Dataset Information

Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme.

ABSTRACT: Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.

SUBMITTER: Stransky J

PROVIDER: S-EPMC4631591 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme.

Stránský Jan J Koval' Tomáš T Podzimek Tomáš T Týcová Anna A Lipovová Petra P Matoušek Jaroslav J Kolenko Petr P Fejfarová Karla K Dušková Jarmila J Skálová Tereza T Hašek Jindřich J Dohnálek Jan J

Acta crystallographica. Section F, Structural biology communications 20151023 Pt 11

Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ...[more]

PMID: 26527269

Similar Datasets

Project description:BackgroundPolymorphisms in the APEX nuclease (multifunctional DNA repair enzyme) 1 gene (APEX1) may be involved in the carcinogenesis by affecting DNA repair. We aimed to summarize available data on the association of the APEX1 Asp148Glu (rs1130409) polymorphism with risk of multiple types of cancer via a meta-analysis.Methods and resultsIn total, 58 qualified articles including 22,398 cancer patients and 26,505 controls were analyzed, and the data were extracted independently by two investigators. Analyses of the full data set indicated a marginally significant association of the APEX1 Asp148Glu polymorphism with cancer risk under allelic (odds ratio (OR)=1.05; 95% confidence interval (95% CI): 0.99-1.11; P=0.071), dominant (OR=1.09; 95% CI: 1.01-1.17; P=0.028), and heterozygous genotypic (OR=1.08; 95% CI: 1.01-1.16; P=0.026) models, with significant heterogeneity and publication bias. In subgroup analyses by cancer type, with a Bonferroni corrected alpha of 0.05/6, significant association was observed for gastric cancer under both dominant (OR=1.74; 95% CI: 1.2-2.51; P=0.003) and heterozygous genotypic (OR=1.66; 95% CI: 1.2-2.31; P=0.002) models. In subgroup analysis by ethnicity, risk estimates were augmented in Caucasians, especially under dominant (OR=1.11; 95% CI: 1.0-1.24; P=0.049) and heterozygous genotypic (OR=1.11; 95% CI: 0.99-1.24; P=0.063) models. By study design, there were no significant differences between population-based and hospital-based studies. In subgroup analysis by sample size, risk estimates were remarkably overestimated in small studies, and no significance was reached in large studies except under the heterozygous genotypic model (OR=1.23; 95% CI: 1.06-1.43; P=0.006, significant at a Bonferroni corrected alpha of 0.05/2). By quality score, the risk estimates, albeit nonsignificant, were higher in low-quality studies than in high-quality studies. Further meta-regression analyses failed to identify any contributory confounders for the associated risk estimates.ConclusionsOur findings suggest that APEX1 Asp148Glu polymorphism might be a genetic risk factor for the development of gastric cancer. Further investigations on large populations are warranted.

Project description:BACKGROUND: The interest in introducing ecologically-clean, and efficient enzymes into modern industry has been growing steadily. However, difficulties associated with controlling their orientation, and maintaining their selectivity and reactivity is still a significant obstacle. We have developed precise immobilization of biomolecules, while retaining their native functionality, and report a new, fast, easy, and reliable procedure of protein immobilization, with the use of Adenylate kinase as a model system. METHODS: Self-assembled monolayers of hexane-1,6-dithiol were formed on gold surfaces. The monolayers were characterized by contact-angle measurements, Elman-reagent reaction, QCM, and XPS. A specifically designed, mutated Adenylate kinase, where cysteine was inserted at the 75 residue, and the cysteine at residue 77 was replaced by serine, was used for attachment to the SAM surface via spontaneously formed disulfide (S-S) bonds. QCM, and XPS were used for characterization of the immobilized protein layer. Curve fitting in XPS measurements used a Gaussian-Lorentzian function. RESULTS AND DISCUSSION: Water contact angle (65-70°), as well as all characterization techniques used, confirmed the formation of self-assembled monolayer with surface SH groups. X-ray photoelectron spectroscopy showed clearly the two types of sulfur atom, one attached to the gold (triolate) and the other (SH/S-S) at the ?-position for the hexane-1,6-dithiol SAMs. The formation of a protein monolayer was confirmed using XPS, and QCM, where the QCM-determined amount of protein on the surface was in agreement with a model that considered the surface area of a single protein molecule. Enzymatic activity tests of the immobilized protein confirmed that there is no change in enzymatic functionality, and reveal activity ~100 times that expected for the same amount of protein in solution. CONCLUSIONS: To the best of our knowledge, immobilization of a protein by the method presented here, with the resulting high enzymatic activity, has never been reported. There are many potential applications for selective localization of active proteins at patterned surfaces, for example, bioMEMS (MEMS--Micro-Electro-Mechanical Systems. Due to the success of the method, presented here, it was decided to continue a research project of a biosensor by transferring it to a high aspect ratio platform--nanotubes.

Project description:In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics-molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water-independent enzyme-catalyzed reaction had a bias-corrected Jarzynski-average barrier height of approximately (6.5 kcal mol(-1) (27.2 kJ mol(-1)) for the carbinolamine formation reaction and 44.5 kcal mol(-1) (186 kJ mol(-1)) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately 15 kcal mol(-1) (62.8 kJ mol(-1)) in the forward (formation) reaction and 19 kcal mol(-1) (79.5 kJ mol(-1)) for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water-independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N-terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site carboxylate and the N-terminal amine.

Dataset Information

Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme.

Publications

Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure