Activation of Anthranilate Phosphoribosyltransferase from <i>Sulfolobus solfataricus</i> by Removal of Magnesium Inhibition and Acceleration of Product Release,

Schlee, Sandra; Deuss, M.; Bruning, Marc; Ivens, Andreas; Schwab, Thomas; Hellmann, Nadja; Mayans, Olga; Sterner, Reinhard

doi:10.1021/bi802335s

Cited by 13 publications

(20 citation statements)

References 60 publications

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“…In agreement with this concept, rational protein design and directed evolution have shown that enzyme mutants with reduced stability often exhibit improved catalytic activity compared to the wild-type form, even though structural alterations are often minimal or u Nde tectable (e.g. [33], [34]). The lack of notable structural differences between the Kisumu 2B and ZAN/U 1C variants and the intrinsic dynamics of the region vicinal to the catalytic site in GSTE2 enzymes led us to speculate an effect of the residue exchanges in protein stability.…”

Section: Discussionmentioning

confidence: 82%

Metabolic and Target-Site Mechanisms Combine to Confer Strong DDT Resistance in Anopheles gambiae

et al. 2014

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The development of resistance to insecticides has become a classic exemplar of evolution occurring within human time scales. In this study we demonstrate how resistance to DDT in the major African malaria vector Anopheles gambiae is a result of both target-site resistance mechanisms that have introgressed between incipient species (the M- and S-molecular forms) and allelic variants in a DDT-detoxifying enzyme. Sequencing of the detoxification enzyme, Gste2, from DDT resistant and susceptible strains of An. gambiae, revealed a non-synonymous polymorphism (I114T), proximal to the DDT binding domain, which segregated with strain phenotype. Recombinant protein expression and DDT metabolism analysis revealed that the proteins from the susceptible strain lost activity at higher DDT concentrations, characteristic of substrate inhibition. The effect of I114T on GSTE2 protein structure was explored through X-ray crystallography. The amino acid exchange in the DDT-resistant strain introduced a hydroxyl group nearby the hydrophobic DDT-binding region. The exchange does not result in structural alterations but is predicted to facilitate local dynamics and enzyme activity. Expression of both wild-type and 114T alleles the allele in Drosophila conferred an increase in DDT tolerance. The 114T mutation was significantly associated with DDT resistance in wild caught M-form populations and acts in concert with target-site mutations in the voltage gated sodium channel (Vgsc-1575Y and Vgsc-1014F) to confer extreme levels of DDT resistance in wild caught An. gambiae.

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Section: Discussionmentioning

confidence: 82%

Metabolic and Target-Site Mechanisms Combine to Confer Strong DDT Resistance in Anopheles gambiae

et al. 2014

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“…In an attempt to rationalize the poor catalytic proficiency of sAnPRT at ambient temperature, the activated D83G+F149S double mutant was isolated from a plasmid‐encoded s trp D gene library by metabolic complementation of an auxotrophic E. coli Δ trp D strain. Steady‐state and transient kinetic analysis showed that the D83G mutation leads to the removal of the inhibition of the wild‐type enzyme by high concentrations of Mg 2+ , whereas the F149S mutation results in accelerated release of the product PRA, which is the rate‐limiting step in the reaction of wild‐type sAnPRT 24. The observed decrease in ${T{{{\rm app}\hfill \atop {\rm M}\hfill}}}$ to 82 °C suggested that the diffusion of the product from the active site is facilitated by an increased conformational flexibility caused by the F149S mutation.…”

Section: Introductionmentioning

confidence: 99%

Stabilization of a Metabolic Enzyme by Library Selection in Thermus thermophilus

Schwab

Sterner

2011

ChemBioChem

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The anthranilate phosphoribosyl transferase from the hyperthermophilic archaeon Sulfolobus solfataricus (sAnPRT, encoded by strpD), which catalyzes the third step in tryptophan biosynthesis, is a thermostable homodimer with low enzymatic activity at room temperature. We have combined two mutations leading to the monomerization and two mutations leading to the activation of sAnPRT. The resulting "activated monomer" sAnPRT-I36E-M47D+D83G-F149S, which is much more labile than wild-type sAnPRT, was stabilized by a combination of random mutagenesis and metabolic library selection using the extremely thermophilic bacterium Thermus thermophilus as host. This approach led to the identification of five mutations that individually increased the thermal stability of sAnPRT-I36E-M47D+D83G-F149S by 1 to 8 °C, and by 13 °C when combined. The beneficial exchanges were located in different parts of the protein structure, but none of them led to the "re-dimerization" of the enzyme. We observed a negative correlation between thermal stability and catalytic activity of the mutants; this suggests that conformational flexibility is required for catalysis by sAnPRT.

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“…Turnover rates as a function of PRPP concentration were obtained in the presence of saturating concentrations of the second substrate anthranilate, and Michaelis‐Menten parameters k cat and K M PRPP were deduced from saturation curves. The inhibitory effects of Mg 2+ concentrations >50 μM, as described for ss AnPRT, were not observed for other AnPRT variants; therefore, Mg 2+ concentration in the activity assays was set to 2 mM. In summary, steady‐state kinetic measurements substantiated that all AnPRT variants are active enzymes.…”

Section: Resultsmentioning

confidence: 99%

Prediction of quaternary structure by analysis of hot spot residues in protein‐protein interfaces: the case of anthranilate phosphoribosyltransferases

et al. 2019

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It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI). This led to the identification of two hydrophobic “hot spot” amino acids in the protein‐protein interface that were predicted to be essential for self‐association. Next, in a comprehensive multiple sequence alignment (MSA), naturally occurring AnPRT variants with hydrophilic or charged amino acids in place of hydrophobic residues in the two hot spot positions were identified. Representative variants were characterized in terms of thermal stability, enzymatic activity, and quaternary structure. We found that AnPRT variants with charged residues in both hot spot positions exist exclusively as monomers in solution. Variants with hydrophilic amino acids in one hot spot position occur in both forms, monomer and dimer. The results of the present study provide a detailed characterization of the determinants of the AnPRT monomer‐dimer equilibrium and show that analysis of hot spots in combination with MSAs can be a valuable tool in prediction of protein quaternary structures.

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Activation of Anthranilate Phosphoribosyltransferase from Sulfolobus solfataricus by Removal of Magnesium Inhibition and Acceleration of Product Release,

Cited by 13 publications

References 60 publications

Metabolic and Target-Site Mechanisms Combine to Confer Strong DDT Resistance in Anopheles gambiae

Metabolic and Target-Site Mechanisms Combine to Confer Strong DDT Resistance in Anopheles gambiae

Stabilization of a Metabolic Enzyme by Library Selection in Thermus thermophilus

Prediction of quaternary structure by analysis of hot spot residues in protein‐protein interfaces: the case of anthranilate phosphoribosyltransferases

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