The <i>XTH</i> Gene Family: An Update on Enzyme Structure, Function, and Phylogeny in Xyloglucan Remodeling

The xyloglucan family of polysaccharides continues to be the focus of much attention due to the central role these highly branched glycans play in contemporary plant cell wall models (Cosgrove, 2005). In such models, xyloglucans figure as key cross-linking molecules, which are responsible for coating and tethering the load-bearing paracrystalline cellulose fibrils (Pauly et al., 1999) in a composite hydrogel of neutral and ionic polysaccharides and structural proteins. In particular, fucogalactoxyloglucans, arabinoxyloglucans, and arabinogalactoxyloglucans are found in the primary walls of all vascular plants analyzed thus far, where they may constitute up to 20% of the dry weight of the wall (graminaceous monocots are a notable exception: xyloglucan is present at less than 5% and arabinoxylans and mixed-linkage β-glucans dominate; Hoffman et al., 2005; Cavalier et al., 2008; Fincher, 2009; Hsieh and Harris, 2009). The presence of xyloglucans in early land plants suggests a pivotal role in the transition from water to drier habitats (Popper, 2008). In addition to wall structural functions, nonfucosylated galactoxyloglucans have been recruited as storage polysaccharides by some species as sources of both hexoses and pentoses for developing embryos (Reid, 1985; Buckeridge et al., 2000). The ubiquity of xyloglucans in the plant kingdom has spurred an ever-growing body of research on the diverse structures and biological roles of these polysaccharides, and recent years have witnessed a significant expansion of our understanding of the enzymology of xyloglucan biosynthesis, rearrangement, and degradation. Elegant work from the Keegstra and Reiter groups has elucidated the identity of a handful of plant glycosyltransferases responsible for xyloglucan assembly, thus providing the first glimpse of the players involved in this complex process (Cavalier et al., 2008; Zabotina et al., 2008, and refs. therein). In the opposite direction, detailed structure-function analyses are highlighting plant and microbial enzymes across a range of glycoside hydrolase (GH) families with a penchant for xyloglucan disassembly, including both backbone- and side chain-cleaving activities (Gilbert et al., 2008; Vlasenko et al., 2010). Microbial enzymes with significant endo-hydrolytic activity toward xyloglucan chains have been identified in no less than six GH families (Gilbert et al., 2008; Vlasenko et al., 2010), a convergent evolution that highlights the importance of foraging this abundant polysaccharide for saprophytes (for a broader perspective on cell wall saccharification by microbes, see the Update by H.J. Gilbert in this issue [Gilbert, 2010]). In contrast, plant enzymes responsible for the cleavage and/or rearrangement of xyloglucan backbones in muro have so far only been identified in GH16, although this limited distribution certainly belies their importance. The XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) genes encode proteins that can potentially have two distinct catalytic activities, with radically different effects on xyloglucan: xyloglucan endo-transglycosylase (XET) activity (formally, xyloglucan:xyloglucosyl transferase; EC 2.4.1.207) results in the nonhydrolytic cleavage and ligation of xyloglucan chains, whereas xyloglucan endo-hydrolase (XEH) activity (formally, xyloglucan-specific endo-β-1,4-glucanase; EC 3.2.151) yields irreversible chain shortening. (Note: XTH has alternately been defined as XYLOGLUCAN ENDO-TRANSGLUCOSYLASE/HYDROLASE [Rose et al., 2002], although this is not strictly correct. An entire glycan chain is transferred, not a single glucosyl residue; compare 4-α-glucanotransferase, EC 2.4.1.25; and 1,4-α-glucan 6-α-glucosyltransferase, EC 2.4.1.24; etc.). Since their initial discovery in the early 1990s, XETs have figured prominently in plant cell wall models, due to the potential of these enzymes to cause transient matrix cleavage without hydrolysis, thus providing a potential molecular mechanism for controlled, turgor-driven wall expansion (Rose et al., 2002). Indeed, XETs have been implicated in both wall-loosening and wall-strengthening roles, gravitropic responses, as well as the incorporation of nascent xyloglucan into the wall during biosynthesis (Rose et al., 2002; Cosgrove, 2005; Mellerowicz et al., 2008). And while the biomechanical roles of XETs are still being debated (Cosgrove, 2005; Van Sandt et al., 2007b), it is doubtless that XTH gene products are important: higher plants maintain large XTH gene families (composed of 20–60 genes; Fig. 1B) whose members are actively transcribed in tissue-, time-, and stimulus-dependent contexts (Rose et al., 2002; Yokoyama et al., 2004; Becnel et al., 2006; Mellerowicz and Sundberg, 2008; Miedes and Lorences, 2009). The evolution of GH16 and grouping of XTH genes in publicly available plant genomes. A, The proposed evolution of clan B containing GH7 and GH16 (updated from Michel et al. [2001]). B, A simplified tree showing the genome representatives of the Plantae (top) and a diagram showing the distribution of XTH genes into groups (bottom), with the number of genes from each organism at the top of each column (only XTH genes predicted to encode functional XTH gene products [i.e. containing a complete active-site motif] are included). The groups are colored as follows: group 1, black; group II, light gray; group III-A, white; group III-B, dark gray. The Chlorophyta are represented by the genomes of Chlamydomonas reinhardtii (http://phytozome.org/), Micromonas pusilla (Worden et al., 2009), Ostreococcus tauri (Derelle et al., 2006), and Ostreococcus lucimarinus (Palenik et al., 2007). All other genome data are available via http://www.phytozome.org/ (accessed March 2010). To understand the physiological effects of individual XTH gene products in diverse processes such as seed germination, organogenesis, cell expansion, and fruit ripening, it is essential to understand their biochemistry. In particular, it is necessary to know whether a regulated transcript encodes a strict XET devoid of kinetically relevant hydrolytic activity, a strict XEH with no capacity to rearrange the xyloglucan network except through irreversible degradation, or an enzyme with mixed function. Since the last major review in 2002 (Rose et al., 2002), there have been significant advances in the understanding of protein structure-function relationships encoded by the XTH gene family, including the first three-dimensional structures of a plant XET (Johansson et al., 2004) and a plant XEH (Baumann et al., 2007). The focus of this Update will thus highlight key aspects of enzyme structure, mechanism, and molecular phylogeny, which underpin ongoing genetic studies of XTH gene products and their individual physiological roles. Other contemporary reviews provide lucid overviews of the literature on primary and secondary wall physiological studies involving XTHs (Cosgrove, 2005; Mellerowicz et al., 2008), which will not be covered further here. The proteins encoded by XTH genes comprise a subfamily of GH16 in the Carbohydrate-Active enZymes (CAZy) classification (Cantarel et al., 2009), which groups enzymes on the basis of structural and mechanistic similarity (Davies and Henrissat, 1995; Davies et al., 2005). GH16 enzymes display a diversity of substrate specificities, with family members cleaving β-1,3 or β-1,4 bonds in various glucans and galactans. GH16 enzymes are also distantly related to the GH7 cellulases within clan GH-B (Michel et al., 2001; Eklöf, 2010). (Clans are groups of GH families with a conserved three-dimensional structure and catalytic machinery despite low amino acid sequence similarity and differing substrate specificity; see http://www.cazy.org/.) Within GH16, the XTH gene products are most closely related to bacterial β-1,3;1,4-glucan hydrolases and fungal CRH (for CONGO RED HYPERSENSITIVE) gene products implicated in cross-linking chitin to β-1,6- and β-1,3-glucans (Cabib et al., 2008; Fig. 1A). The molecular phylogeny of XTH genes and gene products, which was originally divided into three major groups (I, II, and III; Campbell and Braam, 1999a), has undergone continual revision as a consequence of an ever-increasing body of sequence information. Indeed, just over 10 years ago, there were approximately 50 XTH gene product sequences (Campbell and Braam, 1999a). As a result of numerous plant genome sequencing projects (http://www.phytozome.org/), this number has increased to several hundred, with the result that original phylogenetic differences are beginning to become blurred. Indeed, already in 2004, a comparison of the first two public plant genomes, rice (Oryza sativa; a monocot) and Arabidopsis (Arabidopsis thaliana; a dicot), indicated that groups I and II had become indistinguishable (Yokoyama et al., 2004). This observation has been mirrored by the analysis of other large data sets (Baumann et al., 2007; Michailidis et al., 2009). The validation of a subfamily rests in statistical robustness as well as the ability of such groupings to predict enzyme characteristics and/or in vivo functional differences. Within the composite group I/II, there are a number of statistically robust clades, although there is no evidence (thus far) that these clades harbor significantly different activities. Indeed, heterologously expressed XTH genes from this group have all exhibited exclusively XET activity (Supplemental Table S1), although in most cases and substrate have not been XTH to have a an is that of and groupings be on both sequence and analysis (Baumann et al., 2007; et al., 2009). As a in group (Campbell and Braam, can be divided in by both sequence analysis and catalytic The from and from of group to et al., are thus far the only XTH gene products with hydrolytic (XEH) activity et al., et al., et al., 2001; et al., 2007). In contrast, heterologously expressed XTH genes in group III-B, (Campbell and Braam, et al., 2006), and et al., 2010), or exclusively XET activity, thus a functional the A and B As a result of the publicly available land plant genomes (http://www.phytozome.org/), the of XTH gene product family evolution can be and XTH genes are not present in all The of the Chlorophyta are not to xyloglucan and and three available genome sequences that these not XTH genes In other species closely related to land both XET activity and GH16 enzymes have been despite of xyloglucan in their cell walls and in of the to the early land a transcript with sequence similarity to GH16 has been found Sandt et al., is to that such a sequence be the of an early XTH due to the GH16 and XTH gene products 1A). The plants to have both xyloglucan and and XTH genes are the and the early vascular plants Sandt et al., and The of XTH gene products in the genomes of and 1B) that group I is to be the original XTH gene product subfamily and that groups II and in notable in these early plants is the of group III-A, which the XEH enzymes and are in with et al. that XEH activity may have as a of in an XET and also that an of sequences of group to a bacterial A key in the functional of XTH genes whether a protein will have XET or XEH activity, or some of the In 2004, the first three-dimensional structure of a XET was (Johansson et al., that of the which the to this through a of group that is highly in is devoid of hydrolytic activity across a range of xyloglucan et al., 2005; et al., 2007). this first XET structure was by the structure of the XEH from of group et al., 2007). these two structures were in providing the first into the molecular of cell wall and by XTH gene the and structures display the to all members of GH16, with notable differences that the of these enzymes toward their highly branched with the GH16 which chains the XETs and have a much due to a major in the of the active-site compare A and are that the polysaccharide from the of cleavage toward the of the to the of Davies et al. In the a which has been as a of XET sequences in GH16 (Campbell and Braam, the by providing an at the of an et al., 2004). this a sequence across the of the structure which may present a in protein that to the in the of these enzymes in and et al., 2010). of XTH gene products and a closely related GH16 A, of in in with in and and in on data from et al. B, of a in with the the in of showing the structure of the and the catalytic amino with a of a with and an with The (Johansson et al., 2004) and (Baumann et al., structures also the structural importance of highly conserved (Campbell and Braam, 1999a), which to the by the of two the structure highlights the structural importance of the the of which with the chain (Johansson et al., 2004). of this through or protein and in a number of XETs (Campbell and Braam, et al., and refs. therein). This is conserved in all group sequences is in all group enzymes such as (Baumann et al., 2007). In group the is toward the thus it on the other side of the active-site et al., 2005). the of XETs in a GH family may the ability of XETs to is a consequence of the catalytic mechanism by all members of GH16 2000). A key of the mechanism is the of a which can be by or an Gilbert et al., 2008). of the of the has been through and observation of this in by et al., 2008). A of the mechanism by XETs and xyloglucan to XETs and in both and of xyloglucan in and in of Davies et al. the substrate is in a indicated in in the last the is by an water or the of a xyloglucan The catalytic in all XTH gene products is in most amino The first in is as the which the of the in to the in the first of the catalytic alternately as a catalytic acid to the xyloglucan chain the and as a catalytic to the substrate or xyloglucan The role of which on the of the as and in is although (Johansson et al., 2004; et al., that this the of the catalytic as has been for other GH16 enzymes 2000). The of these catalytic conserved in all GH16 are from and structural studies on bacterial (for see Eklöf, 2010). Indeed, the catalytic in the of species are of both and (Johansson et al., 2004; et al., 2007). of the identity of the catalytic in GH16 XETs and from the observation that of the in both and results in the complete of the these are to the of xyloglucans from of xyloglucan et al., 2007; et al., 2009). of in to of the et al., 2008). The side chains of the in the catalytic not play a role in as these into the of the structure, where they a structural function. of the structure with the species in the of in XTH gene products, due to the of key active-site (Johansson et al., 2004). The of the to in is studies the hydrolytic of this at approximately in the complete of a xyloglucan substrate not in while the addition of xyloglucan over the enzyme less than et al., 2008). This in to the hydrolytic of on xyloglucan whose is thus by water (Baumann et al., 2007). The three-dimensional structures of these two enzymes that much of this in catalytic is the result of in active-site as the structures of and the XEH as indicated by of their are The differences these enzymes in two side of the the and while the and A acid in is to members of both group and III-B, to group while the of the in group and group a acid in with group I/II, while this in group is amino with the sequence The importance of the of in the XET and XEH activity was through the of a (Baumann et al., 2007). This in which the of the was on the with to the XEH activity was while XET activity was to that of at substrate (Baumann et al., 2007). while is of the for there are also other the of the In particular, further protein studies are to the hydrolytic capacity of the protein it into a strict on of and with xyloglucan et al., and et al., that may xyloglucan in the while these in the in with in protein are thus to be XET XEH Indeed, there are to be xyloglucan activity in GH16 and the diverse hydrolases and et al., 2002). the potential of to the active-site and to group and substrate et al., 2000). In addition to a active-site which is of xyloglucan chains GH16 XETs and have a number of structural that with the polysaccharide to it for cleavage by the catalytic amino structures of with substrate in the and with substrate in the provide evidence that XTH gene products have a active-site of primary for glucosyl to Fig. A and et al., 2009). The in the through in the to and models from molecular that the glucosyl in the is into a in the complex et al., as a for (Davies et al., structural and data that XETs and have primary it has been that a in et al., et al., 2008), to a transition of only for et al., This be for by and may be due to of the of the active-site in it be to (Rose et al., 2002), there is no evidence to that GH16 XETs and than the et al., et al., 2007; et al., 2008). The of on the xyloglucan on the of with substrate has been a of et al., 2008). The and of this was a in et al., 2006), as was the of and for all initial et al., 2008). The a highly to the products from the from a of the and the of of the of individual to transition in the of the of is of the glucosyl in and A and the by a of or which to less than in et al. The in the [i.e. to is to activity, approximately to which to an in the of than over this et al., 2008). The importance of studies on from group et al., and XET from et al., and thus highlighting that is a major to both XETs and of despite and in xyloglucan side chain a is present two an glucosyl et al., 2005; Hsieh and Harris, 2009). And while in the it be in that the effects of several on the are responsible for xyloglucan in both the and of XTH gene to the of with further side chain the of and has not been to the as In a and have also been to XET activity, to a limited et al., and et al., 2009). substrate of a XET indicated an of although the in catalytic the and was approximately et al., a to transition a protein from Arabidopsis XET activity on xyloglucan from which only and side chains et al., 2004). As with the XET the analysis of enzyme that these results be with some In the the presence of had to no on the of In contrast, and over of et al., 2008). data can be the three-dimensional structures of XET and XEH et al., 2009). As a the is a of a and two side chains and in The to the XTH gene products, of this by with the strictly conserved and A and for structural see et al. The amino acid in the is conserved within groups I and whereas in group II, is by a the functional of this is As is for enzymes and XTH gene products a of and bonds with groups to and their A complete of all active-site is both and et al., the substrate the is of a groups and and each with two to three The species of the have cell walls with low of xyloglucan with the 2009; Hsieh and Harris, 2009). the XTH genes of all are of and XTH gene in the species are abundant in cell et al., 2004). by Fincher, and has that a XET has the ability to xyloglucan with other β-glucans et al., 2007). was to the substrate and at of and of the activity, The enzyme was also to and cellulose as with at and 5% of the the that XTH may that the cell wall in species et al., 2007). the low activities for this with in vivo data may present a significant activities in be a of XTH gene products, due to active-site the activity in from and the enzyme responsible for this activity was not studies indicated that to of xyloglucan (Baumann et al., 2007). the studies that both and as the of thus that is not an for activity et al., 2008). Indeed, the for was approximately of the of the substrate which the results for while that for was of the on the basis of structural data (Johansson et al., 2004; et al., 2007; et al., 2009), there is no XETs and from their active-site although the of side chain be to as of xyloglucan to the the ability of the polysaccharide to as a substrate in the first of the XET et al., 2008). analysis that the presence of a acid may in the substrate and it into a in the complex et al., 2009). The observation that xyloglucans the ability to as is by the observation that the of the active-site (Johansson et al., 2004). An enzyme activity to proposed for mixed-linkage has been found in species as well as in the of land the et al., 2008). activity in the species was in some cases for XET activity in In contrast, activity was in a range of land plants including a of and a an of exhibited a activity of et al., 2008), to from et al., 2009), also a of the A of have been to that the activity is not due to an XTH gene as well as the to this activity into to cell wall et al., 2008), will have to the and of the gene this activity as well as of the is enzyme discovery have identified which have to be Indeed, number of the mechanism side activity, although the low for the substrate certainly for the of a enzyme et al., 2008). not an XTH gene product or GH16 it will be to see this enzyme to GH family et al., family (Gilbert et al., 2008), or a family (Cantarel et al., 2009). The that play a key role in primary cell wall was first in the Since that XETs have by far the most attention in this as other activities are beginning to be In particular, the potential for has been in a et al., from GH family members of which the mechanism (Gilbert et al., 2008). Indeed, the potential of enzymes has been et al., although a structural and analysis is to highlight key active-site et al., that functional on sequence is that of available plant genomes families of et al., 2001; et al., 2006), the numerous members of which all have the potential to Arabidopsis as an GH families the mechanism GH families are to which predicted and GH families GH16 and In contrast, for the members et al., and members from Arabidopsis the mechanism, which only polysaccharide will be to see functional studies families in plants with polysaccharide rearrangement activities. The of in enzyme and structure-function studies to effects in vivo can be a in functional although such are with to know as of as the of an enzyme on plant and this is certainly in the of the activities of polysaccharide and a XET that not xyloglucan in in containing water (Supplemental Table is to so in the hydrogel matrix of the cell where the water activity may be the observation that is et al., and only at xyloglucan (Baumann et al., a product mechanism during seed of across a range of substrate such as and be in a single et al., et al., 2007). and such as on be in of strictly that catalytic such as and of et al., et al., 2006; et al., and of et al., 2005; et al., 2009). can also play a key role in enzymology with and functional in are available for both XET et al., and XEH et al., activity, and has been to plant in vivo et al., 2009). significant has been through enzyme structure-function a complete understanding of the of in the enzymes of GH16 is still will both activities XTH sequence diversity through of enzymes in due to potential from and protein The XTH genes have certainly not all of their family despite into their and activities. In the to the of protein and physiological studies of these The are available in the of this Table XET 2.4.1.207) and XEH activity data for heterologously expressed XTH