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Carbohydrate Esterase Family 2

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Carbohydrate Esterase Family CE2
Clan
Mechanism
Active site residues
CAZy DB link
http://www.cazy.org/CE3.html

Substrate specificities

All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Acc) [1, 2]. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases [3].

Catalytic Residues

Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. CjCE2A is an exception and contains a Ser-His-Asp catalytic triad [1]. The structurally characterized, CtCE2, CjCE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity [1, 2]. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases [1, 2].

Kinetics and Mechanism

Three-dimensional structures

There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include Clostridium thermocellum’s CtCE2 (PDB ID 2WAO), Cellvibrio japonicusCjCE2A (PDB ID 2WAA) and CjCE2B (PDB ID 2W9X), and Butyrivibrio proteoclasticus’ Est2A (PDB ID 3U37). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) [1, 2]. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of CtCE2, CjCE2A, and CjCE2B, the sheet has 5 α-helices in total packed on each side [1], but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively [2].


The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure [1, 2]. The overall structure of CtCE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, CtCel5C (PDB ID 4IM4), which make up a modular protein, called, CtCel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in C. thermocellum [1].

Family Firsts

First characterized
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First mechanistic insight
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First 3-D structure
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References

  1. Montanier C, Money VA, Pires VM, Flint JE, Pinheiro BA, Goyal A, Prates JA, Izumi A, Stålbrand H, Morland C, Cartmell A, Kolenova K, Topakas E, Dodson EJ, Bolam DN, Davies GJ, Fontes CM, and Gilbert HJ. (2009). The active site of a carbohydrate esterase displays divergent catalytic and noncatalytic binding functions. PLoS Biol. 2009;7(3):e71. DOI:10.1371/journal.pbio.1000071 | PubMed ID:19338387 [Montanier2009]
  2. Till M, Goldstone DC, Attwood GT, Moon CD, Kelly WJ, and Arcus VL. (2013). Structure and function of an acetyl xylan esterase (Est2A) from the rumen bacterium Butyrivibrio proteoclasticus. Proteins. 2013;81(5):911-7. DOI:10.1002/prot.24254 | PubMed ID:23345031 [Till2013]
  3. Topakas E, Kyriakopoulos S, Biely P, Hirsch J, Vafiadi C, and Christakopoulos P. (2010). Carbohydrate esterases of family 2 are 6-O-deacetylases. FEBS Lett. 2010;584(3):543-8. DOI:10.1016/j.febslet.2009.11.095 | PubMed ID:19968989 [Topakas2010]
  4. Dalrymple BP, Cybinski DH, Layton I, McSweeney CS, Xue GP, Swadling YJ, and Lowry JB. (1997). Three Neocallimastix patriciarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases. Microbiology (Reading). 1997;143 ( Pt 8):2605-2614. DOI:10.1099/00221287-143-8-2605 | PubMed ID:9274014 [Dalrymple1997]
  5. Hall J, Hazlewood GP, Barker PJ, and Gilbert HJ. (1988). Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity. Gene. 1988;69(1):29-38. DOI:10.1016/0378-1119(88)90375-7 | PubMed ID:3066698 [Hall1988]
  6. Bayer EA, Belaich JP, Shoham Y, and Lamed R. (2004). The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. Annu Rev Microbiol. 2004;58:521-54. DOI:10.1146/annurev.micro.57.030502.091022 | PubMed ID:15487947 [Bayer2004]

All Medline abstracts: PubMed