Substrate Recognition and Specificity of Chitin Deacetylases and Related Family 4 Carbohydrate Esterases

Abstract

Carbohydrate esterases family 4 (CE4 enzymes) includes chitin and peptidoglycan deacetylases, acetylxylan esterases, and poly-N-acetylglucosamine deacetylases that act on structural polysaccharides, altering their physicochemical properties, and participating in diverse biological functions. Chitin and peptidoglycan deacetylases are not only involved in cell wall morphogenesis and remodeling in fungi and bacteria, but they are also used by pathogenic microorganisms to evade host defense mechanisms. Likewise, biofilm formation in bacteria requires partial deacetylation of extracellular polysaccharides mediated by poly-N-acetylglucosamine deacetylases. Such biological functions make these enzymes attractive targets for drug design against pathogenic fungi and bacteria. On the other side, acetylxylan esterases deacetylate plant cell wall complex xylans to make them accessible to hydrolases, making them attractive biocatalysts for biomass utilization. CE4 family members are metal-dependent hydrolases. They are highly specific for their particular substrates, and show diverse modes of action, exhibiting either processive, multiple attack, or patterned deacetylation mechanisms. However, the determinants of substrate specificity remain poorly understood. Here, we review the current knowledge on the structure, activity, and specificity of CE4 enzymes, focusing on chitin deacetylases and related enzymes active on N-acetylglucosamine-containing oligo and polysaccharides.

Keywords: carbohydrate esterases; chitin deacetylases; chitooligosaccharides; chitosan; deacetylation pattern; peptidoglycan; structure; substrate specificity.

Figure 1

Structures of the substrates of CE4 enzymes and representative deacetylated products. (A) Chitin oligosaccharide substrate of chitin deacetylases; (B) Peptidoglycan fragment substrate of peptidoglycan MurNAc deacetylases (a) or peptidoglycan GlcNAc deacetylases (b); (C) Acetyl-d-glucurono-d-xylan substrate of acetylxylan esterases; (D) β-1,6-Glucan substrate of poly-β-1,6-N-acetylglucosamine deacetylases.

Figure 2

Modes of enzymatic action patterns for polysaccharide and oligosaccharide deacetylases: multiple-attack, multiple-chain, and single-chain mechanisms.

Figure 3

Three-dimensional structures by X-ray crystallography of the CE4 enzymes listed in Table 1. The VpCDA structure (3WX7) is essentially identical to that of VcCDA. Loops are colored as in Figure 4.

Figure 4

Multiple sequence alignment of the CE4 enzymes listed in Table 1. Loops are highlighted with colored boxes according to [62]. Conserved catalytic motifs are labelled MT1–5. The “His–His–Asp” metal binding triad (▼), catalytic base (*), and catalytic acid (◊) are labelled. The mark inside Loop 5 for poly-β-1,6-GlcNAc deacetylases (four last sequences) indicates the shuffling point of the circularly permuted CE4 domain.

Figure 5

Conserved catalytic motifs MT1–5 of the CE4 family. (Left) Spatial disposition in the 3D active site structure; (Right) Motif sequences for the enzymes listed in Table 1. Subfamilies separated by a line: CDAs, peptidoglycan GlcNAc deacetylases, peptidoglycan MurNAc deacetylases, unknown, acetylxylan esterases, and poly-β-1,6-GlcNAc deacetylases.

Figure 6

(A) Active site residues in the X-ray structure of the VcCDA·DP2 complex, showing Zn²⁺ coordination and substrate binding; (B) Metal-assisted general acid/base mechanism proposed for CE4 deacetylases. Scheme based on the 3D structure of the enzyme–substrate complex VcCDA_D39S·DP2 [62]. D39 is the general base and His295 is the general acid.

Figure 7

Crystallographic structure of (A) VcCDA in the unliganded form (free enzyme with Zn²⁺ and acetate); (B) Binary complexes with DP2; and (C) DP3 ligands; (D) Superimposition of the three structures. Loop 4 (brown) has different conformations; (E) Magnification of the active site Loop 4 in the unliganded form (blue), and in enzyme–substrate complexes with DP2 (yellow) and DP3 (red) ligands. Only the DP2 ligand is shown.

Figure 8

(A) VcCDA structure with labelled loops 1 to 6. Loops 1, 2, and 6 shape the non-reducing end (negatives) subsites, and Loops 3, 4, and 6 define the reducing end (positives) subsites; (B) Superposition of all 3D structures of CE4 enzymes with solved X-ray structure (Table 1). The core of the proteins (in grey) is highly conserved, and main differences are on the loops surrounding the binding site cleft. Loops colored as in A; (C) Comparison of topology of Loops 1 to 6 for the enzymes overlaid in B.