Carbohydrate Esterase Family 4

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• Author: ^^^Alex Anderson^^^
• Responsible Curator: ^^^Michael Suits^^^

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Carbohydrate Esterase Family 4
Clan	GH-x
Mechanism	retaining/inverting
Active site residues	known
CAZy DB link
https://www.cazy.org/CE4.html

Substrate specificities

CE family 4 esterases catalyze the de-acylation of polysaccharides. Known activities of CE family 4 members include acetylxylan esterases, chitin deacetylases, chitooligosaccharide deacetylases, and peptidoglycan deacetylases. Peptidoglycan, the essential bacterial cell wall polymer, consists of alternating β-(1,4) linked N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-muramic acid (MurNAc) [1]. All but one of the characterized PG deacetylases belonging to CE family 4 deacetylate GlcNAc. The one characterized exception, PdaA from Bacillus subtilis, deacetylates peptidoglycan MurNAc [2].

Kinetics and Mechanism

CE4 enzymes cleave acetyl groups by binding a catalytic water molecule on their metal cofactor [3] (usually Zn²⁺, but Co²⁺ is observed in some structures [4]). The catalytic base residue is responsible for proton abstraction from the catalytic water, and generates a nucleophilic attack on the carbonyl carbon of the acetyl substrate by that water molecule [3]. The resulting intermediate is a tetrahedral oxyanion, stabilized by the metal cofactor [3]. The catalytic acid residue then protonates the nitrogen atom of the intermediate, generating the free amine, along with the acetate product bound to the metal cofactor [3].

Catalytic Residues

Characterized CE4 members have been shown to possess a prototypical NodB conserved domain, containing an Asp-His-His triad responsible for coordinating a Zn²⁺ ion, an Asp residue as the catalytic base, and a His residue as the catalytic acid [3]. These catalytic residues are invariant in all characterized CE4 family members, with the exception of two homologous acetylxylan esterases, the enzymes SlCE4 from Streptomyces lividans and CtCE4 from Clostridium thermocellum [4]. Both SlCE4 and CtCE4 showed preference for Co²⁺ in place of Zn²⁺, and in CtCE4, four water molecules assisted two Asp-His residues in coordination of the Co²⁺ in place of the His-His-Asp triad typical for the family [4].

Three-dimensional structures

All CE4 members contain a NodB domain that houses the catalytic core. This NodB domain contains the triad responsible for coordinating the essential metal cofactor along with the catalytic acid/base pair, all of which are conserved and located in five separate motifs shared across CE4 enzymes. This NodB domain adopts a (β/α)₇ fold in all known structures. A notable exception for the family is PgdA from Streptococcus pneumoniae [3]. PgdA, a peptidoglycan GlcNAc deacetylase, contains the canonical NodB catalytic domain at its C-terminal, but the N and C termini are at opposite ends of the barrel as compared to other known CE4 structures [3]. PgdA is also atypical of the family in that it possesses two accessory domains that are not found in other CE4 members, nor shown any significant predicted to homology to non-CE4 members [3]. Other CE family 4 enzymes show considerable structural diversity outside their catalytic NodB domain, with some representative members also containing accessory CBM [5], β sandwich [6], α-helical [7], and α/β [3] domains, although they are less common. In those CE4 members that do possess accessory domains, they are typically appended to the C-terminal [8], although presence at the N-terminal [7], or flanking the NodB domain has been observed [3]. The length of CE4 enzymes are thus variable, but commonly near 300 residues in a prototypical, unappended CE4 member, but can range to as large as 700 residues in those appended with accessory domain(s).

Family Firsts

First characterized

TLC and HPLC experiments demonstrated that rhizobial NodB was a chitooligosaccharide deacetylase, and that it did not deacetylate GlcNAc monomers, only chitooligomers, and only at their nonreducing end [9].

First mechanistic insight

The highly conserved His-His-Asp triad and His-Asp acid/base pair were first demonstrated to be catalytic in the structure of the SpPdgA peptidoglycan deacetylase from S. pneumoniae [3].

First 3-D structure

The first CE4 structure, the peptidoglycan N-acetylmuramic acid deacetylase from B. subtilis, demonstrates the canonical NodB domain adopting the (β/α)₇ fold, along with the His-His-Asp triad and the catalytic His/Asp acid/base pair [10].

References

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