutions outside the active site for high catalytic efficiency, but these were not rationally predicted, but found with experimental directed evolution. [31] This inability to design efficient enzymes limits new applications of enzymes in medicines, non-polluting manufacture of fine chemicals and pharmaceuticals, food processing, and biodegradation of environmental contaminants.…”

Section: Discussionmentioning

confidence: 99%

“…Previous design of esterase activity achieved only modest catalytic efficiencies; [8][9][10][11][12] the best kcat/KM was 6,600 M -1 min -1 . [11] In contrast, the work below reports an eightsubstitution variant with excellent catalytic efficiency (kcat/KM of 120,000 M -1 min -1 ), which is two-fold higher than that for the target esterase.…”

Section: Introductionmentioning

confidence: 91%

Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase

Casadevall,

Pierce,

Guan

et al. 2023

Preprint

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Hydroxynitrile lyase from rubber tree (HbHNL) shares 45% identical amino acid residues with the homologous esterase from tobacco, SABP2, but the two enzymes catalyze different reactions. The x-ray structures reveal a serine-histidine-aspartate catalytic triad in both enzymes along with several differing amino acid residues within the active site. Previous exchange of three amino acid residues in the active site of HbHNL with the corresponding amino acid residue in SABP2 (T11G-E79H-K236M) created variant HNL3, which showed low esterase activity toward p-nitrophenyl acetate. Further structure comparison reveals additional differences surrounding the active site. HbHNL contains an improperly positioned oxyanion hole residue and differing solvation of the catalytic aspartate. We hypothesized that correcting these structural differences would impart good esterase activity on the corresponding HbHNL variant. To predict the amino acid substitutions needed to correct the structure, we calculated shortest path maps for both HbHNL and SABP2, which reveal correlated movements of amino acids in the two enzymes. Replacing four amino acid residues (C81L-N104T-V106F-G176S) whose movements are connected to the movements of the catalytic residues yielded variant HNL7TV (stabilizing substitution H103V was also added), which showed an esterase catalytic efficiency comparable to that of SABP2. The x-ray structure of an intermediate variant, HNL6V, showed an altered solvation of the catalytic aspartate and a partially corrected oxyanion hole. This dramatic increase in catalytic efficiency demonstrates the ability of shortest path maps to predict which residues outside the active site contribute to catalytic activity.

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“…When necessary, the selected sequences, once their catalytic efficiency has been measured, undergo further refinement to enhance their catalytic performance and improve properties like thermostability. These strategies have been successfully employed to generate de novo enzymes with the capacity to catalyze a range of organic reactions, including Kemp eliminase, retro-aldolase, , Diels–Alderase, esterase, ,, and luciferase . Alternatively, existing enzymes or sequences are repurposed to catalyze increasingly complex chemical reactions, ,,− including poly(ethylene terephthalate) (PET) hydrolysis. − …”

Section: De Novo Enzyme Design and Evolutionmentioning

confidence: 99%

Perspectives on Computational Enzyme Modeling: From Mechanisms to Design and Drug Development

Nam,

Shao,

Major

et al. 2024

ACS Omega

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Understanding enzyme mechanisms is essential for unraveling the complex molecular machinery of life. In this review, we survey the field of computational enzymology, highlighting key principles governing enzyme mechanisms and discussing ongoing challenges and promising advances. Over the years, computer simulations have become indispensable in the study of enzyme mechanisms, with the integration of experimental and computational exploration now established as a holistic approach to gain deep insights into enzymatic catalysis. Numerous studies have demonstrated the power of computer simulations in characterizing reaction pathways, transition states, substrate selectivity, product distribution, and dynamic conformational changes for various enzymes. Nevertheless, significant challenges remain in investigating the mechanisms of complex multistep reactions, large-scale conformational changes, and allosteric regulation. Beyond mechanistic studies, computational enzyme modeling has emerged as an essential tool for computer-aided enzyme design and the rational discovery of covalent drugs for targeted therapies. Overall, enzyme design/engineering and covalent drug development can greatly benefit from our understanding of the detailed mechanisms of enzymes, such as protein dynamics, entropy contributions, and allostery, as revealed by computational studies. Such a convergence of different research approaches is expected to continue, creating synergies in enzyme research. This review, by outlining the ever-expanding field of enzyme research, aims to provide guidance for future research directions and facilitate new developments in this important and evolving field.

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A De Novo Designed Esterase with p-Nitrophenyl Acetate Hydrolysis Activity

Cited by 11 publications

References 40 publications

De novo design of a polycarbonate hydrolase

De novo design of a polycarbonate hydrolase

Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase

Perspectives on Computational Enzyme Modeling: From Mechanisms to Design and Drug Development

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