Crystal structures of wild‐type <i>Trichoderma reesei</i> Cel7A catalytic domain in open and closed states

Bodenheimer, Annette M.; Meilleur, Flora

doi:10.1002/1873-3468.12464

Cited by 12 publications

(12 citation statements)

References 51 publications

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“…[8] Such rigid modelsc an constructively be complemented with molecular dynamics (MD) simulations to obtain information on molecular motionso ver ap eriod of time. [9][10][11][12] Relaxation-matrixc alculations are useful for relating NMR spectroscopic data to structural models from for example, docking poses, crystal structures or molecular dynamics output. [13][14][15] This approach is often ak ey step for the generation of detailed solution-state models bearingi nformation on the dynamics in biomolecular systems.…”

Section: Introductionmentioning

confidence: 99%

Enantioselective Binding of Propranolol and Analogues Thereof to Cellobiohydrolase Cel7A

Hamark

Pendrill

Landström

et al. 2018

Chemistry A European J

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At the catalytic site for the hydrolysis of cellulose the enzyme cellobiohydrolase Cel7A binds the enantiomers of the adrenergic beta-blocker propranolol with different selectivity. Methyl-to-hydroxymethyl group modificationso f propranolol, which result in higher affinity andi mproveds electivity, were herein studied by 1 H, 1 Ha nd 1 H, 13 Cs calar spinspin coupling constants as well as utilizing the nuclearO verhauser effect( NOE) in conjunction with molecular dynamics simulations of the ligandsp er se, whichs howedt he presence of all-antiperiplanar conformations, except for the one containing av icinal oxygen-oxygen arrangementg overned by the gauche effect. For the ligand-protein complexes investigated by NMR spectroscopy using, inter alia, transferred NOESY and saturation-transfer difference( STD) NMR experiments the S-isomers were shown to bind with ah ighera ffin-ity and ac onformation similar to that preferred in solution, in contrastt ot he R-isomer.T he fact that the S-form of the propranolol enantiomer is pre-arrangedf or binding to the protein is also observed for ac rystals tructureo fd ihydroxy-(S)-propranolol and Cel7A presentedh erein.W hereas the binding of propranolol is entropyd riven, the complexation with the dihydroxya naloguei sa nticipated to be favored also by an enthalpic term, such as fori ts enantiomer, that is, dihydroxy-(R)-propranolol, because hydrogen-bond donation replacest he corresponding bondingf rom hydroxyl groups in glucosyl residues of the natural substrate. In addition to a favorable entropy component, albeit lesseri nm agnitude, this represents an effect of enthalpy-to-entropy compensation in ligand-protein interactions.

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Section: Introductionmentioning

confidence: 99%

Enantioselective Binding of Propranolol and Analogues Thereof to Cellobiohydrolase Cel7A

Hamark

Pendrill

Landström

et al. 2018

Chemistry A European J

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“…Product inhibition is a topical question in CBH catalysis. On the one hand cellobiose negatively affects the overall reaction rate and its inhibition should be alleviated by means of selection of CBHs from other sources or respective mutations of the downstream sites in HjCel7A . On the other, strong cellobiose affinity for downstream (expulsion) sites drives the processive hydrolysis by CBH and provides decrystallization free energy to overcome cellulose recalcitrance .…”

Section: Resultsmentioning

confidence: 99%

Predominant Nonproductive Substrate Binding by Fungal Cellobiohydrolase I and Implications for Activity Improvement

et al. 2018

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Enzymatic conversion of the most abundant renewable source of organic compounds, cellulose to fermentable sugars is attractive for production of green fuels and chemicals. The major component of industrial enzyme systems, cellobiohydrolase I from Hypocrea jecorina (Trichoderma reesei) (HjCel7A) processively splits disaccharide units from the reducing ends of tightly packed cellulose chains. HjCel7A consists of a catalytic domain (CD) and a carbohydrate-binding module (CBM) separated by a linker peptide. A tunnel-shaped substrate-binding site in the CD includes nine subsites for β-d-glucose units, seven of which (-7 to -1) precede the catalytic center. Low catalytic activity of Cel7A is the bottleneck and the primary target for improvement. Here it is shown for the first time that, in spite of much lower apparent k of HjCel7A at the hydrolysis of β-1,4-glucosidic linkages in the fluorogenic cellotetra- and -pentaose compared to the structurally related endoglucanase I (HjCel7B), the specificity constants (catalytic efficiency) k /K for both enzymes are almost equal in these reactions. The observed activity difference appears from strong nonproductive substrate binding by HjCel7A, particularly significant for MU-β-cellotetraose (MUG ). Interaction of substrates with the subsites -6 and -5 proximal to the nonconserved Gln101 residue in HjCel7A decreases K by >1500 times. HjCel7A can be nonproductively bound onto cellulose surface with K ≈2-9 nM via CBM and CD that captures six terminal glucose units of cellulose chain. Decomposition of this nonproductive complex can determine the rate of cellulose conversion. MUG is a promising substrate to select active cellobiohydrolase I variants with reduced nonproductive substrate binding.

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“…Stahlberg et al reported that divalent metal ions, as Ni 2+ , Co 2+ , Ca 2+ , Mn 2+ , and Mg 2+ , seem to be required by crystallization [24]. There are several CBHI structures containing the different ions, e.g., Sm 3+ and Gd 3+ , than divalent ones [30,31]. Before deciding on the swapped region for Co 2+ coordination, the crystal structures displaying more than 95% sequence similarity and also the high level of structural similarity within our reference structure (CBHI enzyme) are carefully examined.…”

Section: Construction Of Egi_swapped and Cloning Approachmentioning

confidence: 99%

“…Before deciding on the swapped region for Co 2+ coordination, the crystal structures displaying more than 95% sequence similarity and also the high level of structural similarity within our reference structure (CBHI enzyme) are carefully examined. Among 26 reported crystal structures, only 7 ones contain different metal ions than Co 2+ , e.g., Sm 3+ in PDB id: 4P1H, Gd 3+ in PDB id: 5TC9, and Ca 2+ in PDB id: 1CEL, 2CEL, 3CEL and 4CEL [30][31][32][33]. To make a true decision for swapping, we also perform the structural alignments between EGI (PDB id: 1EG1) with them.…”

Section: Construction Of Egi_swapped and Cloning Approachmentioning

confidence: 99%

“…Among 26 reported crystal structures, only 7 ones contain different metal ions than Co 2+ , e.g. Sm 3+ in PDB id: 4P1H, Gd 3+ in PDB id: 5TC9, and Ca 2+ in PDB id: 1CEL, 2CEL, 3CEL and 4CEL [30][31][32][33]. To make a true decision for swapping, we also perform the structural alignments between EGI (PDB id: 1EG1) with them.…”

Section: Construction Of Egi_swapped and Cloning Approachmentioning

confidence: 99%

See 1 more Smart Citation

Enhancing Enzymatic Properties of Endoglucanase I Enzyme from Trichoderma Reesei via Swapping from Cellobiohydrolase I Enzyme

2019

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Utilizing plant-based materials as a biofuel source is an increasingly popular attempt to redesign the global energy cycle. This endeavour underlines the potential of cellulase enzymes for green energy production and requires the structural and functional engineering of natural enzymes to enhance their utilization. In this work, we aimed to engineer enzymatic and functional properties of Endoglucanase I (EGI) by swapping the Ala43-Gly83 region of Cellobiohydrolase I (CBHI) from Trichoderma reesei. Herein, we report the enhanced enzymatic activity and improved thermal stability of the engineered enzyme, called EGI_swapped, compared to EGI. The difference in the enzymatic activity profile of EGI_swapped and the EGI enzymes became more pronounced upon increasing metal-ion concentrations in the reaction media. Notably, the engineered enzyme retained a considerable level of enzymatic activity after thermal incubation for 90 min at 70 °C while EGI completely lost its enzymatic activity. Circular Dichroism spectroscopy studies revealed distinctive conformational and thermal susceptibility differences between EGI_swapped and EGI enzymes, confirming the improved structural integrity of the swapped enzyme. This study highlights the importance of swapping the metal-ion coordination region in the engineering of EGI enzyme for enhanced structural and thermal stability.

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Crystal structures of wild‐type Trichoderma reesei Cel7A catalytic domain in open and closed states

Cited by 12 publications

References 51 publications

Enantioselective Binding of Propranolol and Analogues Thereof to Cellobiohydrolase Cel7A

Enantioselective Binding of Propranolol and Analogues Thereof to Cellobiohydrolase Cel7A

Predominant Nonproductive Substrate Binding by Fungal Cellobiohydrolase I and Implications for Activity Improvement

Enhancing Enzymatic Properties of Endoglucanase I Enzyme from Trichoderma Reesei via Swapping from Cellobiohydrolase I Enzyme

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