Corrigendum to: Tryptophan 272: an essential determinant of crystalline cellulose degradation by <i>Trichoderma reesei</i> cellobiohydrolase Cel6A (FEBS 20361)

Koivula, Anu; Kinnari, Tiina; Harjunpää, Vesa; Ruohonen, Laura; Teleman, Anita; Drakenberg, Torbjörn; Rouvinen, Juha; Jones, T. Alwyn; Teeri, Tuula T.

doi:10.1016/s0014-5793(99)00213-6

Cited by 19 publications

(24 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…3a). Similar conclusions have been reached for aromatic residue function in a number of cellulase and chitinase studies (Igarashi et al, 2009;Koivula et al, 1998;Nakamura et al, 2013;Uchiyama et al, 2001;Zakariassen et al, 2009Zakariassen et al, , 2010.…”

Section: The Role Of Aromatic Residues In the Hydrolysis Of Bcsupporting

confidence: 74%

Cel48A from Thermobifida fusca: Structure and site directed mutagenesis of key residues

Kostylev

Alahuhta

Chen

et al. 2013

Biotech & Bioengineering

View full text Add to dashboard Cite

Lignocellulosic biomass is a potential source of sustainable transportation fuels, but efficient enzymatic saccharification of cellulose is a key challenge in its utilization. Cellulases from the glycoside hydrolase (GH) family 48 constitute an important component of bacterial biomass degrading systems and structures of three enzymes from this family have been previously published. We report a new crystal structure of TfCel48A, a reducing end directed exocellulase from Thermobifida fusca, which shows that this enzyme shares important structural features with the other members of the GH48 family. The active site tunnel entrance of the known GH48 exocellulases is enriched in aromatic residues, which are known to interact well with anhydroglucose units of cellulose. We carried out site-directed mutagenesis studies of these aromatic residues (Y97, F195, Y213, and W313) along with two non-aromatic residues (N212 and S311) also located around the tunnel entrance and a W315 residue inside the active site tunnel. Only the aromatic residues located around the tunnel entrance appear to be important for the ability of TfCel48A to access individual cellulose chains on bacterial cellulose (BC), a crystalline substrate. Both Trp residues were found to be important for the processivity of TfCel48A on BC and phosphoric acid swollen cellulose (PASC), but only W313 appears to play a role in the ability of the enzyme to access individual cellulose chains in BC. When acting on BC, reduced processivity was found to correlate with reduced enzyme activity. The reverse, however, is true when PASC is the substrate. Presumably, higher density of available cellulose chain ends and the amorphous nature of PASC explain the increased initial activity of mutants with lower processivity.

show abstract

Section: The Role Of Aromatic Residues In the Hydrolysis Of Bcsupporting

confidence: 74%

Cel48A from Thermobifida fusca: Structure and site directed mutagenesis of key residues

Kostylev

Alahuhta

Chen

et al. 2013

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…This suggests that the relatively low specific activity (typically < 1 s À1 ) observed for cellulolytic enzymes under normal experimental conditions is not associated with slow conversion of the b-1,4 bond, but must rely on other slow steps including non-covalent changes. It is also interesting to note that the minimum value for k hyd found here is slightly faster than k cat for the conversion of soluble cellooligosaccharides by Cel6A [8,32]. This faster conversion of a solid substrate may appear counterintuitive in light of the intermolecular interactions in the cellulose particle.…”

Section: Rate-limiting Stepmentioning

confidence: 66%

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

et al. 2018

View full text Add to dashboard Cite

The cellobiohydrolase (CBH) Cel6A is an important component of enzyme cocktails for industrial degradation of lignocellulosic biomass. However, the kinetics of this enzyme acting on its natural, insoluble substrate remains sparsely investigated. Here, we studied Cel6A from Trichoderma reesei with respect to adsorption, processivity, and kinetics both in the steady‐state and pre‐steady‐state regimes, on microcrystalline and amorphous cellulose. We found that slow dissociation (koff) was limiting the overall reaction rate, and we suggest that this leads to an accumulation of catalytically inactive complexes in front of obstacles and irregularities on the cellulose surface. The processivity number of Cel6A was low on both investigated substrates (5–10), and this suggested a rugged surface with short obstacle‐free path lengths. The turnover of the inner catalytic cycle (the reactions of catalysis in one processive step) was too fast to be fully resolved, but a minimum value of about 20 s−1 could be established. This is among the highest values reported hitherto for a cellulase, and it underscores the catalytic efficiency of Cel6A. Conversely, we found that Cel6A had a poor ability to recognize attack sites on the cellulose surface. On amorphous cellulose, for example, Cel6A was only able to initiate hydrolysis on about 4% of the sites to which it could adsorb. This probably reflects high requirements of Cel6A to the architecture of the site. We conclude that compared to the other CBH, Cel7A, secreted by T. reesei, Cel6A is catalytically more efficient but less capable of attacking a broad range of structurally distinct sites on the cellulose surface. Enzymes TrCel6A, nonreducing end‐acting cellobiohydrolase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/91.html) from Trichoderma reesei; TrCel7A, reducing end‐acting cellobiohydrolase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/176.html) from T. reesei.

show abstract

“…The W332Y mutation reduced activity on BMCC more than on the other substrates. The corresponding T. reesei Cel6A mutations (W272A/D) [49] also specifically reduced hydrolysis of BMCC but not of CMC and SC. This is further evidence that the W332 residue, located near the entrance of the active-site tunnel, may assist in hydrolysis of crystalline cellulose by helping a chain enter the active site.…”

Section: Discussionmentioning

confidence: 98%

Site‐directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B

Zhang¹,

Irwin

Wilson

2000

European Journal of Biochemistry

View full text Add to dashboard Cite

Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2,4-dinitrophenyl-b-d-cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites 22, 11 and 12, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B.

show abstract

Corrigendum to: Tryptophan 272: an essential determinant of crystalline cellulose degradation by Trichoderma reesei cellobiohydrolase Cel6A (FEBS 20361)

Cited by 19 publications

References 9 publications

Cel48A from Thermobifida fusca: Structure and site directed mutagenesis of key residues

Cel48A from Thermobifida fusca: Structure and site directed mutagenesis of key residues

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

Site‐directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B

Contact Info

Product

Resources

About