Enzymatic Activity of Xyloglucan Xylosyltransferase 5

Abstract

ORCID IDs: 0000-0002-5107-9009 (R.F.); 0000-0001-6938-6923 (O.A.Z.).Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a b-(1,4)-glucan backbone decorated with D-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher K m and 9-fold lower k cat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone.

Structure of xyloglucan xylosyltransferase 1 reveals simple steric rules that define biological patterns of xyloglucan polymers

“…S9). Hence, as long as 6 GGXXGG 1 is the preferred product in the pool of doubly modified cellohexaoses, the addition of a third xylosyl moiety will never exceed 50%, as experimentally observed (33).…”

Metatranscriptomic and metabolite profiling reveals vertical heterogeneity within a Zygnema green algal mat from Svalbard (High Arctic)

“…Similar to expansins, xyloglucan endotransglucosylases act on the xyloglucans of the cell wall to loosen those polymers and enable modifications (Van Sandt et al ., ). Xyloglucan xylosyltransferases, on the other hand, are involved in biosynthesis of xyloglucan, one of the most abundant hemicellulosic components of the cell wall (Culbertson et al ., ). In Z .…”

“…Similar to expansins, xyloglucan endotransglucosylases act on the xyloglucans of the cell wall to loosen those polymers and enable modifications (Van Sandt et al, 2007). Xyloglucan xylosyltransferases, on the other hand, are involved in biosynthesis of xyloglucan, one of the most abundant hemicellulosic components of the cell wall (Culbertson et al, 2016). In Z. circumcarinatum, the occurrence of xyloglucan: xyloglucan endotransglucosylase, for instance, was observed in young longitudinal cell walls (1 month) but not in old cells (1 year).…”

Analyzing Xyloglucan Endotransglycosylases by Incorporating Synthetic Oligosaccharides into Plant Cell Walls

“…It is important to note that At XXT2 is part of a five‐member gene family in A. thaliana ; therefore, the possibility remains that one of the other XXTs might be capable of catalyzing the addition of xylose at the terminal glucose. However, previous reports suggested that XXT1, XXT2, and XXT5 have similar substrate specificities . We therefore hypothesize that the terminal glucose residue at the nonreducing end of xyloglucan polysaccharides is not xylosylated during biosynthesis, but might be trimmed later by β‐glucosidases .…”