scholarly journals A Case Study of the Glycoside Hydrolase Enzyme Mechanism Using an Automated QM-cluster Model Building Toolkit

2021 ◽  
Author(s):  
Qianyi Cheng ◽  
Nathan J. DeYonker
Keyword(s):  
Free Energy ◽  
Glycoside Hydrolase ◽  
Model Building ◽  
Enzyme Mechanism ◽  
Protein Glycosylation ◽  
Cluster Models ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Residue Interaction

Glycoside hydrolase enzymes are important for hydrolyzing the β-1,4 glycosidic bond in polysaccharides for deconstruction of carbohydrates. The two-step retaining reaction mechanism was explored with different sized QM-cluster models built by the Residue Interaction Network ResidUe Selector (RINRUS) software using both the wild-type protein and its E217Q mutant. The first step is the glycosylation, in which the acidic residue 217 donates a proton to the glycosidic oxygen leading to bond cleavage. In the subsequent deglycosylation step, one water molecule migrates into the active site and attacks the anomeric carbon. Residue interaction-based QM-cluster models lead to reliable structural and energetic results for proposed glycoside hydrolase mechanisms. The free energies of activation for glycosylation in the largest QM-cluster models were predicted to be 19.5 and 31.4 kcal mol for the wild-type protein and its E217Q mutant, which agree with experimental trends that mutation of the acidic residue Glu217 to Gln will slow down the reaction, and are higher in free energy than the deglycosylation transition states (13.8 and 25.5 kcal mol for the wild-type protein and its mutant, respectively). For the mutated protein, glycosylation led to a low-energy product. This thermodynamic sink may correspond to the intermediate state which was isolated in the X-ray crystal structure. Hence, the glycosylation is validated to be the rate-limiting step in both the wild-type and mutated enzyme. The E217Q mutation led to a higher glycosylation activation free energy that also agrees with experimental observation that mutation of E217 will slow down the reaction, but not deactivate catalysis.

scholarly journals Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0128954 ◽  
Author(s):  
Saara Laulumaa ◽  
Tuomo Nieminen ◽  
Mari Lehtimäki ◽  
Shweta Aggarwal ◽  
Mikael Simons ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Neutron Scattering ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Peripheral Membrane Protein

Two novel mutations in Gli-similar 3 in patients with congenital hypothyroidism and thyroid dysgenesis

2021 ◽  
Author(s):  
Jie Lan ◽  
Chunhui Sun ◽  
Xinping Liang ◽  
Ruixin Ma ◽  
Yuhua Ji ◽  
...  
Keyword(s):  
Congenital Hypothyroidism ◽  
Transcriptional Activation ◽  
Luciferase Assay ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Thyroid Dysgenesis ◽  
Type Protein ◽  
Wild Type Protein

Abstract Background: Thyroid dysgenesis (TD) is the main cause of congenital hypothyroidism (CH). As variants of the transcription factor Gli-similar 3 (GLIS3) have been associated with CH and GLIS3 is one of candidate genes of TD, we screened and characterized GLIS3 mutations in Chinese patients with CH and TD.Methods: To detect mutations, we sequenced all GLIS3 exons in the peripheral blood genomic DNA isolated from 50 patients with TD and 100 healthy individuals. Wild-type and mutant expression vectors of Glis3 were constructed. Quantitative real-time PCR, western blotting, and double luciferase assay were performed to investigation the effect of the mutations on GLIS3 protein function and transcriptional activation.Results: Two novel heterozygous missense mutations, c.2710G>A (p.G904R) and c.2507C>A (p.P836Q), were detected in two unrelated patients. Functional studies revealed that p.G904R expression was 59.95% lower and p.P836Q was 31.23% lower than wild-type GLIS3 mRNA expression. The p.G904R mutation also resulted in lower GLIS3 protein expression compared with that encoded by wild-type GLIS3. Additionally, the luciferase reporter assay revealed that p.G904R mediated impaired transcriptional activation compared with the wild-type protein (p < 0.05) but did not have a dominant-negative effect on the wild-type protein.Conclusions: We for the first time screened and characterized the function of GLIS3 mutations in Chinese individuals with CH and TD. Our study not only broadens the GLIS3 mutation spectrum, but also provides further evidence that GLIS3 defects cause TD.

scholarly journals Effect of Structural Changes Induced by Deletion of 54FLRAPSWF61 Sequence in αB-Crystallin on Chaperone Function and Anti-Apoptotic Activity

2021 ◽  
Vol 22 (19) ◽  
pp. 10771
Author(s):  
Sundararajan Mahalingam ◽  
Srabani Karmakar ◽  
Puttur Santhoshkumar ◽  
Krishna K. Sharma
Keyword(s):  
Deletion Mutant ◽  
Mutant Protein ◽  
Wild Type ◽  
Chaperone Function ◽  
Type Protein ◽  
Wild Type Protein ◽  
Cytotoxicity Studies ◽  
Αb Crystallin ◽  
Apoptotic Activity ◽  
Subunit Organization

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein.

scholarly journals Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion

1998 ◽  
Vol 329 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Esther YÁÑEZ ◽  
A. Teresa CARMONA ◽  
Mercedes TIEMBLO ◽  
Antonio JIMÉNEZ ◽  
María FERNÁNDEZ-LOBATO
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Efficiency ◽  
Extracellular Enzyme ◽  
Site Directed Mutagenesis ◽  
Activity Levels ◽  
Wild Type ◽  
Schwanniomyces Occidentalis ◽  
Type Protein ◽  
Wild Type Protein

The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 α-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that α-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. α-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50 = 50 °C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 °C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 α-amylase in both its wild-type and mutant forms.

scholarly journals Regio- and stereo-chemical oxidation of linoleic acid by human myoglobin and hydrogen peroxide: Tyr103 affects rate and product distribution

2004 ◽  
Vol 381 (2) ◽  
pp. 365-372 ◽  
Author(s):  
Benjamin S. RAYNER ◽  
Roland STOCKER ◽  
Peter A. LAY ◽  
Paul K. WITTING
Keyword(s):  
Linoleic Acid ◽  
Lipid Oxidation ◽  
Octadecadienoic Acid ◽  
Total Yield ◽  
Product Distribution ◽  
Chemical Oxidation ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Lipid Oxidation Products

Mb (myoglobin) plus H2O2 catalyses the oxidation of various substrates via a peroxidase-like activity. A Y103F (Tyr103→Phe) variant of human Mb has been constructed to assess the effect of exchanging an electron-rich oxidizable amino acid on the peroxidase activity of human Mb. Steady-state analyses of reaction mixtures containing Y103F Mb, purified linoleic acid and H2O2 revealed a lower total yield of lipid oxidation products than mixtures containing the wild-type protein, consistent with the reported decrease in the rate constant for reaction of Y103F Mb with H2O2 [Witting, Mauk and Lay (2002) Biochemistry 41, 11495–11503]. Irrespective of the Mb employed, lipid oxidation yielded 9(R/S)-HODE [9(R,S)-hydroxy-10E,12Z-octadecadienoic acid] in preference to 13(R/S)-HODE [13(R,S)-hydroxy-9Z,11E-octadecadienoic acid], while 9- and 13-keto-octadecadienoic acid were formed in trace amounts. However, lipid oxidation by the Y103F variant of Mb proceeded with a lower Vmax value and an increased Km value relative to the wild-type control. Consistent with the increased Km, the product distribution from reactions with Y103F Mb showed decreased selectivity compared with the wild-type protein, as judged by the decreased yield of 9(S)-relative to 9(R)-HODE. Together, these data verify that Tyr103 plays a significant role in substrate binding and orientation in the haem pocket of human Mb. Also, the midpoint potential for the Fe(III)/(II) one-electron reduction was shifted slightly, but significantly, to a higher potential, confirming the importance of Tyr103 to the hydrogen-bonding network involving residues that line the haem crevice of human Mb.

scholarly journals S279 Point Mutations in Candida albicans Sterol 14-α Demethylase (CYP51) ReduceIn VitroInhibition by Fluconazole

2012 ◽  
Vol 56 (4) ◽  
pp. 2099-2107 ◽  
Author(s):  
Andrew G. S. Warrilow ◽  
Jonathan G. L. Mullins ◽  
Claire M. Hull ◽  
Josie E. Parker ◽  
David C. Lamb ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Point Mutations ◽  
Triazole Ring ◽  
Wild Type ◽  
Protein Activity ◽  
Content Type ◽  
Mutant Proteins ◽  
Type Protein ◽  
Wild Type Protein ◽  
Binding Spectra

ABSTRACTThe effects of S279F and S279Y point mutations inCandida albicansCYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities (Ks, 7.1 and 8.0 μM, respectively) compared to the wild-type CaCYP51 protein (Ks, 13.5 μM). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type II binding spectra. However, the S279F and S279Y mutants had 4- to 5-fold lower affinities for fluconazole, 3.5-fold lower affinities for voriconazole, and 3.5- to 4-fold lower affinities for itraconazole than the wild-type CaCYP51 protein. The S279F and S279Y mutants gave 2.3- and 2.8-fold higher 50% inhibitory concentrations (IC50s) for fluconazole in a CYP51 reconstitution assay than the wild-type protein did. The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for fluconazole. In addition, lanosterol displaced fluconazole from the S279F and S279Y mutants but not from the wild-type protein. Molecular modeling of the wild-type protein indicated that the oxygen atom of S507 interacts with the second triazole ring of fluconazole, assisting in orientating fluconazole so that a more favorable binding conformation to heme is achieved. In contrast, in the two S279 mutant proteins, this S507-fluconazole interaction is absent, providing an explanation for the higherKdvalues observed.

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