Xyloglucan Xylosyltransferase 1 Displays Promiscuity Toward Donor Substrates During in Vitro Reactions

Abstract

Glycosyltransferases (GTs) are a large family of enzymes that add sugars to a broad range of acceptor substrates, including polysaccharides, proteins, and lipids, by utilizing a wide variety of donor substrates in the form of activated sugars. Individual GTs have generally been considered to exhibit a high level of substrate specificity, but this has not been thoroughly investigated across the extremely large set of GTs. Here we investigate Xyloglucan Xylosyltransferase 1 (XXT1), a GT involved in synthesis of the plant cell wall polysaccharide, xyloglucan. Xyloglucan has a glucan backbone, with initial side chain substitutions exclusively composed of xylose from UDP-Xylose. While this conserved substitution pattern suggests a high substrate specificity for XXT1, our in vitro kinetic studies elucidate a more complex set of behavior. Kinetic studies demonstrate comparable kcat values for reactions with UDP-Xylose and UDP-Glucose, while reactions with UDP-Arabinose and UDP-Galactose are over 10-fold slower. Using kcat/Km as a measure of efficiency, UDP-Xylose is 8-fold more efficient as a substrate than the next best alternative, UDP-Glucose. To the best of our knowledge, we are the first to demonstrate that not all plant XXTs are highly substrate specific, and some do show significant promiscuity in their in vitro reactions. Kinetic parameters alone likely do not explain the high substrate selectivity in planta, suggesting there are additional control mechanisms operating during polysaccharide biosynthesis. Improved understanding of substrate specificity of the GTs will aid in protein engineering, development of diagnostic tools, and understanding of biological systems.

The biosynthesis, degradation, and function of cell wall β‐xylosylated xyloglucan mirrors that of arabinoxyloglucan

“…Supporting this idea, the same phenomenon has been observed in the unrelated GT61 family (Cenci et al ., 2018 ; Zhong et al ., 2022 ). Generally, many glycosyltransferases exhibit some level of donor substrate promiscuity (Laursen et al ., 2018 ; Ohashi et al ., 2018 ; Biswas & Thattai, 2020 ; Ehrlich et al ., 2021 ), so the effective activity could depend to some extent on the availability of the relevant sugar nucleotides. Interestingly, however, and in contrast to glycosyl hydrolases, there is not yet any evidence for Ara f /Xyl promiscuity in any member of the GT47 or GT61 families.…”

A chromosome-scale genome assembly of turmeric provides insights into curcumin biosynthesis and tuber formation mechanism

“…Our RNA-seq analysis revealed numerous genes were significantly up-regulated expressed in Tu1-Tu3 stage involved in carbohydrate metabolism, such as XylT, GBSS and SWEET. XylT participates in the biosynthesis of plant cell wall xyloglucan, which is the abundant hemicellulosic component of the primary cell wall (Ehrlich et al;Ding et al, 2020). A high correlation between starch and sucrose content and tuber size.…”

Xyloglucan Biosynthesis: From Genes to Proteins and Their Functions

“…Another study also using in vitro assays demonstrated that XXT1 is capable of using multiple activated sugars as donor substrates: UDP-Glc, UDP-Gal, and UDP-Ara in addition to UDP-Xyl ( Ehrlich et al, 2021 ). It is worth to note that the rates of XXT1 catalysis using other nucleotide sugars were significantly lower in comparison to UDP-Xyl.…”

“…Combined with localization evidence, the GTs likely interchange throughout different cisternae. It is highly likely these multiprotein complexes are not required for GT catalytic activity, as many of them are active alone in solution in in vitro assays ( Culbertson et al, 2016 , 2018 ; Rocha et al, 2016 ; Urbanowicz et al, 2017 ; Zhong et al, 2018 , 2020 , 2021 ; Ehrlich et al, 2021 ). In context to complexes, the formation of multiprotein structures likely increases their efficiency and the overall rate of XyG synthesis.…”

“…Acceptors have been found to include the four building blocks of life: nucleotides, proteins, lipids, and other glycans, producing glycosylated RNA, glycoproteins, glycolipids, and polysaccharides ( Reichmann and Gründling, 2011 ; Riley et al, 2019 ; Flynn et al, 2021 ). GTs have been believed to be very specific in the binding of both donors and acceptors in vivo ; however, more recent in vitro studies have demonstrated that their specificity to be more promiscuous than originally thought ( Ohashi et al, 2019 ; Ruprecht et al, 2020 ; Ehrlich et al, 2021 ). GTs have been divided into more than 110 families within the CAZy database, 1 demonstrating a vast variety between GTs ( Drula et al, 2022 ).…”