Structural stability of an enzyme biocatalyst

Dalby, Paul A.; Aucamp, Jean P.; George, Roger; Martinez-Torres, R.J.

doi:10.1042/bst0351606

Cited by 7 publications

(5 citation statements)

References 40 publications

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“…Surprisingly, heating the enzyme to 40-55 °C for 1 hour, and re-cooling, increased its activity by up to 3-fold [17]. The deactivation and aggregation of E. coli TK at extreme pH, high temperature, and in the presence of co-solvents is strongly linked to the binding of cofactors and formation of the two cofactor-binding loops at residues 185-192 and 382-392 (E. coli numbering) [17][18][19].…”

Section: Introductionmentioning

confidence: 99%

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase

Morris

Rios‐Solis

García-Arrazola

et al. 2016

Enzyme and Microbial Technology

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Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185-192 and 382-392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60°C and 65°C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55°C or 60°C for 1h. Interestingly, H192P was also 2-times more active than wild-type TK at 25°C. Optimal activity was achieved at 60°C for H192P compared to 55°C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.

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Section: Introductionmentioning

confidence: 99%

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase

Morris

Rios‐Solis

García-Arrazola

et al. 2016

Enzyme and Microbial Technology

Self Cite

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“…In addition, reduced gene expression levels of acat2 (which protects hepatocytes from excess cholesterol [99]), as well as of ALDH9a1a and ALDH2b (aldehydes are irreversibly converted into acids in the liver, and large increases in aldehydes may result in enzyme inactivation and DNA damage, among other things [100,101]), and echs1 (enoyl-coa hydratase 1 is an important gene in fatty acid metabolism [102]) further confirmed that MP exposure can affect glucose, lipid, and amino acid metabolism. In addition, it provides substantial information regarding MPS-induced metabolic disorders in aquatic animals [47].…”

Section: Toxicological Mechanisms Of Mnps Causing Metabolic Disordersmentioning

confidence: 99%

Advances in the Utilization of Zebrafish for Assessing and Understanding the Mechanisms of Nano-/Microparticles Toxicity in Water

Lei

Zhang

et al. 2023

Toxics

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A large amount of nano-/microparticles (MNPs) are released into water, not only causing severe water pollution, but also negatively affecting organisms. Therefore, it is crucial to evaluate MNP toxicity and mechanisms in water. There is a significant degree of similarity between the genes, the central nervous system, the liver, the kidney, and the intestines of zebrafish and the human body. It has been shown that zebrafish are exceptionally suitable for evaluating the toxicity and action mechanisms of MNPs in water on reproduction, the central nervous system, and metabolism. Providing ideas and methods for studying MNP toxicity, this article discusses the toxicity and mechanisms of MNPs from zebrafish.

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“…A number of structural features are thought to confer thermostability to proteins and these include higher number and clustering of salt bridges, the shortening of surface loops and an increase in hydrophobicity at domain and monomer interfaces (Littlechild et al, 2007(Littlechild et al, , 2013. Previous studies have shown that the TPP cofactor binding plays an important role in preventing deactivation and aggregation of the EcTK at extreme pH, temperature and in the presence of organic solvents (Dalby et al, 2007;Martinez-Torres et al, 2007;Jahromi et al, 2011). This TPP binding is controlled by the formation of two cofactor binding loops containing residues 185-192 and 382-392 (EcTK numbering which correspond to 178-185 and 311-321 in ChTK-F numbering).…”

Section: Structural Basis For Thermostabilitymentioning

confidence: 99%

A ‘Split-Gene’ Transketolase From the Hyper-Thermophilic Bacterium Carboxydothermus hydrogenoformans: Structure and Biochemical Characterization

et al. 2020

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A novel transketolase has been reconstituted from two separate polypeptide chains encoded by a 'split-gene' identified in the genome of the hyperthermophilic bacterium, Carboxydothermus hydrogenoformans. The reconstituted active α 2 β 2 tetrameric enzyme has been biochemically characterized and its activity has been determined using a range of aldehydes including glycolaldehyde, phenylacetaldehyde and cyclohexanecarboxaldehyde as the ketol acceptor and hydroxypyruvate as the donor. This reaction proceeds to near 100% completion due to the release of the product carbon dioxide and can be used for the synthesis of a range of sugars of interest to the pharmaceutical industry. This novel reconstituted transketolase is thermally stable with no loss of activity after incubation for 1 h at 70 • C and is stable after 1 h incubation with 50% of the organic solvents methanol, ethanol, isopropanol, DMSO, acetonitrile and acetone. The X-ray structure of the holo reconstituted α 2 β 2 tetrameric transketolase has been determined to 1.4 Å resolution. In addition, the structure of an inactive tetrameric β 4 protein has been determined to 1.9 Å resolution. The structure of the active reconstituted α 2 β 2 enzyme has been compared to the structures of related enzymes; the E1 component of the pyruvate dehydrogenase complex and D-xylulose-5phosphate synthase, in an attempt to rationalize differences in structure and substrate specificity between these enzymes. This is the first example of a reconstituted 'splitgene' transketolase to be biochemically and structurally characterized allowing its potential for industrial biocatalysis to be evaluated.

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Structural stability of an enzyme biocatalyst

Cited by 7 publications

References 40 publications

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase

Advances in the Utilization of Zebrafish for Assessing and Understanding the Mechanisms of Nano-/Microparticles Toxicity in Water

A ‘Split-Gene’ Transketolase From the Hyper-Thermophilic Bacterium Carboxydothermus hydrogenoformans: Structure and Biochemical Characterization

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