Enzymatic characterization of ancestral/group‐IV clade xyloglucan endotransglycosylase/hydrolase enzymes reveals broad substrate specificities

Abstract

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play important roles in cell wall remodelling. Although previous studies have shown a pathway of evolution for XTH genes from bacterial licheninases, through plant endoglucanases (EG16), the order of development within the phylogenetic clades of true XTHs is yet to be elucidated. In addition, recent studies have revealed interesting and potentially useful patterns of transglycosylation beyond the standard xyloglucan-xyloglucan donor/acceptor substrate activities.To study evolutionary relationships and to search for enzymes with useful broad substrate specificities, genes from the 'ancestral' XTH clade of two monocots, Brachypodium distachyon and Triticum aestivum, and two eudicots, Arabidopsis thaliana and Populus tremula, were investigated. Specific activities of the heterologously produced enzymes showed remarkably broad substrate specificities. All the enzymes studied had high activity with the cellulose analogue HEC (hydroxyethyl cellulose) as well as with mixed-link bglucan as donor substrates, when compared with the standard xyloglucan. Even more surprising was the wide range of acceptor substrates that these enzymes were able to catalyse reactions with, opening a broad range of possible roles for these enzymes, both within plants and in industrial, pharmaceutical and medical fields. Genome screening and expression analyses unexpectedly revealed that genes from this clade were found only in angiosperm genomes and were predominantly or solely expressed in reproductive tissues. We therefore posit that this phylogenetic group is significantly different and should be renamed as the group-IV clade.

In silico analysis of XTH gene family from barley (Hordeum vulgare L.) and their comparative expression analysis during germination

“…This finding may indicate that these gene may play some small but specific role in these tissues, or that their highest expression and hence most important role, is in tissues not studied here. HvXET27 is a member of the Group IV clade, which as previously mentioned shows highly tissue specific expression in reproductive tissues (Seven et al, 2021). Two of the Arabidopsis members of Group IV, AtXTH1 & 3, show their highest expression in floral tissues (Becnel et al 2006), suggesting some highly specific role.…”

“…XTH enzymes align closely with each other and form a phylogenetic tree that is comprised of six distinguishable groups; Group I, Group II, Group IIIA, Group IIIB, Group IV, and the XTHprecursor EG16 endoglucanase group (Seven et al 2021). The phylogenetic groups differ from each other not only by their amino acid sequence similarities, but also by their enzymatic activities and substrate preferences.…”

“…Two of the Arabidopsis members of Group IV, AtXTH1 & 3, show their highest expression in floral tissues (Becnel et al 2006), suggesting some highly specific role. Some of these Group IV clade members have been shown to have very high levels of heterotransglycosylation ability (Seven et al 2021) which may indicate that they utilise substrates other than xyloglucan.…”

“…This may be explained by highly specific tissue expression for which the tissue was not represented in these experiments. Indeed, HvXET27 and HvXET30 are members of phylogenetic clade IV, which we have recently shown to contain genes that are preferentially or only expressed in floral tissues (Seven et al 2021), for which there are no barley RNA-seq libraries.…”

“…Despite their transferase ability, XTH enzymes are classified by their structure as members of the Glycoside Hydrolase Family 16 (GH16) in the CAZy database (Lombard et al 2014). When XTH sequences are compared in a phylogenetic tree, several major branches are typically present, including Groups I, II, IIIA, IIIB, and IV (Figure 1; Rose et al 2002;Yokoyama et al 2004;Baumann et al 2007;Seven et al 2021). More recently another clade, Endoglucanase 16 (EG16), has been examined and appears to contain strict endoglucanases that likely represent evolutionary links from a progenitor bacterial gene (Eklöf et al 2013).…”