Engineering the acceptor substrate specificity in the xyloglucan endotransglycosylase TmXET6.3 from nasturtium seeds (Tropaeolum majus L.)

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin

“…We used affinity‐purified barley XET isoforms (Figure S1) and high‐performance liquid chromatography (HPLC) with an inert size‐exclusion chromatography column coupled to fluorescent and refractive index detectors to analyse transglycosylation activities. Reactions were monitored via a gradual increase of fluorescence in the XG or hydroxyethyl cellulose (HEC) regions (Stratilová et al ., 2019), where XG and HEC were used as donors, and a variety of oligosaccharides with defined chemistries as acceptors (Figure 2; Scheme S1).…”

“…We identified hetero‐transglycosylation activities in all investigated barley isoforms with the XG‐donor/[α(1‐4)GalA p ] 5 ‐acceptor substrate pair (Figures 3 and S4), except previously characterised HvXET5 (Hrmova et al ., 2007) and nasturtium TmXET6.3 (Stratilová et al ., 2019; Figure 5). To find out if this hetero‐transglycosylation activity could be localised to barley tissues, we fine‐sectioned young barley roots and localised it to the cell walls, using the Alexa Fluor 488‐labelled [α(1‐4)GalA p ] 7 /[α(1‐4)GalA p ] 8 mixture (Figure 1); this validated the RT‐qPCR analyses that HvXET4 and HvXET3 transcripts occurred in root tips (Figure 6).…”

“…It has been postulated that pectin may hinder the unfolding of XG chains and cause deformations of primary cell walls through the mechanical interactions of polymeric networks, as hypocotyl Arabidopsis cells elongate (Abasolo et al ., 2009). These XG‐cellulose or XG‐pectin network associations could be formed through backbone or side‐chain residue hydrogen bonds, van der Waals and electrostatic interactions (Zykwinska et al ., 2008), or covalent linkages between XG and pectins (Cumming et al ., 2005; Popper and Fry, 2008; Cornuault et al ., 2014), cellulose or (1,3;1,4)‐β‐ d ‐glucans (Hrmova et al ., 2007; Mohler et al ., 2013; Simmons et al ., 2015; Stratilová et al ., 2019; Viborg et al ., 2019). These more recent findings explain how the underlying mechanisms facilitate wall elongation, and how expansins may contribute to these processes (Cosgrove, 2005; Abasolo et al ., 2009; Nonogaki, 2019).…”

“…The non‐reducing fragment of the polysaccharide remains covalently bound in the enzyme active site. In the next step, XET enzymes transfer the bound portion of the initial XG substrate onto the non‐reducing end of another XG polysaccharide (Farkaš et al ., 1992; Fry et al ., 1992; Nishitani and Tominaga, 1992), or XG‐derived or alternate XG‐non‐derived oligosaccharides (Ait Mohand and Farkaš, 2006; Hrmova et al ., 2007, 2009; Simmons et al ., 2015; Shinohara et al ., 2017; Stratilová et al ., 2019). The initial polymer that is cleaved by the enzyme is referred to as a donor substrate, while the polysaccharide or oligosaccharide to which the cleaved product is transferred is known as an acceptor substrate.…”

“…In this work, we focus on the barley HvXET3 and HvXET4 isozymes (Kaewthai et al ., 2010; Vaaje‐Kolstad et al ., 2010a,b) to define their homo‐ and hetero‐transglycosylation activities, and extend our previous data on nasturtium TmXET6.3 (Stratilová et al ., 2019). We discover that the barley isoforms exhibited significant hetero‐transglycosylation activities with chemically defined donor and acceptor substrates, explicitly with the fragment of homogalacturonan, the penta‐galacturonide [α(1‐4)GalA p ] 5 as the acceptor.…”