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 Evolving Bacterial Fitness with an Expanded Genetic Code

2017 ◽  
Author(s):  
Drew S. Tack ◽  
Austin C. Cole ◽  
R. Shroff ◽  
B.R. Morrow ◽  
Andrew D. Ellington
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acid ◽  
Noncanonical Amino Acids ◽  
Bacterial Fitness ◽  
Genetic Code Expansion ◽  
Encoding Strategy ◽  
Translation Systems ◽  
Expanded Genetic Code

AbstractEvolution has for the most part used the canonical 20 amino acids of the natural genetic code to construct proteins. While several theories regarding the evolution of the genetic code have been proposed, experimental exploration of these theories has largely been restricted to phylogenetic and computational modeling. The development of orthogonal translation systems has allowed noncanonical amino acids to be inserted at will into proteins. We have taken advantage of these advances to evolve bacteria to accommodate a 21 amino acid genetic code in which the amber codon ambiguously encodes either 3-nitro-L-tyrosine or stop. Such an ambiguous encoding strategy recapitulates numerous models for genetic code expansion, and we find that evolved lineages first accommodate the unnatural amino acid, and then begin to evolve on a neutral landscape where stop codons begin to appear within genes. The resultant lines represent transitional intermediates on the way to the fixation of a functional 21 amino acid code.

scholarly journals Structure of CARB-4 and AER-1 CarbenicillinHydrolyzing β-Lactamases

1998 ◽  
Vol 42 (8) ◽  
pp. 1966-1972 ◽  
Author(s):  
François Sanschagrin ◽  
Noureddine Bejaoui ◽  
Roger C. Levesque
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Dna Sequences ◽  
Active Site ◽  
Multiple Alignment ◽  
Dna Homology ◽  
Class A ◽  
Canonical Amino Acid ◽  
Multiple Alignment Analysis ◽  
Alignment Analysis

ABSTRACT We determined the nucleotide sequences ofblaCARB-4 encoding CARB-4 and deduced a polypeptide of 288 amino acids. The gene was characterized as a variant of group 2c carbenicillin-hydrolyzing β-lactamases such as PSE-4, PSE-1, and CARB-3. The level of DNA homology between thebla genes for these β-lactamases varied from 98.7 to 99.9%, while that between these genes andblaCARB-4 encoding CARB-4 was 86.3%. TheblaCARB-4 gene was acquired from some other source because it has a G+C content of 39.1%, compared to a G+C content of 67% for typical Pseudomonas aeruginosa genes. DNA sequencing revealed that blaAER-1 shared 60.8% DNA identity with blaPSE-3 encoding PSE-3. The deduced AER-1 β-lactamase peptide was compared to class A, B, C, and D enzymes and had 57.6% identity with PSE-3, including an STHK tetrad at the active site. For CARB-4 and AER-1, conserved canonical amino acid boxes typical of class A β-lactamases were identified in a multiple alignment. Analysis of the DNA sequences flankingblaCARB-4 and blaAER-1 confirmed the importance of gene cassettes acquired via integrons inbla gene distribution.

scholarly journals Comparative Analyses of the Transcriptome and Proteome of Escherichia coli C321.△A and Further Improving Its Noncanonical Amino Acids Containing Protein Expression Ability by Integration of T7 RNA Polymerase

2021 ◽  
Vol 12 ◽  
Author(s):  
Huawei Yi ◽  
Jing Zhang ◽  
Famin Ke ◽  
Xiurong Guo ◽  
Jian Yang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Expression ◽  
Rna Polymerase ◽  
T7 Rna Polymerase ◽  
Noncanonical Amino Acids ◽  
E Coli ◽  
Growth Defects ◽  
Genetic Code Expansion ◽  
And Function

Incorporation of noncanonical amino acids (ncAAs) into proteins has been proven to be a powerful tool to manipulate protein structure and function, and to investigate many biological processes. Improving the yields of ncAA-containing proteins is of great significance in industrial-scale applications. Escherichia coli C321.ΔA was generated by the replacement of all known amber codons and the deletion of RF1 in the genome and has been proven to be an ideal host for ncAA-containing protein expression using genetic code expansion. In this study, we investigated the transcriptome and proteome profiles of this first codon reassignment strain and found that some functions and metabolic pathways were differentially expressed when compared with those of its parent strain. Genes involved in carbohydrate and energy metabolism were remarkably downregulated. Our results may provide important clues about the growth defects in E. coli C321.ΔA. Furthermore, we improved the yields of ncAA-containing proteins in E. coli C321.ΔA by integrating the T7 RNA polymerase system.

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.

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