Distinct catalytic capacities of two aluminium-repressed Arabidopsis thaliana xyloglucan endotransglucosylase/hydrolases, XTH15 and XTH31, heterologously produced in Pichia

XTH15 and XTH31, two highly basic Arabidopsis XTHs, were very predominantly a transglycanase (XET) and a glycanase (XEH), respectively. XTH31 had a pH optimum of 4.5 and may contribute to ‘acid growth’. Their substrate preferences are compared and contrasted with those of other XTHs. [Display omitted] •XTH31 has very predominantly glycanase (XEH) activity but also measurable transglycanase (XET).•XTH15 has undetectable XEH activity but high XET activity, with an unexceptional pH optimum of ∼6.0.•XTH31 has a low pH optimum (∼4.5) for both XEH and XET activity and may have an ‘acid growth’ role.•XTH31’s XET activity is compromised by galactosylation on the 2nd Xyl residue, unlike other XTHs.•These XTHs contrast with other Arabidopsis XTHs in pH optimum and acceptor substrate specificity. Xyloglucan plays an important structural role in primary cell walls, possibly tethering adjacent microfibrils and restraining cell expansion. There is therefore considerable interest in understanding the role of xyloglucan endotransglucosylase/hydrolases (XTHs), which are encoded in Arabidopsis by a 33-member gene family. We compared the key catalytic properties of two very different Arabidopsis XTHs (heterologously produced in Pichia), both of which are aluminium-repressed. Reductively tritiated oligosaccharides of xyloglucan were used as model acceptor substrates. Untransformed Pichia produced no xyloglucan-acting enzymes; therefore purification of the XTHs was unnecessary. XTH15, a classical group-I/II XTH, had high XET and undetectable XEH activity in vitro; its XET Km values were 31μM XXXGol (acceptor substrate) and 2.9mg/ml xyloglucan (donor substrate). In contrast, XTH31, a group-III-A XTH, showed predominant XEH activity and only slight XET activity in vitro; its XET Km was 86μM XXXGol (acceptor), indicating a low affinity of this predominantly hydrolytic protein for a transglycosylation acceptor substrate. The Km of XTH31’s XEH activity was 1.6mg/ml xyloglucan. For both proteins, the preferred XET acceptor substrate, among five cellotetraitol-based oligosaccharides tested, was XXXGol. XTH31’s XET activity was strongly compromised when the second Xyl residue was galactosylated. XTH15’s XET activity, in contrast, tolerated substitution at the second Xyl residue. The two enzymes also showed different pH preferences, XTH31 exhibiting an unusually low pH optimum and XTH15 an unusually broad optimum. XTH31’s hydrolase activity increased almost linearly with decreasing pH