Improving the activity of <i>Trichoderma reesei</i> cel7B through stabilizing the transition state

Wang, Yefei; Song, Xiangfei; Zhang, Shujun; Li, Jingwen; Shu, Zhiyu; He, Chaoliang; Huang, Qingshan; Yao, Lishan

doi:10.1002/bit.25887

Cited by 6 publications

(4 citation statements)

References 51 publications

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“…The ΔG value was determined according to the equation: ΔG ϭ G complex Ϫ G receptor Ϫ G ligand . The internal, electrostatic, and van der Waals contributions to ΔG were obtained from the force field (http://ambermd.org/tutorials/ advanced/tutorial3/py_script/section2.htm) (32)(33)(34). Accession number(s).…”

Section: Strainsmentioning

confidence: 99%

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Yang

Shi

Liu

et al. 2017

Appl Environ Microbiol

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Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using NfEG12A from Neosartorya fischeri P1 and EG (PDB 1KS4) from Aspergillus niger. Acidophilic and thermophilic NfEG12A had the highest catalytic efficiency (k cat /K m , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into NfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (k cat /K m ) of these enzymes (in descending order, NfEG12A ). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (k cat ). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis.IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the Ϫ1 or ϩ1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (k cat ). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.KEYWORDS Neosartorya fischeri, GH12 ␤-endoglucanase, loop, catalytic efficiency, hydrogen bond I n recent decades, bioethanol as a clean energy source has become a hot spot of research and industrial application (1). The bioconversion processes of producing fermentable sugars from biomass need a few glycoside hydrolases (GH), especially the

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Section: Strainsmentioning

confidence: 99%

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Yang

Shi

Liu

et al. 2017

Appl Environ Microbiol

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“…MMPBSA calculations were performed with the ambertools package [ 68 ] using snapshots from the first 500 ps of the production simulations at an interval of 1 ps. As in previous studies, the ionic strength, external dielectric, and internal dielectric constants were set to 0.15 m , 4.0, and 80.0, respectively [ 69 , 70 ], and the entropy term was excluded in the calculations [ 70 ]. Structural visualization and analyses and trajectories were done with pymol [ 71 ] and vmd [ 72 ].…”

Section: Methodsmentioning

confidence: 99%

Enzyme kinetics by GH7 cellobiohydrolases on chromogenic substrates is dictated by non‐productive binding: insights from crystal structures and MD simulation

et al. 2022

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Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) (EC3.2.1.176) are the major cellulose degrading enzymes both in industrial settings and in the context of carbon cycling in nature. Small carbohydrate conjugates such as p‐nitrophenyl‐β‐d‐cellobioside (pNPC), p‐nitrophenyl‐β‐d‐lactoside (pNPL) and methylumbelliferyl‐β‐d‐cellobioside have commonly been used in colorimetric and fluorometric assays for analysing activity of these enzymes. Despite the similar nature of these compounds the kinetics of their enzymatic hydrolysis vary greatly between the different compounds as well as among different enzymes within the GH7 family. Through enzyme kinetics, crystallographic structure determination, molecular dynamics simulations, and fluorometric binding studies using the closely related compound o‐nitrophenyl‐β‐d‐cellobioside (oNPC), in this work we examine the different hydrolysis characteristics of these compounds on two model enzymes of this class, TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium. Protein crystal structures of the E212Q mutant of TrCel7A with pNPC and pNPL, and the wildtype TrCel7A with oNPC, reveal that non‐productive binding at the product site is the dominating binding mode for these compounds. Enzyme kinetics results suggest the strength of non‐productive binding is a key determinant for the activity characteristics on these substrates, with PcCel7D consistently showing higher turnover rates (kcat) than TrCel7A, but higher Michaelis–Menten (KM) constants as well. Furthermore, oNPC turned out to be useful as an active‐site probe for fluorometric determination of the dissociation constant for cellobiose on TrCel7A but could not be utilized for the same purpose on PcCel7D, likely due to strong binding to an unknown site outside the active site.

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“…MMPBSA calculations were performed with the AMBER-TOOLS package [68] using snapshots from the first 500 ps of the production simulations at an interval of 1 ps. As in previous studies, the ionic strength, external dielectric, and internal dielectric constants were set to 0.15 M, 4.0, and 80.0, respectively [69,70], and the entropy term was excluded in the calculations [70]. Structural visualization and analyses and trajectories were done with PYMOL [71] and VMD [72].…”

Section: Molecular Dynamics Simulations and Free Energy Calculationsmentioning

confidence: 99%

Structure-function studies of GH7 cellulases, key enzymes in the global carbon cycle

Haataja¹

2023

Acta Universitatis Agriculturae Sueciae

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Enzyme mixtures used for lignocellulosic ethanol production are most commonly derived from filamentous fungi, and enzymes from the glycoside hydrolase family 7 (GH7) constitute the most abundant components in these cocktails. In this thesis I have aimed to increase our understanding of this enzyme family, with focus on the interrelation between their structure and function. In a study of the two model enzymes Trichoderma reesei Cel7A (TreCel7A) and Phanerochaete chrysosporium Cel7D (Pch7D), we determined factors governing the idiosyncratic behavior of these enzymes on commonly used model compounds, and by using fluorescence titration, enzyme kinetics, structure determination and molecular dynamics simulations found specific structural features connected to nonproductive binding, playing a major role in enzyme activity on these compounds. We also determined the molecular structure of a GH7 enzyme RsSymEG1, belonging to a group of smaller GH7 endoglucanases with previously unknown structure architecture, and originating from symbiotic protozoa of wood eating lower termites. The X-ray crystal structure revealed a configuration with several key differences to previously known GH7 structures, and will aid in modelling and engineering of enzymes in this so far little-known group of enzymes. A further look into this group of sequences, as well as other GH7 enzymes found in the termite symbiont protists, also revealed previously unknown details about the evolution of this ancient enzyme family. Furthermore, we explored single molecule imaging of the model enzyme TreCel7A with novel imaging methods, providing a first proof-of-concept of using fluorescence resonance energy transfer (FRET) for the study of inter-domain dynamics of this enzyme, as well as total internal reflection dark-field microscopy (TIRDFM) for imaging enzyme movement on cellulose surface at ultra-high temporal resolutions.

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Improving the activity of Trichoderma reesei cel7B through stabilizing the transition state

Cited by 6 publications

References 51 publications

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Enzyme kinetics by GH7 cellobiohydrolases on chromogenic substrates is dictated by non‐productive binding: insights from crystal structures and MD simulation

Structure-function studies of GH7 cellulases, key enzymes in the global carbon cycle

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