scholarly journals Fine‐tuning the activity and stability of an evolved enzyme active‐site through noncanonical amino‐acids

FEBS Journal ◽  
2020 ◽  
Author(s):  
Henry C. Wilkinson ◽  
Paul A. Dalby
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Fine Tuning ◽  
Noncanonical Amino Acids ◽  
Enzyme Active Site

Active amino acids of the Kell blood group protein and model of the ectodomain based on the structure of neutral endopeptidase 24.11

Blood ◽  
2003 ◽  
Vol 102 (8) ◽  
pp. 3028-3034 ◽  
Author(s):  
Soohee Lee ◽  
Asim K. Debnath ◽  
Colvin M. Redman
Keyword(s):  
Amino Acids ◽  
Blood Group ◽  
Active Site ◽  
Neutral Endopeptidase ◽  
Site Directed Mutagenesis ◽  
Peptide Hydrolysis ◽  
Enzyme Active Site ◽  
Endopeptidase 24.11 ◽  
Outer Domain ◽  
Group Protein

Abstract In addition to its importance in transfusion, Kell protein is a member of the M13 family of zinc endopeptidases and functions as an endothelin-3–converting enzyme. To obtain information on the structure of Kell protein we built a model based on the crystal structure of the ectodomain of neutral endopeptidase 24.11 (NEP). Similar to NEP, the Kell protein has 2 globular domains consisting mostly of α-helical segments. The domain situated closest to the membrane contains both the N- and C-terminal sequences and the enzyme-active site. The outer domain contains all of the amino acids whose substitutions lead to different Kell blood group phenotypes. In the model, the zinc peptidase inhibitor, phosphoramidon, was docked in the active site. Site-directed mutagenesis of amino acids in the active site was performed and the enzymatic activities of expressed mutant Kell proteins analyzed and compared with NEP. Our studies indicate that Kell and NEP use the same homologous amino acids in the coordination of zinc and in peptide hydrolysis. However, Kell uses different amino acids than NEP in substrate binding and appears to have more flexibility in the composition of amino acids allowed in the active site.

Improving Nature's Enzyme Active Site with Genetically Encoded Unnatural Amino Acids

2006 ◽  
Vol 128 (34) ◽  
pp. 11124-11127 ◽  
Author(s):  
Jennifer C. Jackson ◽  
Sean P. Duffy ◽  
Kenneth R. Hess ◽  
Ryan A. Mehl
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Unnatural Amino Acids ◽  
Enzyme Active Site

scholarly journals Enhancement of Activity and Thermostability of Keratinase From Pseudomonas aeruginosa CCTCC AB2013184 by Directed Evolution With Noncanonical Amino Acids

2021 ◽  
Vol 9 ◽  
Author(s):  
Xianchao Pan ◽  
Jian Yang ◽  
Peijuan Xie ◽  
Jing Zhang ◽  
Famin Ke ◽  
...  
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Local Structure ◽  
Noncanonical Amino Acids ◽  
Practical Applications ◽  
Enhanced Activity ◽  
Reducing Conditions ◽  
Genetic Code Expansion ◽  
New Interactions

A keratinase from Pseudomonas aeruginosa (KerPA), which belongs to the M4 family of metallopeptidases, was characterised in this study. This enzyme was engineered with non-canonical amino acids (ncAAs) using genetic code expansion. Several variants with enhanced activity and thermostability were identified and the most prominent, Y21pBpF/Y70pBpF/Y114pBpF, showed an increase in enzyme activity and half-life of approximately 1.3-fold and 8.2-fold, respectively. Considering that keratinases usually require reducing agents to efficiently degrade keratin, the Y21pBpF/Y70pBpF/Y114pBpF variant with enhanced activity and stability under reducing conditions may have great significance for practical applications. Molecular Dynamics (MD) was performed to identify the potential mechanisms underlying these improvements. The results showed that mutation with pBpF at specific sites of the enzyme could fill voids, form new interactions, and reshape the local structure of the active site of the enzyme.

scholarly journals Plasticity and Constraints of tRNA Aminoacylation Define Directed Evolution of Aminoacyl-tRNA Synthetases

2019 ◽  
Vol 20 (9) ◽  
pp. 2294 ◽  
Author(s):  
Ana Crnković ◽  
Oscar Vargas-Rodriguez ◽  
Dieter Söll
Keyword(s):  
Amino Acids ◽  
Directed Evolution ◽  
Active Site ◽  
Nonsense Suppression ◽  
Natural Amino Acid ◽  
Noncanonical Amino Acids ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Genetic Incorporation

Genetic incorporation of noncanonical amino acids (ncAAs) has become a powerful tool to enhance existing functions or introduce new ones into proteins through expanded chemistry. This technology relies on the process of nonsense suppression, which is made possible by directing aminoacyl-tRNA synthetases (aaRSs) to attach an ncAA onto a cognate suppressor tRNA. However, different mechanisms govern aaRS specificity toward its natural amino acid (AA) substrate and hinder the engineering of aaRSs for applications beyond the incorporation of a single l-α-AA. Directed evolution of aaRSs therefore faces two interlinked challenges: the removal of the affinity for cognate AA and improvement of ncAA acylation. Here we review aspects of AA recognition that directly influence the feasibility and success of aaRS engineering toward d- and β-AAs incorporation into proteins in vivo. Emerging directed evolution methods are described and evaluated on the basis of aaRS active site plasticity and its inherent constraints.

scholarly journals An enzymatic activation of formaldehyde for nucleotide methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charles Bou-Nader ◽  
Frederick W. Stull ◽  
Ludovic Pecqueur ◽  
Philippe Simon ◽  
Vincent Guérineau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Crystallographic Structure ◽  
De Novo Synthesis ◽  
Active Site Residues ◽  
Enzymatic Activation ◽  
Enzyme Cofactors ◽  
Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

Noncanonical Amino Acids for Hypoxia-Responsive Peptide Self-Assembly and Fluorescence

2021 ◽  
Author(s):  
Binbin Hu ◽  
Na Song ◽  
Yawei Cao ◽  
Mingming Li ◽  
Xin Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Self Assembly ◽  
Noncanonical Amino Acids

scholarly journals Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1679
Author(s):  
Vishnu Mohan ◽  
Jean P. Gaffney ◽  
Inna Solomonov ◽  
Maxim Levin ◽  
Mordehay Klepfish ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Drug Targets ◽  
Fas Ligand ◽  
Specific Activity ◽  
Matrix Metalloproteases ◽  
Ductal Adenocarcinoma ◽  
Enzyme Active Site ◽  
Induced Apoptosis

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7′s enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

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