Computational Investigation of the pH Dependence of Loop Flexibility and Catalytic Function in Glycoside Hydrolases

Bu, Lintao; Crowley, Michael F.; Himmel, Michael E.; Beckham, Gregg T.

doi:10.1074/jbc.m113.462465

Cited by 34 publications

(60 citation statements)

References 82 publications

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“…As argued above, pK a values obtained from measurements at very low pNPL concentrations may be taken as representative of the apoenzyme, and values in Table are in line with earlier suggestions based on the experimental (Becker et al, ) and computational (Bu, Crowley, Himmel, & Beckham, ) studies. For Cel7A, these earlier works assigned the low pK a value (pK a1 in Table ) to the nucleophile in the catalytic reaction (Glu212 in TrCel7A numbering), while the higher pK a (pK a2 ) was assigned to the catalytic acid/base, Glu217.…”

Section: Discussionsupporting

confidence: 87%

“…Earlier work on Cel7A has revealed small differences in pH profiles made with different soluble substrate analogs (Becker et al, ), and this has been attributed to interactions in the Michaelis complex that shifts the pK a of key catalytic residues (Bu et al, ). Such shifts due to local interactions of catalytic residues (including larger shifts than those observed here) are common in enzymes (Harris & Turner, ) and undoubtedly also relevant for the pH profiles observed in this study.…”

Section: Discussionmentioning

confidence: 99%

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pH profiles of cellulases depend on the substrate and architecture of the binding region

Røjel

Kari

Sørensen

et al. 2019

Biotech & Bioengineering

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Understanding the pH effect of cellulolytic enzymes is of great technological importance. In this study, we have examined the influence of pH on activity and stability for central cellulases (Cel7A, Cel7B, Cel6A from Trichoderma reesei, and Cel7A from Rasamsonia emersonii). We systematically changed pH from 2 to 7, temperature from 20°C to 70°C, and used both soluble (4‐nitrophenyl β‐ d‐lactopyranoside [pNPL]) and insoluble (Avicel) substrates at different concentrations. Collective interpretation of these data provided new insights. An unusual tolerance to acidic conditions was observed for both investigated Cel7As, but only on real insoluble cellulose. In contrast, pH profiles on pNPL were bell‐shaped with a strong loss of activity both above and below the optimal pH for all four enzymes. On a practical level, these observations call for the caution of the common practice of using soluble substrates for the general characterization of pH effects on cellulase activity. Kinetic modeling of the experimental data suggested that the nucleophile of Cel7A experiences a strong downward shift in pKa upon complexation with an insoluble substrate. This shift was less pronounced for Cel7B, Cel6A, and for Cel7A acting on the soluble substrate, and we hypothesize that these differences are related to the accessibility of water to the binding region of the Michaelis complex.

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Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

pH profiles of cellulases depend on the substrate and architecture of the binding region

Røjel

Kari

Sørensen

et al. 2019

Biotech & Bioengineering

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“…As mentioned, three carboxylic residues of Glu209, Asp211, and Glu214 are important to the catalytic mechanism in Re Cel7A. Based on the investigation of carboxylic p K a in Tr Cel7A, the p K a values of Glu212, Asp214, and Glu217 (corresponding to the Glu209, Asp211, and Glu214 in Re Cel7A) are 2.41, 7.63, and 7.12, respectively . The values of Asp214 and Glu217 are greater than the optimum pH, thus they should be protonated.…”

Section: Theory and Methodscontrasting

confidence: 49%

Investigation of an “alternate water supply system” in enzymatic hydrolysis in the processive endocellulase Cel7A from Rasamsonia emersonii by molecular dynamics simulation

et al. 2016

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Cel7A from Rasamsonia emersonii is one of the processive endocellulases classified under family 7 glycoside hydrolase. Molecular dynamics simulations were carried out to obtain the optimized sliding and hydrolyzing conformations, in which the reducing ends of sugar chains are located on different sites. Hydrogen bonds are investigated to clarify the interactions between protein and substrate in either conformation. Nine hydrogen bonding interactions are identified in the sliding conformation, and six similar interactions are also found correspondingly in the hydrolyzing conformation. In addition, four strong hydrophobic interactions are also determined. The domain cross-correlation map analysis shows movement correlation of protein including autocorrelation between residues. The root mean square fluctuations analysis represents the various flexibilities of different fragment in the two conformations. Comparing the two conformations reveals the water-supply mechanism of selective hydrolysis of cellulose in Cel7A. The mechanism can be described as follow. When the reducing end of substrate slides from the unhydrolyzing site (sliding conformation) to the hydrolyzing site (hydrolyzing conformation), His225 is pushed down and rotated, the rotation leads to the movement of Glu209 with the interstrand hydrogen bonding in β-sheet. It further makes Asp211 close to the hydrolysis center and provides a water molecule bounding on its carboxyl in the previous unhydrolyzing site. After the hydrolysis takes place and the product is excluded from the enzyme, the Asp211 comes back to its initial position. In summary, Asp211 acts as an elevator to transport outer water molecules into the hydrolysis site for every other glycosidic bond.

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“…Influence of the net charge on the structural stability and enzymatic activity Previous study indicates enzymes which activity is directly related by the balance between protonation and conformational changes (Bu et al 2013). Interestingly, at acidic pH, our results indicated that there were no significant changes in the structures of the β-glucosidases at temperatures below 80 °C.…”

Section: Recombinant β-Glucosidases From T Petrophila Are Stable Hommentioning

confidence: 56%

Oligomeric state and structural stability of two hyperthermophilic β-glucosidases from Thermotoga petrophila

et al. 2015

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The β-glucosidases are enzymes essential for several industrial applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts. In a recent study, we have provided a biochemical characterization of two hyperthermostable β-glucosidases from Thermotoga petrophila belonging to the families GH1 (TpBGL1) and GH3 (TpBGL3). Here, as part of a continuing investigation, the oligomeric state, the net charge, and the structural stability, at acidic pH, of the TpBGL1 and TpBGL3 were characterized and compared. Enzymatic activity is directly related to the balance between protonation and conformational changes. Interestingly, our results indicated that there were no significant changes in the secondary, tertiary and quaternary structures of the β-glucosidases at temperatures below 80 °C. Furthermore, the results indicated that both the enzymes are stable homodimers in solution. Therefore, the observed changes in the enzymatic activities are due to variations in pH that modify protonation of the enzymes residues and the net charge, directly affecting the interactions with ligands. Finally, the results showed that the two β-glucosidases displayed different pH dependence of thermostability at temperatures above 80 °C. TpBGL1 showed higher stability at pH 6 than at pH 4, while TpBGL3 showed similar stability at both pH values. This study provides a useful comparison of the structural stability, at acidic pH, of two different hyperthermostable β-glucosidases and how it correlates with the activity of the enzymes. The information described here can be useful for biotechnological applications in the biofuel and food industries.

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Computational Investigation of the pH Dependence of Loop Flexibility and Catalytic Function in Glycoside Hydrolases

Cited by 34 publications

References 82 publications

pH profiles of cellulases depend on the substrate and architecture of the binding region

pH profiles of cellulases depend on the substrate and architecture of the binding region

Investigation of an “alternate water supply system” in enzymatic hydrolysis in the processive endocellulase Cel7A from Rasamsonia emersonii by molecular dynamics simulation

Oligomeric state and structural stability of two hyperthermophilic β-glucosidases from Thermotoga petrophila

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