Cellulose 1,4-beta-cellobiosidase (non-reducing end)

 

Cellobiohydrolase enzymes depolymerise cellulose into its fundamental repeating unit of two glucose molecules (cellobiose). These enzymes are key components of fungal cellulase multienzyme systems; together with randomly-acting endo-1,4-beta-glucanases (which cut internal beta-1,4-glucosidic bonds) they hydrolyse cellulose via a processive mechanism, forming cellobiose as a major product. Cellobiose is then converted by beta-glucosidases to glucose; the latter product may further be fermented to ethanol or other liquid fuels and chemicals.

These enzymes suffer from inhibition by this product, which lingers in the enzymes’ active sites and thus delays their catalytic cycles. Cellobiose accumulates over the course of the reaction unless removed by an enzyme such as beta-glucosidase, which cleaves cellobiose yielding two glucose molecules.

This entry represents the Glycoside hydrolase, family 7 (GH7).

 

Reference Protein and Structure

Sequence
P62694 UniProt (3.2.1.91) IPR001722 (Sequence Homologues) (PDB Homologues)
Biological species
Trichoderma reesei (Fungus) Uniprot
PDB
1cel - THE THREE-DIMENSIONAL CRYSTAL STRUCTURE OF THE CATALYTIC CORE OF CELLOBIOHYDROLASE I FROM TRICHODERMA REESEI (1.8 Å) PDBe PDBsum 1cel
Catalytic CATH Domains
2.70.100.10 CATHdb (see all for 1cel)
Click To Show Structure

Enzyme Reaction (EC:3.2.1.91)

water
CHEBI:15377ChEBI
+
cellotetraose
CHEBI:62974ChEBI
→
cellobiose
CHEBI:17057ChEBI
Alternative enzyme names: 1,4-beta-glucan cellobiosidase, Beta-1,4-glucan cellobiohydrolase, Beta-1,4-glucan cellobiosylhydrolase, C1 cellulase, CBH 1, Avicelase, Cellobiohydrolase, Cellobiohydrolase I, Cellobiosidase, Exo-beta-1,4-glucan cellobiohydrolase, Exo-cellobiohydrolase, Exoglucanase, Exocellobiohydrolase, 1,4-beta-cellobiohydrolase, Exo-1,4-beta-D-glucanase,

Enzyme Mechanism

Introduction

Mechanism thought to be similar (but not identical) to that of the non-homologous alpha-amylase.

The two glutamate residues act as the catalytic nucleophile (Glu212) and general acid/base (Glu217), whereas the aspartate and histidine are thought to modulate the pKa of the catalytic nucleophile. It has been determined that Asp214 and His228 are also critical in shuttling the catalytic water molecule from the bulk solvent into the active site. The water channel has been shown to involve Glu212, Asp214, Thr226, and His228.

In the protein's ground state, Glu212 is negatively charged; Asp214 and Glu217 are protonated.

Catalytic Residues Roles

UniProt PDB* (1cel)
Asp231 Asp214A Thought to modulate the pKa of the catalytic nucleophile. It has also been shown to act as "an elevator" to transport outer water molecules into the hydrolysis site for every other glycosidic bond. Finally,, this residue has been implicated in the enantioselectivity of the protein. modifies pKa
Glu229 Glu212A Acts as the catalytic nucleophile. In the enzyme's ground state, this residue is negatively charged (approx pKa of 2.4). covalent catalysis
Glu234 Glu217A Acts as a general acid/base. proton shuttle (general acid/base)
His245 His228A Thought to be involved in the perturbation of the general acid/base's pKa. Also shown to be involved in shuttling the catalytic water into the active site. modifies pKa
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Ståhlberg J et al. (2001), J Mol Biol, 305, 79-93. Structural basis for enantiomer binding and separation of a common β-blocker: crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 Å resolution. DOI:10.1006/jmbi.2000.4237. PMID:11114249.
  2. Sun X et al. (2017), Biopolymers, 107, 46-60. Investigation of an “alternate water supply system” in enzymatic hydrolysis in the processive endocellulase Cel7A from R asamsonia emersonii by molecular dynamics simulation. DOI:10.1002/bip.22991. PMID:27696356.
  3. Dotsenko AS et al. (2016), Biotechnol Bioeng, 113, 283-291. N-linked glycosylation of recombinant cellobiohydrolase I (Cel7A) fromPenicillium verruculosumand its effect on the enzyme activity. DOI:10.1002/bit.25812. PMID:26301455.
  4. Bodenheimer AM et al. (2016), FEBS Lett, 590, 4429-4438. Crystal structures of wild-type Trichoderma reesei Cel7A catalytic domain in open and closed states. DOI:10.1002/1873-3468.12464. PMID:27943301.
  5. Atreya ME et al. (2016), Biotechnol Bioeng, 113, 330-338. Alleviating product inhibition in cellulase enzyme Cel7A. DOI:10.1002/bit.25809. PMID:26302366.
  6. Olsen JP et al. (2016), Biotechnol Bioeng, 113, 1178-1186. Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose. DOI:10.1002/bit.25900. PMID:26636743.
  7. Moroz OV et al. (2015), Acta Crystallogr F Struct Biol Commun, 71, 114-120. The three-dimensional structure of the cellobiohydrolase Cel7A fromAspergillus fumigatusat 1.5 Å resolution. DOI:10.1107/s2053230x14027307. PMID:25615982.
  8. Kari J et al. (2014), J Biol Chem, 289, 32459-32468. Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity. DOI:10.1074/jbc.m114.604264. PMID:25271162.
  9. Qi F et al. (2014), Appl Environ Microbiol, 80, 3962-3971. Deciphering the Effect of the Different N-Glycosylation Sites on the Secretion, Activity, and Stability of Cellobiohydrolase I from Trichoderma reesei. DOI:10.1128/aem.00261-14. PMID:24747898.
  10. Nakamura A et al. (2013), J Biol Chem, 288, 13503-13510. The Tryptophan Residue at the Active Site Tunnel Entrance of Trichoderma reesei Cellobiohydrolase Cel7A Is Important for Initiation of Degradation of Crystalline Cellulose. DOI:10.1074/jbc.m113.452623. PMID:23532843.
  11. Textor LC et al. (2013), FEBS J, 280, 56-69. Joint X-ray crystallographic and molecular dynamics study of cellobiohydrolase I fromTrichoderma harzianum: deciphering the structural features of cellobiohydrolase catalytic activity. DOI:10.1111/febs.12049. PMID:23114223.
  12. Pingali SV et al. (2011), J Biol Chem, 286, 32801-32809. Small Angle Neutron Scattering Reveals pH-dependent Conformational Changes in Trichoderma reesei Cellobiohydrolase I: IMPLICATIONS FOR ENZYMATIC ACTIVITY. DOI:10.1074/jbc.m111.263004. PMID:21784865.
  13. Igarashi K et al. (2009), J Biol Chem, 284, 36186-36190. High Speed Atomic Force Microscopy Visualizes Processive Movement of Trichoderma reesei Cellobiohydrolase I on Crystalline Cellulose. DOI:10.1074/jbc.m109.034611. PMID:19858200.
  14. Mulakala C et al. (2005), Proteins, 60, 598-605. Hypocrea jecorina (Trichoderma reesei) Cel7A as a molecular machine: A docking study. DOI:10.1002/prot.20547. PMID:16001418.
  15. Boer H et al. (2003), Eur J Biochem, 270, 841-848. The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A. PMID:12603317.
  16. Becker D et al. (2001), Biochem J, 356, 19-30. Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/ A224H/L225V/T226A/D262G mutant. PMID:11336632.
  17. Hedeland M et al. (1999), J Chromatogr A, 864, 1-16. Chromatographic evaluation of structure selective and enantioselective retention of amines and acids on cellobiohydrolase I wild type and its mutant D214N. DOI:10.1016/s0021-9673(99)00968-1.
  18. Divne C et al. (1998), J Mol Biol, 275, 309-325. High-resolution crystal structures reveal how a cellulose chain is bound in the 50 Å long tunnel of cellobiohydrolase I from Trichoderma reesei. DOI:10.1006/jmbi.1997.1437. PMID:9466911.
  19. Ståhlberg J et al. (1996), J Mol Biol, 264, 337-349. Activity Studies and Crystal Structures of Catalytically Deficient Mutants of Cellobiohydrolase I fromTrichoderma reesei. DOI:10.1006/jmbi.1996.0644. PMID:8951380.
  20. Divne C et al. (1994), Science, 265, 524-528. The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei. DOI:10.2210/pdb1cel/pdb. PMID:8036495.
  21. Konstantinidis AK et al. (1993), Biochem J, 291 ( Pt 3), 883-888. Hydrolyses of alpha- and beta-cellobiosyl fluorides by cellobiohydrolases of Trichoderma reesei. PMID:8489514.
  22. Tomme P et al. (1989), FEBS Lett, 243, 239-243. Identification of a functionally important carboxyl group in cellobiohydrolase I fromTrichoderma reesei. DOI:10.1016/0014-5793(89)80136-x.

Step 1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu212A covalent catalysis
Glu217A proton shuttle (general acid/base)
Asp214A modifies pKa
His228A modifies pKa

Chemical Components

Step 1.

Contributors

Nozomi Nagano, Gemma L. Holliday, Craig Porter