scholarly journals Dissecting the evolvability landscape of the CalB active site toward aromatic substrates

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yossef López de los Santos ◽  
Ying Lian Chew-Fajardo ◽  
Guillaume Brault ◽  
Nicolas Doucet
Keyword(s):  
Active Site ◽  
Rational Design ◽  
Interaction Network ◽  
Saturation Mutagenesis ◽  
Rational Approach ◽  
Library Size ◽  
Aromatic Substrates ◽  
Enzyme Substrate ◽  
Residue Interaction ◽  
Enzyme Recognition

Abstract A key event in the directed evolution of enzymes is the systematic use of mutagenesis and selection, a process that can give rise to mutant libraries containing millions of protein variants. To this day, the functional analysis and identification of active variants among such high numbers of mutational possibilities is not a trivial task. Here, we describe a combinatorial semi-rational approach to partly overcome this challenge and help design smaller and smarter mutant libraries. By adapting a liquid medium transesterification assay in organic solvent conditions with a combination of virtual docking, iterative saturation mutagenesis, and residue interaction network (RIN) analysis, we engineered lipase B from P. antarctica (CalB) to improve enzyme recognition and activity against the bulky aromatic substrates and flavoring agents methyl cinnamate and methyl salicylate. Substrate-imprinted docking was used to target active-site positions involved in enzyme-substrate and enzyme-product complexes, in addition to identifying ‘hot spots’ most likely to yield active variants. This iterative semi-rational design strategy allowed selection of CalB variants exhibiting increased activity in just two rounds of site-saturation mutagenesis. Beneficial replacements were observed by screening only 0.308% of the theoretical library size, illustrating how semi-rational approaches with targeted diversity can quickly facilitate the discovery of improved activity variants relevant to a number of biotechnological applications.

How carbonyl reductases control stereoselectivity: Approaching the goal of rational design

2010 ◽  
Vol 82 (1) ◽  
pp. 117-128 ◽  
Author(s):  
Dunming Zhu ◽  
Ling Hua
Keyword(s):  
Rational Design ◽  
Reaction Medium ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Rational Approach ◽  
Reaction Conditions ◽  
In Silico Docking ◽  
Enzyme Substrate ◽  
Hydrophobic Binding ◽  
Binding Pockets

Although "Prelog’s rule" and "two hydrophobic binding pockets" model have been used to predict and explain the stereoselectivity of enzymatic ketone reduction, the molecular basis of stereorecognition by carbonyl reductases has not been well understood. The stereoselectivity is not only determined by the structures of enzymes and substrates, but also affected by the reaction conditions such as temperature and reaction medium. Structural analysis coupled with site-directed mutagenesis of stereocomplementary carbonyl reductases readily reveals the key elements of controlling stereoselectivity in these enzymes. In our studies, enzyme-substrate docking and molecular modeling have been engaged to understand the enantioselectivity diversity of the carbonyl reductase from Sporobolomyces salmonicolor (SSCR), and to guide site-saturation mutagenesis for altering the enantioselectivity of this enzyme. These studies provide valuable information for our understanding of how the residues involved in substrate binding affect the orientation of bound substrate, and thus control the reaction stereoselectivity. The in silico docking-guided semi-rational approach should be a useful methodology for discovery of new carbonyl reductases.

Engineering of ωTransaminase for Effective Production of Chiral Amines

2020 ◽  
Vol 17 (6) ◽  
pp. 2827-2832
Author(s):  
Mitra Mirzaei ◽  
Per Berglund
Keyword(s):  
Active Site ◽  
Rational Design ◽  
Enzyme Stability ◽  
Building Blocks ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Rational Approach ◽  
Chiral Amines ◽  
Enzyme Specificity ◽  
Screening Assay

ωTransaminases are pyridoxal-5-phosphat (PLP) dependent enzymes having the ability to catalyze the transference of an amino group to a keto compound. These enzymes are used for production of chiral amines which are important building blocks in pharmaceutical industry. There is often a need to improve enzyme properties such as enzyme stability, enzyme specificity and to decrease substrate-product inhibition. Here, protein engineering was applied to improve the enzyme activity of the enzyme from Chromobacterium violaceum Rational-design and site-directed mutagenesis were applied on position of (W60) in the active site of the enzyme. Different mutated enzyme variants such as W60H, W60F and W60Y were made. Also, the enantiopreference of the wild type enzyme was reversed to produce (R)-chiral amines. For this aim, a screening assay was followed by semi-rational approach and saturation mutagenesis in the active site of the enzyme. Creating the mutated enzyme libraries resulted to obtaining two enzyme variants. Their properties were low enantiopreference towards formations of (R)-enantiopreference and low specific constant ratio between fast and slow enantiomers (Evalue around one).

Crystallographic analysis of 1,2,3-trichloropropane biodegradation by the haloalkane dehalogenase DhaA31

2014 ◽  
Vol 70 (2) ◽  
pp. 209-217 ◽  
Author(s):  
Maryna Lahoda ◽  
Jeroen R. Mesters ◽  
Alena Stsiapanava ◽  
Radka Chaloupkova ◽  
Michal Kuty ◽  
...  
Keyword(s):  
Active Site ◽  
Rational Design ◽  
Chloride Ions ◽  
Saturation Mutagenesis ◽  
Hydrolytic Cleavage ◽  
Wild Type ◽  
Haloalkane Dehalogenase ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Resolution Structure

Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon–halogen bonds, which is a key step in the aerobic mineralization of many environmental pollutants. One important pollutant is the toxic and anthropogenic compound 1,2,3-trichloropropane (TCP). Rational design was combined with saturation mutagenesis to obtain the haloalkane dehalogenase variant DhaA31, which displays an increased catalytic activity towards TCP. Here, the 1.31 Å resolution crystal structure of substrate-free DhaA31, the 1.26 Å resolution structure of DhaA31 in complex with TCP and the 1.95 Å resolution structure of wild-type DhaA are reported. Crystals of the enzyme–substrate complex were successfully obtained by adding volatile TCP to the reservoir after crystallization at pH 6.5 and room temperature. Comparison of the substrate-free structure with that of the DhaA31 enzyme–substrate complex reveals that the nucleophilic Asp106 changes its conformation from an inactive to an active state during the catalytic cycle. The positions of three chloride ions found inside the active site of the enzyme indicate a possible pathway for halide release from the active site through the main tunnel. Comparison of the DhaA31 variant with wild-type DhaA revealed that the introduced substitutions reduce the volume and the solvent-accessibility of the active-site pocket.

scholarly journals Towards a cheminformatic design for quantum mechanical enzyme models: the case of catechol-O-methyltransferase

2020 ◽  
Author(s):  
Thomas Summers ◽  
Qianyi Cheng ◽  
Manuel Palma ◽  
Diem-Trang Pham ◽  
Dudley Kelso III ◽  
...  
Keyword(s):  
Active Site ◽  
Ad Hoc ◽  
Interaction Network ◽  
Quantum Mechanical ◽  
Model Design ◽  
Residue Contact ◽  
Enzyme Models ◽  
Residue Interaction ◽  
Adenosyl Methionine ◽  
Automate Construction

The efficiency, accuracy, and replicability of enzyme simulations is often hampered by ad hoc model design. To address this problem, we have developed the Residue Interaction Network ResidUe Selector (RINRUS) toolkit. RINRUS utilizes residue contact networks to automate construction of rational quantum mechanical cluster models. This work examines this problem by computing the reaction kinetics and thermodynamics for 508 models of the active site of catechol-o-methyltransferase, an enzyme which catalyzes the methyl transfer from S-adenosyl methionine cofactor to catechol substrates. Our results demonstrate using RINRUS to rationally design small and accurate active site models.<br>

scholarly journals Rational, Reproducible, and Rigorous Computational Enzymology: The Case of Catechol-O-Methyltransferase

2020 ◽  
Author(s):  
Thomas Summers ◽  
Qianyi Cheng ◽  
Manuel Palma ◽  
Diem-Trang Pham ◽  
Dudley Kelso III ◽  
...  
Keyword(s):  
Active Site ◽  
Ad Hoc ◽  
Interaction Network ◽  
Quantum Mechanical ◽  
Model Design ◽  
Residue Contact ◽  
Residue Interaction ◽  
Adenosyl Methionine ◽  
Computational Enzymology ◽  
Automate Construction

The efficiency, accuracy, and replicability of enzyme simulations is often hampered by ad hoc model design. To address this problem, we have developed the Residue Interaction Network ResidUe Selector (RINRUS) toolkit. RINRUS utilizes residue contact networks to automate construction of rational quantum mechanical cluster models. This work examines this problem by computing the reaction kinetics and thermodynamics for 508 models of the active site of catechol-o-methyltransferase, an enzyme which catalyzes the methyl transfer from S-adenosyl methionine cofactor to catechol substrates. Our results demonstrate using RINRUS to rationally design small and accurate active site models.<br>

scholarly journals Towards a cheminformatic design for quantum mechanical enzyme models: the case of catechol-O-methyltransferase

2020 ◽  
Author(s):  
Thomas Summers ◽  
Qianyi Cheng ◽  
Manuel Palma ◽  
Diem-Trang Pham ◽  
Dudley Kelso III ◽  
...  
Keyword(s):  
Active Site ◽  
Ad Hoc ◽  
Interaction Network ◽  
Quantum Mechanical ◽  
Model Design ◽  
Residue Contact ◽  
Enzyme Models ◽  
Residue Interaction ◽  
Adenosyl Methionine ◽  
Automate Construction

The efficiency, accuracy, and replicability of enzyme simulations is often hampered by ad hoc model design. To address this problem, we have developed the Residue Interaction Network ResidUe Selector (RINRUS) toolkit. RINRUS utilizes residue contact networks to automate construction of rational quantum mechanical cluster models. This work examines this problem by computing the reaction kinetics and thermodynamics for 508 models of the active site of catechol-o-methyltransferase, an enzyme which catalyzes the methyl transfer from S-adenosyl methionine cofactor to catechol substrates. Our results demonstrate using RINRUS to rationally design small and accurate active site models.<br>

Thermostability of Lipase A and Dynamic Communication Based on Residue Interaction Network

2019 ◽  
Vol 26 (9) ◽  
pp. 702-716 ◽  
Author(s):  
Qian Xia ◽  
Yanrui Ding
Keyword(s):  
Time Series ◽  
Active Site ◽  
Interaction Network ◽  
Signal Propagation ◽  
Secondary Structures ◽  
Tight Contact ◽  
Residue Interaction ◽  
Spatio Temporal ◽  
The Relationship ◽  
310 Helix

Objective: Dynamic communication caused by mutation affects protein stability. The main objective of this study is to explore how mutations affect communication and to provide further insight into the relationship between heat resistance and signal propagation of Bacillus subtilis lipase (Lip A). Methods: The relationship between dynamic communication and Lip A thermostability is studied by long-time MD simulation and residue interaction network. The Dijkstra algorithm is used to get the shortest path of each residue pair. Subsequently, time-series frequent paths and spatio-temporal frequent paths are mined through an Apriori-like algorithm. Results: Time-series frequent paths show that the communication between residue pairs, both in wild-type lipase (WTL) and mutant 6B, becomes chaotic with an increase in temperature; however, more residues in 6B can maintain stable communication at high temperature, which may be associated with the structural rigidity. Furthermore, spatio-temporal frequent paths reflect the interactions among secondary structures. For WTL at 300K, β7, αC, αB, the longest loop, αA and αF contact frequently. The 310-helix between β3 and αA is penetrated by spatio-temporal frequent paths. At 400K, only αC can be frequently transmitted. For 6B, when at 300K, αA and αF are in more tight contact by spatio-temporal frequent paths though I157M and N166Y. Moreover, the rigidity of the active site His156 and the C-terminal of Lip A are increased, as reflected by the spatio-temporal frequent paths. At 400K, αA and αF, 310-helix between β3 and αA, the longest loop, and the loop where the active site Asp133 is located can still maintain stable communication. Conclusion: From the perspective of residue dynamic communication, it is obviously found that mutations cause changes in interactions between secondary structures and enhance the rigidity of the structure, contributing to the thermal stability and functional activity of 6B.

scholarly journals Key Residues for Controlling Enantioselectivity of Halohydrin Dehalogenase from Arthrobacter sp. Strain AD2, Revealed by Structure-Guided Directed Evolution

2012 ◽  
Vol 78 (8) ◽  
pp. 2631-2637 ◽  
Author(s):  
Lixia Tang ◽  
Xuechen Zhu ◽  
Huayu Zheng ◽  
Rongxiang Jiang ◽  
Maja Majerić Elenkov
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Saturation Mutagenesis ◽  
Active Site Residues ◽  
Agrobacterium Radiobacter ◽  
Content Type ◽  
Halohydrin Dehalogenase ◽  
Aromatic Substrates ◽  
Key Residues ◽  
Selection Of

ABSTRACTHalohydrin dehalogenase fromAgrobacterium radiobacterAD1 (HheC) is a valuable tool in the preparation ofRenantiomers of epoxides and β-substituted alcohols. In contrast, the halohydrin dehalogenase fromArthrobactersp. AD2 (HheA) shows a lowSenantioselectivity toward most aromatic substrates. Here, three amino acids (V136, L141, and N178) located in the two neighboring active-site loops of HheA were proposed to be the key residues for controlling enantioselectivity. They were subjected to saturation mutagenesis aimed at evolving anS-selective enzyme. This led to the selection of two outstanding mutants (the V136Y/L141G and N178A mutants). The double mutant displayed an inverted enantioselectivity (fromSenantioselectivity [ES] = 1.7 toRenantioselectivity [ER] = 13) toward 2-chloro-1-phenylethanol without compromising enzyme activity. Strikingly, the N178A mutant showed a large enantioselectivity improvement (ES> 200) and a 5- to 6-fold-enhanced specific activity toward (S)-2-chloro-1-phenylethanol. Further analysis revealed that those mutations produced some interference for the binding of nonfavored enantiomers which could account for the observed enantioselectivities. Our work demonstrated that those three active-site residues are indeed crucial in modulating the enantioselectivity of HheA and that a semirational design strategy has great potential for rapid creation of novel industrial biocatalysts.

scholarly journals Next Step in Gene Delivery: Modern Approaches and Further Perspectives of AAV Tropism Modification

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 750
Author(s):  
Maxim A. Korneyenkov ◽  
Andrey A. Zamyatnin
Keyword(s):  
Gene Therapy ◽  
Genome Organization ◽  
Rational Design ◽  
Structural Data ◽  
Rational Approach ◽  
Tissue Tropism ◽  
Adeno Associated Virus ◽  
Chemical Conjugation ◽  
The Usa ◽  
Therapy Delivery

Today, adeno-associated virus (AAV) is an extremely popular choice for gene therapy delivery. The safety profile and simplicity of the genome organization are the decisive advantages which allow us to claim that AAV is currently among the most promising vectors. Several drugs based on AAV have been approved in the USA and Europe, but AAV serotypes’ unspecific tissue tropism is still a serious limitation. In recent decades, several techniques have been developed to overcome this barrier, such as the rational design, directed evolution and chemical conjugation of targeting molecules with a capsid. Today, all of the abovementioned approaches confer the possibility to produce AAV capsids with tailored tropism, but recent data indicate that a better understanding of AAV biology and the growth of structural data may theoretically constitute a rational approach to most effectively produce highly selective and targeted AAV capsids. However, while we are still far from this goal, other approaches are still in play, despite their drawbacks and limitations.

scholarly journals Ketoamide Resistance and Hepatitis C Virus Fitness in Val55 Variants of the NS3 Serine Protease

2012 ◽  
Vol 56 (4) ◽  
pp. 1907-1915 ◽  
Author(s):  
Christoph Welsch ◽  
Sabine Schweizer ◽  
Tetsuro Shimakami ◽  
Francisco S. Domingues ◽  
Seungtaek Kim ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Molecular Mechanisms ◽  
Interaction Network ◽  
Rna Replication ◽  
Viral Fitness ◽  
Dynamics Simulation ◽  
Structural Flexibility ◽  
Residue Interaction

ABSTRACTDrug-resistant viral variants are a major issue in the use of direct-acting antiviral agents in chronic hepatitis C. Ketoamides are potent inhibitors of the NS3 protease, with V55A identified as mutation associated with resistance to boceprevir. Underlying molecular mechanisms are only partially understood. We applied a comprehensive sequence analysis to characterize the natural variability at Val55 within dominant worldwide patient strains. A residue-interaction network and molecular dynamics simulation were applied to identify mechanisms for ketoamide resistance and viral fitness in Val55 variants. An infectious H77S.3 cell culture system was used for variant phenotype characterization. We measured antiviral 50% effective concentration (EC50) and fold changes, as well as RNA replication and infectious virus yields from viral RNAs containing variants. Val55 was found highly conserved throughout all hepatitis C virus (HCV) genotypes. The conservative V55A and V55I variants were identified from HCV genotype 1a strains with no variants in genotype 1b. Topology measures from a residue-interaction network of the protease structure suggest a potential Val55 key role for modulation of molecular changes in the protease ligand-binding site. Molecular dynamics showed variants with constricted binding pockets and a loss of H-bonded interactions upon boceprevir binding to the variant proteases. These effects might explain low-level boceprevir resistance in the V55A variant, as well as the Val55 variant, reduced RNA replication capacity. Higher structural flexibility was found in the wild-type protease, whereas variants showed lower flexibility. Reduced structural flexibility could impact the Val55 variant's ability to adapt for NS3 domain-domain interaction and might explain the virus yield drop observed in variant strains.

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