Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase

Unspecific peroxygenases (UPOs) enable oxyfunctionalisations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed a protein engineering in S. cerevisiae with the novel MthUPO. Experimental approaches were combined with computational modelling resulting in the screening of more than 5,300 transformants. This protein engineering led to a significant reshaping of the active site as elucidated by molecular dynamics. The kcat/Km was improved by 16.5-fold. Variants were identified with high chemo- and regioselectivities in the oxyfunctionalisation of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with excellent enantioselectivities of 95 % ee. Additionally, the first reported effective exchange of the conserved catalytic Glu residue was observed.

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Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one)

Applied and Environmental Microbiology ◽

10.1128/aem.01780-17 ◽

2017 ◽

Vol 84 (1) ◽

Cited By ~ 9

Author(s):

Shuaihua Gao ◽

Shaozhou Zhu ◽

Rong Huang ◽

Hongxia Li ◽

Hao Wang ◽

...

Keyword(s):

Molecular Dynamics ◽

Protein Engineering ◽

Root Mean Square ◽

Active Site ◽

Kinetic Resolution ◽

Mean Square ◽

Content Type ◽

Enantiomerically Pure ◽

Mean Square Fluctuation ◽

Microbacterium Hydrocarbonoxydans

ABSTRACT To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (−)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (−)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and thermostability) of a γ-lactamase identified from Microbacterium hydrocarbonoxydans were tackled using a semirational design. The protein engineering was initialized by combinatorial active-site saturation test to improve the enantioselectivity. At the same time, two strategies were applied to identify mutation candidates to enhance the thermostability based on calculations from both a static (B-FITTER based on the crystal structure) and a dynamic (root mean square fluctuation [RMSF] values based on molecular dynamics simulations) way. After combining the mutants, we successfully obtained the final mutants showing better properties in both properties. The engineered (+)-lactamase could be a candidate for the preparation of (−)-Vince lactam.

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Computational modelling of nanotube delivery of anti-cancer drug into glutathione reductase enzyme

Scientific Reports ◽

10.1038/s41598-021-84006-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Saheen Shehnaz Begum ◽

Dharitri Das ◽

Nand Kishor Gour ◽

Ramesh Chandra Deka

Keyword(s):

Molecular Dynamics ◽

Glutathione Reductase ◽

Active Site ◽

Density Functional ◽

Computational Modelling ◽

Density Functional Theory Method ◽

Docking Simulation ◽

Drug Binding ◽

Dynamics Simulation ◽

Cancer Drug

AbstractDensity functional theory method combined with docking and molecular dynamics simulations are used to understand the interaction of carmustine with human glutathione reductase enzyme. The active site of the enzyme is evaluated by docking simulation is used for molecular dynamics simulation to deliver the carmustine molecule by (5,5) single walled carbon nanotube (SWCNT). Our model of carmustine in the active site of GR gives a negative binding energy that is further refined by QM/MM study in gas phase and solvent phase to confirm the stability of the drug molecule inside the active site. Once released from SWCNT, carmustine forms multiple polar and non-polar hydrogen bonding interactions with Tyr180, Phe209, Lys318, Ala319, Leu320, Leu321, Ile350, Thr352 and Val354 in the range of 2–4 Å. The SWCNT vehicle itself is held fix at its place due to multiple pi-pi stacking, pi-amide, pi-sigma interactions with the neighboring residues. These interactions in the range of 3–5 Å are crucial in holding the nanotube outside the drug binding region, hence, making an effective delivery. This study can be extended to envisage the potential applications of computational studies in the modification of known drugs to find newer targets and designing new and improved controlled drug delivery systems.

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In Silico Molecular Dynamics Docking of Drugs to the Inhibitory Active Site of SARS-CoV-2 Protease and Their Predicted Toxicology and ADME

SSRN Electronic Journal ◽

10.2139/ssrn.3580951 ◽

2020 ◽

Author(s):

Leif Peterson

Keyword(s):

Molecular Dynamics ◽

Active Site ◽

In Silico

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WHICH IS THE PROTON-SHUTTLE IN ISOXANTHOPTERIN DEAMINASE? QM/MM MD UNDERSTANDING

Journal of Theoretical and Computational Chemistry ◽

10.1142/s0219633613410022 ◽

2013 ◽

Vol 12 (08) ◽

pp. 1341002 ◽

Cited By ~ 1

Author(s):

XIN ZHANG ◽

MING LEI

Keyword(s):

Crystal Structure ◽

Molecular Dynamics ◽

Hydrogen Bonds ◽

Proton Transfer ◽

Glutamic Acid ◽

Active Site ◽

Nucleophilic Attack ◽

Zinc Ions ◽

Initial Model ◽

Dynamics Simulations

The deamination process of isoxanthopterin catalyzed by isoxanthopterin deaminase was determined using the combined QM(PM3)/MM molecular dynamics simulations. In this paper, the updated PM3 parameters were employed for zinc ions and the initial model was built up based on the crystal structure. Proton transfer and following steps have been investigated in two paths: Asp336 and His285 serve as the proton shuttle, respectively. Our simulations showed that His285 is more effective than Aap336 in proton transfer for deamination of isoxanthopterin. As hydrogen bonds between the substrate and surrounding residues play a key role in nucleophilic attack, we suggested mutating Thr195 to glutamic acid, which could enhance the hydrogen bonds and help isoxanthopterin get close to the active site. The simulations which change the substrate to pterin 6-carboxylate also performed for comparison. Our results provide reference for understanding of the mechanism of deaminase and for enhancing the deamination rate of isoxanthopterin deaminase.

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In Silico Molecular Dynamics Docking of Drugs to the Inhibitory Active Site of SARS-CoV-2 Protease and Their Predicted Toxicology and ADME

African Journal of Pharmaceutical Sciences ◽

10.51483/afjps.1.1.2021.16-39 ◽

2021 ◽

Vol 1 (2) ◽

pp. 16

Author(s):

Leif E. Peterson

Keyword(s):

Molecular Dynamics ◽

Active Site ◽

In Silico

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Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1812204115 ◽

2018 ◽

Vol 115 (52) ◽

pp. E12192-E12200 ◽

Cited By ~ 13

Author(s):

Haoran Yu ◽

Paul A. Dalby

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Active Site ◽

Dynamic Networks ◽

Aromatic Aldehydes ◽

Active Site Residues ◽

Trade Off ◽

Highly Correlated ◽

Dynamics Simulations ◽

The Impact

The directed evolution of enzymes for improved activity or substrate specificity commonly leads to a trade-off in stability. We have identified an activity–stability trade-off and a loss in unfolding cooperativity for a variant (3M) of Escherichia coli transketolase (TK) engineered to accept aromatic substrates. Molecular dynamics simulations of 3M revealed increased flexibility in several interconnected active-site regions that also form part of the dimer interface. Mutating the newly flexible active-site residues to regain stability risked losing the new activity. We hypothesized that stabilizing mutations could be targeted to residues outside of the active site, whose dynamics were correlated with the newly flexible active-site residues. We previously stabilized WT TK by targeting mutations to highly flexible regions. These regions were much less flexible in 3M and would not have been selected a priori as targets using the same strategy based on flexibility alone. However, their dynamics were highly correlated with the newly flexible active-site regions of 3M. Introducing the previous mutations into 3M reestablished the WT level of stability and unfolding cooperativity, giving a 10.8-fold improved half-life at 55 °C, and increased midpoint and aggregation onset temperatures by 3 °C and 4.3 °C, respectively. Even the activity toward aromatic aldehydes increased up to threefold. Molecular dynamics simulations confirmed that the mutations rigidified the active-site via the correlated network. This work provides insights into the impact of rigidifying mutations within highly correlated dynamic networks that could also be useful for developing improved computational protein engineering strategies.

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