Exchange of active site residues alters substrate specificity in extremely thermostable β-glycosidase from Thermococcus kodakarensis KOD1

Hwa, Kuo-Yuan; Subramani, Boopathi; Shen, Sheng‐Feng; Lee, Yu‐May

doi:10.1016/j.enzmictec.2015.05.002

Cited by 5 publications

(6 citation statements)

References 23 publications

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“…These reactions can happen between two carbohydrates or between a carbohydrate and a noncarbohydrate moiety (amino acids, e.g., in glycoproteins) . Due to the critical role of carbohydrates in human health as well as their possible use as fuel, there has been an increased interest in the study of glycosidases in recent years …”

Section: Introductionmentioning

confidence: 99%

Benchmarking of density functionals for the kinetics and thermodynamics of the hydrolysis of glycosidic bonds catalyzed by glycosidases

Pereira

Ribeiro

Fernandes

et al. 2017

Int J of Quantum Chemistry

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In recent years, there has been an increased interest in understanding the enzymatic mechanism of glycosidases resorting mostly to DFT and DFT/MM calculations. However, the performance of density functionals (DFs) is well known to be system and property dependent. Trends drawn from general studies, despite important to evaluate the quality of the DFs and to pave the way for the development of new DFs, may be misleading when applied to a single specific system/property. To overcome this issue, we carried out a benchmarking study of 40 DFs applied to the geometry optimization and to the electronic barrier height (EBarrier) and electronic energy of reaction (ER) of prototypical glycosidase‐catalyzed reactions. Additionally, we report calculations with SCC‐DFTB and four semiempirical MO methods applied to the same problem. We have used a universal molecular model for retaining glycosidases, comprising only a 22‐atoms system that mimics the active site and substrate. High accuracy reference geometries and energies were calculated at the CCSD(T)/CBS//MP2/aug‐cc‐pVTZ level of theory. Most DFs reproduce the reference geometries extremely well, with mean unsigned errors (MUE) smaller than 0.05 Å for bond lengths and 3° for bond angles. Among the DFs, wB97X‐D, CAM‐B3LYP, B3P86, and PBE1PBE have the best performance in geometry optimizations (MUE = 0.02 Å). Conversely, semiempirical MO and SCC‐DFTB methods yielded less accurate geometries (MUE between 0.09 and 0.17 Å). The inclusion of D3 correction has a small, but still relevant, influence in the geometry predicted by some DFs. Regarding EBarrier, 11 DFs (MPW1B95, CAM‐B3LYP, M06 ‐ 2X, PBE1PBE, wB97X ‐ D, B1B95, BMK, MN12 – SX, M05, M06, and M11) presented errors below 1 kcal.mol−1, in relation to the reference energy. Most of these functionals belong to the family of hybrid functionals (H‐GGA, HH‐GGA, and HM‐GGA), which shows a positive influence of HF exchange in the determination of EBarrier. The inclusion of D3 correction has not affected significantly the EBarrier and ER. The use of geometries at the accurate but expensive MP2/aug‐cc‐pVTZ level of theory has a small, albeit not insignificant, influence in the EBarrier when compared with energies calculated with geometries determined with the DFs (usually a few tenths of kcal.mol−1, with exceptions). In general, semiempirical MO methods and DFTB are associated with larger errors in the determination of EBarrier, with unsigned errors from 6.9 to 24.7 kcal.mol−1.

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Section: Introductionmentioning

confidence: 99%

Benchmarking of density functionals for the kinetics and thermodynamics of the hydrolysis of glycosidic bonds catalyzed by glycosidases

Pereira

Ribeiro

Fernandes

et al. 2017

Int J of Quantum Chemistry

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“…A 3D model structure of MBSP belonging to the group of trypsin‐like serine proteinases with 222 amino acid residues shows that His40, Asp86 and Ser176 comprise the catalytic triad and the composition of the substrate‐binding pocket is similar to that of trypsin at three key positions, Asp170, Gly193 and Gly203 [Fig. (a)].…”

Section: Resultsmentioning

confidence: 99%

“…To date, many successful examples of improvements in substrate specificity through protein engineering studies have been reported. For instance, the substrate specificity of a β‐glycosidase mutant was altered due to its electrostatic effect and extending the side chains, which affected substrate binding during catalysis . Similarly, G414W displayed a shift in substrate preference compared to Candida rugosa lipase 1 because of strong steric hindrance that blocked the entrance to the binding site .…”

Section: Introductionmentioning

confidence: 99%

“…For instance, the substrate specificity of a -glycosidase mutant was altered due to its electrostatic effect and extending the side chains, which affected substrate binding during catalysis. 6 Similarly, G414W displayed a shift in substrate preference compared to Candida rugosa lipase 1 because of strong steric hindrance that blocked the entrance to the binding site. 7 Such successful transformations of enzyme properties have laid the foundation for our experiment, as it is feasible that the substrate specificity of MBSP can be changed by gene modification.…”

Section: Introductionmentioning

confidence: 99%

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Altering the substrate specificity of a myofibril‐bound serine proteinase from Crucian carp by site‐directed mutagenesis

Mei

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUND Myofibril‐bound serine proteinase (MBSP) comprises a class of trypsin‐type serine proteinases that can specifically cleave the carboxyl side of peptide bonds of arginine or lysine residues. To obtain higher specificity MBSPs, mutants were designed based on the substrate‐binding pocket and constructed through site‐directed mutagenesis. RESULTS The wild‐type (WT) and mutants were expressed in Pichia pastoris, and its enzymatic properties indicated that the mutants D170S, D170T, D170K and D170T/G203A were not biologically active. However, S171A specifically cleaved arginine residues and G203A preferably hydrolyzed lysine residues. The secondary structures of mutants were approximately similar to that of the WT according to the results of circular dichroism spectroscopy. Additionally, molecular docking showed that substrates were able to combine with S171A within the critical areas through hydrophobic interaction, hydrogen bonds and van der Waals electrostatic force. CONCLUSION The structural stability of MBSP was consistent using amino acid substitution. Due to the interaction pattern of substrates and mutant enzyme changes, only S171A was selective in cutting Arg residues, which illustrates how the catalytic triad and substrate‐binding pocket of MBSP plays an important role during enzymatic hydrolysis of substrates. © 2018 Society of Chemical Industry

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“…Interestingly, the PF1208 enzyme also shows moderate cleavage ability both for methyl α‐ l ‐fucopyranoside and for methyl β‐ l ‐fucopyranoside substrates, thus suggesting a possible secondary fucosidase activity. d ‐Mannose and l ‐fucose share a certain structural similarity, and such secondary fucosidase activity is not uncommon for previously characterized mannosidases, especially from thermophilic origins . Additionally, the archaeon domain is considered to have glycosidases with broader specificity towards substrates than those of human or mammalian origins .…”

Section: Methodsmentioning

confidence: 99%

A High‐Throughput Mass‐Spectrometry‐Based Assay for Identifying the Biochemical Functions of Putative Glycosidases

et al. 2017

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The most common glycosidase assays rely on bulky ultraviolet or fluorescent tags at the anomeric position of potential carbohydrate substrates, thereby limiting the utility of these assays for broad substrate characterization. Here we report a mass spectrometry-based glycosidase assay amenable to high-throughput screening for the identification of the biochemical functions of putative glycosidases. The assay utilizes a library of methyl glycosides and is demonstrated on a high-throughput robotic liquid handling system for enzyme substrate screening. Identification of glycosidase biochemical function is achieved by observing a correct mass-loss between a potential sugar substrate and its corresponding product using electrospray ionization mass spectrometry (ESI-MS). In addition to screening known glycosidases, the assay was demonstrated to characterize the biochemical function and enzyme substrate competency of the recombinantly-expressed product of a putative glycosidase gene from the thermophilic bacterium Thermus thermophilus.

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Exchange of active site residues alters substrate specificity in extremely thermostable β-glycosidase from Thermococcus kodakarensis KOD1

Cited by 5 publications

References 23 publications

Benchmarking of density functionals for the kinetics and thermodynamics of the hydrolysis of glycosidic bonds catalyzed by glycosidases

Benchmarking of density functionals for the kinetics and thermodynamics of the hydrolysis of glycosidic bonds catalyzed by glycosidases

Altering the substrate specificity of a myofibril‐bound serine proteinase from Crucian carp by site‐directed mutagenesis

A High‐Throughput Mass‐Spectrometry‐Based Assay for Identifying the Biochemical Functions of Putative Glycosidases

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