Crystallographic insight into the evolutionary origins of xyloglucan endotransglycosylases and endohydrolases

Abstract

SUMMARY The xyloglucan endo-transglycosylase/hydrolase (XTH) gene family encodes enzymes of central importance to plant cell wall remodelling. The evolutionary history of plant XTH gene products is incompletely understood vis-à-vis the larger body of bacterial endo-glycanases in Glycoside Hydrolase Family 16 (GH16). To provide molecular insight into this issue, high-resolution X-ray crystal structures and detailed enzyme kinetics of an extant transitional plant endo-glucanase (EG) were determined. Functionally intermediate between plant XTH gene products and bacterial licheninases of GH16, Vitis vinifera EG16 (VvEG16) effectively catalyzes the hydrolysis of the backbones of two dominant plant cell wall matrix glycans, xyloglucan (XyG) and β(1,3)/β(1,4)-mixed-linkage glucan (MLG). Crystallographic complexes with extended oligosaccharide substrates reveal the structural basis for the accommodation of both unbranched, mixed-linked (MLG) and highly decorated, linear (XyG) polysaccharide chains in a broad, extended active-site cleft. Structural comparison with representative bacterial licheninases, a xyloglucan endo-tranglycosylase (XET), and a xyloglucan endo-hydrolase (XEH) outline the functional ramifications of key sequence deletions and insertions across the phylogenetic landscape of GH16. Although the biological role(s) of EG16 orthologs remains to be fully resolved, the present biochemical and tertiary structural characterization provides key insight into plant cell wall enzyme evolution, which will continue to inform genomic analyses and functional studies across species.

Molecular Mechanism by which Prominent Human Gut Bacteroidetes Utilize Mixed-Linkage Beta-Glucans, Major Health-Promoting Cereal Polysaccharides

“…Thus, BoGH16 MLG specifically hydrolyzes β(1,4)-linkages of glycosyl residues that are immediately preceded by a β(1,3)-linked glucosyl residue (toward the non-reducing chain end). This specificity is typical of bacterial endo -MLGases within GH16 (Gaiser et al, 2006; Mcgregor et al, 2017; Planas, 2000). …”

Comprehensive cross‐genome survey and phylogeny of glycoside hydrolase family 16 members reveals the evolutionary origin of EG16 and XTH proteins in plant lineages

“…The genomic databases were limited with regard to lycophyte genomes, with only two available, Selaginella kraussiana and Selaginella moellendorffii , of which S. moellendorffi had a known EG16 homolog (McGregor et al ., ). Of 21 lycophyte transcriptomes, 12 had an EG16 homolog (57%) whereas all had XTH homologs (Figure ).…”

“…Maximum‐likelihood phylogeny of EG 16 members: 337 EG 16s, 38 EG 16‐2, 28 XTH s (a minimal random selection representing the diversity of plant clades), three red algae putative MLG ases and three bacterial MLG ase high‐quality sequences were aligned with muscle and manual structure‐guided refinement (see Appendix 4 for all input sequences and accessions) prior to RA x ML analysis with 406 rapid bootstraps. The bacterial MLG ases, the ancestors of which are proposed to have given rise to XTH s and EG 16s (Planas, ; Eklöf et al ., ; McGregor et al ., ), were used as an outgroup to root the phylogeny. The output file in nexus format is provided as Appendix S7.…”

“…The original hypothesis was that a possible evolutionary pathway to extant plant GH16 members could be: bacterial MLGase → ancestral plant EG16 (via loop deletion) → plant XTHs (via C‐terminal extension and gene duplication) (Eklöf et al ., ). Such a pathway might reasonably be anticipated from three‐dimensional structural analysis of these proteins (Eklöf et al ., ; McGregor et al ., ; Figure ) and consideration of gene copy numbers in species. The evolution of a comparatively restricted number of XEHs as a result of key active‐site loop extension within the larger group of XETs was previously revealed by combined biochemical, structural and phylogenetic analyses (Baumann et al ., ).…”

“…Second, in plants that encode an EG16, only a single ortholog is present in the genome, whereas XTH homologs are usually present in groups of 20–60 genes per species (Figure ; Eklöf and Brumer, ). An additional curiosity is that EG16 members lack apoplastic secretion signal peptides, which are ubiquitous in XETs and XEHs, thus precluding the definitive attribution of a cell‐wall modifying role (Eklöf et al ., ; McGregor et al ., ). As such, their biological role remains unclear.…”