Rate‐limiting step and substrate accessibility of cellobiohydrolase Cel6A from <i>Trichoderma reesei</i>

Christensen, Stefan Jarl; Kari, Jeppe; Badino, Silke Flindt; Borch, Kim; Westh, Peter

doi:10.1111/febs.14668

Cited by 27 publications

(26 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The biological basis of the molecular selection for processivity across a wide range of Group 5 members is not immediately intuited. Processivity is generally considered to be advantageous for enzymes acting on crystalline substrates such as cellulose or chitin, where initial chain engagement is thought to be rate-limiting (42)(43)(44)(45)(46)(47). However, this would not be expected for soluble polysaccharides, such as XyG, especially under dilute assay conditions in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…Endoxyloglucanases can be further delineated into endo-dissociative enzymes, which hydrolyze the backbone and immediately release both new chain ends, and endo-processive enzymes, which perform multiple hydrolytic events, releasing short oligosaccharides before disengaging. The ability of some GH74 enzymes (26,28,29,35) to act processively on soluble XyG is notable, considering that this mode of action is more commonly associated with GHs acting on crystalline cellulose or chitin (42)(43)(44)(45)(46)(47).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Substrate specificity, regiospecificity, and processivity in glycoside hydrolase family 74

Arnal

Stogios

Asohan

et al. 2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

Glycoside hydrolase family 74 (GH74) is a historically important family of endo-␤-glucanases. On the basis of early reports of detectable activity on cellulose and soluble cellulose derivatives, GH74 was originally considered to be a "cellulase" family, although more recent studies have generally indicated a high specificity toward the ubiquitous plant cell wall matrix glycan xyloglucan. Previous studies have indicated that GH74 xyloglucanases differ in backbone cleavage regiospecificities and can adopt three distinct hydrolytic modes of action: exo, endo-dissociative, and endo-processive. To improve functional predictions within GH74, here we coupled in-depth biochemical characterization of 17 recombinant proteins with structural biology-based investigations in the context of a comprehensive molecular phylogeny, including all previously characterized family members. Elucidation of four new GH74 tertiary structures, as well as one distantly related dual seven-bladed ␤propeller protein from a marine bacterium, highlighted key structure-function relationships along protein evolutionary trajectories. We could define five phylogenetic groups, which delineated the mode of action and the regiospecificity of GH74 members. At the extremes, a major group of enzymes diverged to hydrolyze the backbone of xyloglucan nonspecifically with a dissociative mode of action and relaxed backbone regiospecificity. In contrast, a sister group of GH74 enzymes has evolved a large hydrophobic platform comprising 10 subsites, which facilitates processivity. Overall, the findings of our study refine our understanding of catalysis in GH74, providing a framework for future experimentation as well as for bioinformatics predictions of sequences emerging from (meta)genomic studies. Terrestrial plants harbor ϳ80% of the biomass on Earth, some 450 gigatons of carbon, in the form of lignocellulose (cell walls comprised of cellulose, matrix glycans, lignin, and other polymers) (1). Although terrestrial biomass represents an attractive renewable resource for the production of fuels, chemicals, and materials for human consumption, the controlled degradation of lignocellulose, whether (thermo)chemical or enzymatic, is hindered by its heterogeneous composition and complex organization (2). Hence, significant efforts have been made to identify enzymes able to efficiently modify and deconstruct this complex material. Xyloglucans (XyGs) 3 comprise a prominent family of cell wall matrix glycans (hemicelluloses). XyGs are ubiquitous in land plants, in which they constitute up to 20% of the dry weight of cell walls (3, 4). Notably, XyGs are secreted by roots of diverse plant species and are therefore likely to actively influence rhizobiota (5). XyGs are also found as storage polysaccharides comprising ϳ50% of the mass of some seeds (e.g. tamarind and nasturtium) and therefore represent important agricultural byproducts with applications in the food, biomaterial, and medical sectors (6, 7). XyGs have a ␤-1,4-linked glucosyl backbone ("G" unit), some of which a...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Substrate specificity, regiospecificity, and processivity in glycoside hydrolase family 74

Arnal

Stogios

Asohan

et al. 2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…This has the noticeable consequence that the free energy of the (rate-limiting) TS is quite similar for all tested enzymes, and that the main kinetic diversity lies in different affinities for the substrate. Experimental studies have suggested that the ratelimiting step for some cellulases is slow dissociation [32][33][34][35][36] . Since weaker binding is associated with a lower activation barrier for dissociation ( Fig 5B-3), a dissociation limited mechanism would explain the inverse correlation of binding strength and maximal turnover.…”

Section: Discussionmentioning

confidence: 99%

Physical constrains and functional plasticity of cellulases: Linear scaling relationships for a heterogeneous enzyme reaction

Kari

Schaller

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. We have produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the strength of substrate binding and the activation barrier. This common scaling occurred despite the investigated enzymes were structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection have condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes. On a more general note, the LFER may identify a link to inorganic heterogeneous catalysis, and hence open for the implementation of approaches from this field within interfacial enzymology.

show abstract

“…Enzyme activity and binding a nity were measured on three different cellulosic substrates: regenerated amorphous cellulose (RAC), Avicel PH101 (Sigma-Aldrich, St. Louis, MO) and bacterial microcrystalline cellulose (BMCC). RAC was prepared from Avicel as described earlier [39,40]. BMCC was prepared from bacterial cellulose (BC) as described in [34,35].…”

Section: Substratesmentioning

confidence: 99%

Systematic modification of N-glycosylation sites in cellobiohydrolase Cel7A from Trichoderma reesei reveals higher activity and binding affinity on crystalline cellulose

Kołaczkowski

Schaller

Sørensen

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Background: Cellobiohydrolase from glycoside hydrolase family 7 is a major component of commercial enzymatic mixtures for lignocellulosic biomass degradation. For many years, Trichoderma reesei Cel7A (TrCel7A) has served as a model to understand structure-function relationships of processive cellobiohydrolases. The architecture of TrCel7A includes an N-glycosylated catalytic domain, which is connected to a carbohydrate-binding module through a flexible, O-glycosylated linker. Depending on the fungal expression host, glycosylation can vary not only in glycoforms, but also in site occupancy, leading to a complex pattern of glycans, which can affect the enzyme’s stability and kinetics. Results: Two expression hosts, Aspergillus oryzae and Trichoderma reesei, were utilized to successfully express wild-types TrCel7A (WTAo and WTTr) and the triple N-glycosylation site deficient mutants TrCel7A N45Q, N270Q, N384Q (ΔN-glycAo and ΔN-glycTr). Also, we expressed single N-glycosylation site deficient mutants TrCel7A (N45QAo, N270QAo, N384QAo). The TrCel7A enzymes were studied by steady-state kinetics under both substrate- and enzyme-saturating conditions using different cellulosic substrates. The Michaelis constant (KM) was consistently found to be lowered for the variants with reduced N-glycosylation content, and for the triple deficient mutants, it was less than half of the WTs value on some substrates. The ability of the enzyme to combine productively with sites on the cellulose surface followed a similar pattern on all tested substrates. Thus, site density (number of sites per gram cellulose) was 30-60 % higher for the single deficient variants compared to the WT, and about two-fold larger for the triple deficient enzyme. Molecular dynamic simulation of the N-glycan mutants TrCel7A reveled higher number of contacts between CD and cellulose crystal upon removal of glycans at position N45 and N384. Conclusions: The kinetic changes of TrCel7A imposed by removal of N-linked glycans reflected modifications of substrate accessibility. The presence of N-glycans with extended structures increased KM and decreased attack site density of TrCel7A likely due to steric hindrance effect and distance between the enzyme and the cellulose surface, preventing the enzyme from achieving optimal conformation. This knowledge could be applied to modify enzyme glycosylation to engineer enzyme with higher activity on the insoluble substrates.

show abstract

Rate‐limiting step and substrate accessibility of cellobiohydrolase Cel6A from Trichoderma reesei

Cited by 27 publications

References 44 publications

Substrate specificity, regiospecificity, and processivity in glycoside hydrolase family 74

Substrate specificity, regiospecificity, and processivity in glycoside hydrolase family 74

Physical constrains and functional plasticity of cellulases: Linear scaling relationships for a heterogeneous enzyme reaction

Systematic modification of N-glycosylation sites in cellobiohydrolase Cel7A from Trichoderma reesei reveals higher activity and binding affinity on crystalline cellulose

Contact Info

Product

Resources

About