Research Article: Structure and function of a glycoside hydrolase family 8 endoxylanase from Teredinibacter turnerae

Date Published: October 01, 2018

Publisher: International Union of Crystallography

Author(s): Claire A. Fowler, Glyn R. Hemsworth, Fiona Cuskin, Sam Hart, Johan Turkenburg, Harry J. Gilbert, Paul H. Walton, Gideon J. Davies.


The symbionts of marine shipworms provide a rich reservoir of potential carbohydrate-active enzymes. Here, the 1.5 Å resolution three-dimensional structure of a T. turnerae GH8 xylanase is revealed and its potential in biomass degradation is highlighted.

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The demand for biofuels is increasing amid positive shifts in political and public opinion regarding the growing need for more sustainable fuel sources (discussed, for example, in Somerville, 2007 ▸; Pauly & Keegstra, 2008 ▸). A barrier towards the sustainable and efficient usage of plant biomass for fuel conversion lies in the complexity and recalcitrance of plant cell walls (Himmel et al., 2007 ▸; Bomble et al., 2017 ▸). The heterogeneous matrix of various carbohydrate compounds hinders the enzymatic breakdown of plant cell walls into energy-rich carbohydrate monomers. Crystalline cellulose regions are interspersed with a web of more soluble polysaccharides, known as hemicelluloses. The plant cell-wall matrix is strengthened by a hydrophobic and insoluble barrier: a mixture of phenolic compounds known as lignin (Li et al., 2015 ▸). In nature, the breakdown of lignocellulosic material is achieved through the synergistic action of a wide variety of different enzymes, including glycoside hydrolases and polysaccharide oxygenases (Hemsworth et al., 2015 ▸; Walton & Davies, 2016 ▸). A single organism can produce a consortium of enzymes capable of lignocellulosic degradation or can utilize the symbiotic behaviour of other smaller organisms such as bacteria and fungi (Cragg et al., 2015 ▸). One organism of increasing interest is the marine symbiont Teredinibacter turnerae, which has been found in the gills (Fig. 1 ▸) of at least 24 species of bivalve molluscs (Ekborg et al., 2007 ▸; Horak & Montoya, 2014 ▸).

As with recent studies on xylan utilization by the human microbiota (Rogowski et al., 2015 ▸), the digestive system of bivalve molluscs such as marine wood-boring shipworms has the potential to provide a wealth of carbohydrate-active enzymes with potentially beneficial applications. Here, we have studied a potential GH8 xylanase from the shipworm symbiont T. turnerae, the genome sequence of which (Yang et al., 2009 ▸) unveiled a treasure chest of carbohydrate-active enzymes. We have shown that TtGH8 is a single-domain endo-xylanase with six catalytically relevant subsites that hydrolyses X6 to yield predominantly xylotriose. The enzyme is active on diverse classical β-1,4-xylans, likely reflecting its role in the host digestion of woody biomass after the possible translocation of bacterial proteins from the gills to the connected gastrointestinal tract of its Teredinidae shipworm host (O’Connor et al., 2014 ▸). The enzyme thus bears similarities to the well studied P. haloplanktis ‘cold-adapted’ xylanase, with which TtGH8 shares 33% identity. Intriguingly, TtGH8 shows the highest activity on mixed-linkage β-1,3,β-1,4 xylans, which may reflect a genuine biological adaption to, or at least an accommodation of, these marine substrates. The mixed-linkage marine xylan used in this study is found as a component of the red alga Palmaria palmata, and is a polysaccharide that is involved in mechanical support, development and defence (Viana et al., 2011 ▸). Analysis of this polysaccharide suggests a 1:3 ratio of β-1,3:β-1,4 moieties. Whilst pure β-1,4 bonding of xylose residues would result in a threefold screw-axis helical structure, the irregular distribution of β-1,3 sections between variable lengths of β-1,4 sections may cause disruption of this regular conformation (Viana et al., 2011 ▸). Optical rotation alignment further suggests that mixed-linkage xylan exhibits a ‘random-coil’ structure; unlike linear β-1,4-xylan, which may form interactions with other chains, the presence of β-1,3 linkages introduces flexibility which may assist in the solubility (Viana et al., 2011 ▸; Cerezo et al., 1971 ▸). Flexibility may improve the fitting of the polysaccharide into the V-shaped binding site of TtGH8. Improvement in solu­bility owing to the flexible nature of the xylan chain may be a factor in the increased degradation rate exhibited by TtGH8. We would argue, therefore, that the increased activity on MLX may not necessarily reflect the requirement for a β-1,3-linked xylose at one or more of the subsites, but confers increased solubility and thus enzyme access. It is also possible, given its marine environment, that the specificity of the enzyme has adapted to potential terrestrial xylans (β-1,4-xylans) and marine xylans (MLXs). It should be noted, however, that only kcat/Km values were determined and not the individual kinetic constants. This was because the maximum soluble substrate concentration was much lower than Km, as indicated by the linear relationship between the rate and the substrate concentration. It is possible, therefore, that the difference in activity reflects a variation in Km values that may not reflect the binding affinities but the actual concentrations of available substrate in two very different xylans. MLX has not been extensively used as a substrate in the analysis of xylanase activity. Exploring whether MLXs feature as the optimum substrate for all xylanases, or only those enzymes exposed to a marine system, will provide insight into the environmental selection pressures that influence glycoside hydrolase activity. In a discussion of shipworm larvae, Turner states that whilst shipworm larvae are quick to settle into burrows after extrusion from the adult, most wooden structures are covered in a ‘protective forest’ of various organisms, including algae, in which the young shipworm larvae may swim before settlement (Turner, 1966 ▸). Bacterial symbionts are passed onto shipworm young, so it is possible that algal particles are digested using enzymes such as TtGH8 before or during larvae settlement. Such analyses highlight how shipworms and their symbionts offer a plethora of possibilities for novel enzyme discovery and application for biotechnology and biofuels.




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