Location: Sustainable Biofuels and Co-products ResearchTitle: The mechanism by which arabinoxylanases can recognize highly decorated xylans
|LABOUREL, A - Newcastle University|
|CROUCH, L - Newcastle University|
|BRÁSB, J - Avenida Da Universidade Tecnica|
|JACKSON, A - Newcastle University|
|ROGOWSKI, A - Newcastle University|
|GRAV, J - Newcastle University|
|HENRISSAT, B - Aix-Marseille University|
|FONTES, C - Avenida Da Universidade Tecnica|
|GILBERT, H - Newcastle University|
|NAJMUDIN, S - Avenida Da Universidade Tecnica|
|BASLE, A - Newcastle University|
|CUSKIN, F - Newcastle University|
Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2016
Publication Date: 10/14/2016
Publication URL: https://handle.nal.usda.gov/10113/5695457
Citation: Labourel, A., Crouch, L.I., Brásb, J.L., Jackson, A., Rogowski, A., Grav, J., Yadav, M.P., Henrissat, B., Fontes, C.M., Gilbert, H.J., Najmudin, S., Basle, A., Cuskin, F. 2016. The mechanism by which arabinoxylanases can recognize highly decorated xylans. Journal of Biological Chemistry. 291(42):22149-22159.
Interpretive Summary: The degradation of plant cell walls by enzymes is an important biological process of increasing industrial significance. Corn arabinoxylan (AX) is a major component of corn fiber (a cell wall component) consisting of a backbone of xylose units, which are usually decorated with arabinose side chains. It has been shown previously that an arabinoxylanase (enzyme) cleaves arabinoxylans. However, the mechanism of AX recognition by this enzyme has not been well studied. Thus we have studied the specificity of this enzyme towards substrate (arabinoxylans) in detail and provided the molecular basis for its substrate recognition. We have found that this enzyme has an open xylan binding cleft (pocket), which probably allows it to associate with a highly decorated (branched) forms of AX. The study of such specificity of the enzyme has the potential to contribute to the toolbox of biocatalysts required by industries. Thus these studies can exploit corn fiber as a sustainable substrate for generating functional prebiotic oligosaccharides, which promote the growth of beneficial bacteria present in human gut. Corn fiber is a low value by-product of the corn milling process and is currently used as a component of low-value animal feeds. So its utilization for making highly functional products will benefit U.S. corn processors and corn growers. The generation and recovery of additional valuable product from corn milling by-products will also indirectly help to reduce the overall cost of fuel ethanol produced from corn kernels.
Technical 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 beta 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 mature arabinoxylanase, and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilises the catalytic domain. The C-terminal noncatalytic carbohydrate binding module could not be observed in the crystal structure indicating positional flexibility. The structure of the enzyme in complex with Xylp-beta-1,4-Xylp-beta-1,4-Xylp-[alpha-1,3-Araf]-beta- 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 centre. 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 abrogate 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 hydrolysing xylans that are extensively decorated, and which are recalcitrant to classic endo-xylanase attack.