Structural basis for substrate recognition by Erwinia chrysanthemi GH30 glucuronoxylanase

Abstract

Xylanase A from the phytopathogenic bacterium Erwinia chrysanthemi is classified as a glycoside hydrolase family 30 enzyme (previously in family 5) and is specialized for degradation of glucuronoxylan. The recombinant enzyme was crystallized with the aldotetraouronic acid β‐d‐xylopyranosyl‐(1→4)‐[4‐O‐methyl‐α‐d‐glucuronosyl‐(1→2)]‐β‐d‐xylopyranosyl‐(1→4)‐d‐xylose as a ligand. The crystal structure of the enzyme–ligand complex was solved at 1.39 Å resolution. The ligand xylotriose moiety occupies subsites −1, −2 and −3, whereas the methyl glucuronic acid residue attached to the middle xylopyranosyl residue of xylotriose is bound to the enzyme through hydrogen bonds to five amino acids and by the ionic interaction of the methyl glucuronic acid carboxylate with the positively charged guanidinium group of Arg293. The interaction of the enzyme with the methyl glucuronic acid residue appears to be indispensable for proper distortion of the xylan chain and its effective hydrolysis. Such a distortion does not occur with linear β‐1,4‐xylooligosaccharides, which are hydrolyzed by the enzyme at a negligible rate. Database Structural and experimental data are available in the Protein Data Bank database under accession number http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2y24 [45].

Modular Glucuronoxylan-Specific Xylanase with a Family CBM35 Carbohydrate-Binding Module

“…These enzymes have an absolute requirement of methyl-glucuronosyl (MeGlcA) side residues for hydrolysis of xylan, releasing as products xylooligosaccharides substituted in the penultimate xylose from the reducing end (38,47). The crystalline structure of XynA from Erwinia chrysanthemi (43) and XynC from Bacillus subtilis (40) reveals that the accommodation of MeGlcA in the Ϫ2 subsite of the catalytic site is a determinant for substrate recognition of these enzymes. GH30 xylanases are targeted to glucuronic acid branched regions of xylans, unlike the majority of xylanases belonging to families GH10 or GH11, which are not active on densely substituted xylan (32).…”

“…are specific for glucuronoxylan, as they hydrolyze this polymer but are not active on arabinose branched xylan. Analysis of the three-dimensional (3D) structure of these enzymes suggests that the accommodation of a xylopyranosyl residue substituted with methyl-glucuronic acid in the Ϫ2 subsite of the catalytic cleft is a specificity determinant (40,43). GH30 xylanases must play an important role in complementing the action of GH10 and GH11 enzymes in the depolymerization of glucuronoxylans in plant biomass.…”

Evolution of Xylan Substitution Patterns in Gymnosperms and Angiosperms: Implications for Xylan Interaction with Cellulose

“…Xylan was hydrolyzed with Erwinia chrysanthemi GH30 (at concentration 0.1 -0.5 mM) in 0.1 M ammonium acetate buffer, pH 6.0, for 60 min. EcGH30 (Urbániková et al, 2011) was obtained from Novozymes.…”

Structural Analysis of Glucuronoxylan-specific Xyn30D and Its Attached CBM35 Domain Gives Insights into the Role of Modularity in Specificity*

“…2c) and EcXynA (Fig. 2e) have been also reported (28,29). These two complex structures have shown how the MeGXn chain is attached to the active site cavity at the aglycon moiety of the substrate and allowed a detailed description of the different binding subsites.…”

“…Like the few examples of characterized glucuronoxylanases of family GH30, the enzyme shows requirement of methylglucuronic substitutions for catalysis and is not active on arabinoxylans (27). However, unlike the structurally characterized GH30 glucuronoxylanases from Bacillus subtilis and Erwinia chrysanthemi (28,29), which are single domain enzymes, Xyn30D is a modular enzyme. The glucuronoxylanase contains a carbohydrate binding module of the CBM35 family, which is rarely found in xylanases.…”