Abstract
The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center. The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.
Highlights
The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance
With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, they are present in GH8 [1, 7]
The GH30 glucuronoxylanases require the Xylp bound at the Ϫ2 to contain a glucuronic acid (GlcA) side chain [9] (the scissile bond targeted by glycoside hydrolases is between subsites Ϫ1 and ϩ1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively [10])
Summary
The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains [11]. In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (Ϫ1 subsite). CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1. This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity. The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases
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