Carbohydrate Esterase Family 3

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• Author: ^^^Stefen Stanglerhin^^^
• Responsible Curators: ^^^Joel Weadge^^^ and ^^^Michael Suits^^^

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Carbohydrate Esterase Family CE3
Clan	None
Mechanism	N/A
Active site residues	known
CAZy DB link
https://www.cazy.org/CE3.html

Substrate specificities

Carbohydrate Esterase Family 3 (https://www.cazy.org/CE3.html) is currently comprised entirely of de-O-acetylxylan esterases. Xylan is a plant cell-wall polysaccharide composed of β-1,4-linked xylose decorated with α-arabinofuranose and α-glucuronic acid substituents.[1].

Catalytic Residues

Characterized CE3 members are all known to contain the classical catalytic triad of Ser-His-Asp, typical of the SGNH hydrolase family of enzymes.2,3 The active site residues are spread across four conserved consensus sequences (Blocks I-III and V), and lack the GXSXG nucleophilic “elbow” turn motif typical of the related α/β-hydrolase family.4 The catalytic triad, along with the Block II Gly and Block III Asn residues, that comprise the oxyanion hole, are universally conserved across all characterized CE3 enzymes. The Block V Asp residue mediates the amphoteric nature of the Block V His residue, which abstracts a proton from the Block I Ser rendering it nucleophilic.

Kinetics and Mechanism

CE3 esterases catalyze the hydrolysis of O-linked acetyl groups from xylan oligo- and poly-saccharides. The Block V His residue abstracts a proton from the Block I Ser, rendering it nucleophilic, which attacks the electrophilic carbonyl carbon of the substrate’s acetyl group, generating a tetrahedral oxyanion transition state that is stabilized by the backbone amides of the Block I Ser and Block II Gly, as well as the sidechain amide of the Block III Asn, together forming the oxyanion hole in the active site.5 Collapse of the oxyanion intermediate generates an acyl-enzyme intermediate and alcohol by-product.5 A hydrolytic water molecule is then deprotonated by the Block V His residue, and attacks the acyl-enzyme intermediate, hydrolyzing the bond and releasing acetate and the free enzyme.5 In the process, the Ser (Block I) is re-protonated and ready for another catalytic cycle.

Three-dimensional structures

Two members of the CE3 family have been structurally resolved, TcAE206 from Talaromyces cellulolyticus,^5,6 and CtCes3-1 from Clostridium thermocellum.⁷ Both structures adopt an (α/β/α)-sandwich fold typical of the SGNH hydrolase family. The (α/β/α)-sandwich contains five central parallel β-strands forming a curved β-sheet, which is flanked by 5-6 α-helices.⁷ Additionally, both structures contain a calcium binding loop motif (DxVG and DxD/N, respectively) located above the N-terminal end of the central β-strand (β2).⁶ This binding motif is conserved across all currently characterized CE3s. A coordinated zinc ion was also observed next to a calcium ion in a TcAE206_S10A variant, however this was attributed to the use of ZnSO₄ in the crystallization conditions.⁵ Unique to TcAE206 is a disulfide bond formed near the N-terminus that is thought to position the catalytic Ser by stabilizing neighbouring areas, including a β-turn (β1) that involves the catalytic Ser.⁶

Family Firsts

First characterized

In 1994, the sequence of XynB from Ruminococcus flavefaciens 17 was found to be related to family G xylanases.⁸ Later, in 1997, BnaC from Neocallimastix patriciarum was found to have close relation to XynB, and other enzymes known to be members of a diverse family of esterases.⁹ It wasn’t until 2000 that CesA from R. flavefaciens 17, which was shown to have significant sequence identity to XynB, was characterized with the ability to deacetylate acetylated xylans, representing the first characterized enzymes of family 3 CEs.¹⁰

First mechanistic insight

In 2000, CesA, XynB, and BnaC were aligned and shown to contain what was thought to be a Ser-His-Asp catalytic triad responsible for the observed esterase activity.10 This was later confirmed by the structural resolution of CtCes3-1.7

First 3-D structure

The first resolved structure belongs to CtCes3-1 from C. thermocellum, displaying the (α/β/α)-sandwich fold, and Ser-His-Asp catalytic triad typical of SGNH hydrolases.7

References

1. Faik A (2010). Xylan biosynthesis: news from the grass. Plant Physiol. 2010;153(2):396-402. DOI:10.1104/pp.110.154237 |
1. Faik A (2010). Xylan biosynthesis: news from the grass. Plant Physiol. 2010;153(2):396-402. DOI:10.1104/pp.110.154237 |
1. Faik A (2010). Xylan biosynthesis: news from the grass. Plant Physiol. 2010;153(2):396-402. DOI:10.1104/pp.110.154237 |

2. ár2005 pmid=20375115

3. ølgaardet.al2000 pmid=20375115

4. Faik A (2010). Xylan biosynthesis: news from the grass. Plant Physiol. 2010;153(2):396-402. DOI:10.1104/pp.110.154237 |

5. Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. The Biochemist, vol. 30, no. 4., pp. 26-32. Download PDF version.