Engineering the substrate specificity of a thermophilic penicillin acylase from thermus thermophilus
Leticia L. Torres,Ángel Cantero,Mercedes del Valle,Anabel Marina,Fernando López-Gallego,Jose M. Guisan,José Berenguer,Aurelio Hidalgo +7 more
TLDR
Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.Abstract:
A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified Through rational redesign, residues were replaced to mimic the aromatic binding site of the E coli penicillin G acylase A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G The introduction of a single phenylalanine residue in position α188, α189, or β24 improved the K(m) for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 66-fold Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active siteread more
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A review of metabolic and enzymatic engineering strategies for designing and optimizing performance of microbial cell factories.
TL;DR: Computational tools such as de novo biosynthetic pathway builders, docking, molecular dynamics and steered MD, and genome-scale metabolic flux modeling all play critical roles in the rational design of an MCF.
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Modular optimization of multi-gene pathways for fumarate production.
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Penicillin acylases revisited: importance beyond their industrial utility
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Intracellular calcium mishandling leads to cardiac dysfunction and ventricular arrhythmias in a mouse model of propionic acidemia
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CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells.
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