Inside the Matrix: Crystal Structure of a Xyloglucan Endotransglycosylase

The cell wall is an integral component of plant cells and is a dynamic entity that is continually undergoing chemical changes throughout plant growth and development that directly alter wall structure and load bearing capacity. The primary cell wall of most dicotyledonous plants is composed of cellulose microfibrils embedded in a matrix of hemicellulosic and pectic polysaccharides, of which the hemicellulose xyloglucan is a major component. Xyloglucan and cellulose together make up about two-thirds of the dry weight of primary cell walls and are the major tension-bearing components of the matrix.

During cell expansion and elongation, the cell wall continually undergoes temporary loosening followed by rapid reinforcement of wall structure. Xyloglucan endotransglycosylases (XETs) are unique enzymes in plants that are capable of modulating the chemistry of the matrix and therefore performing both of these functions. XETs catalyze cleavage of a xyloglucan chain and subsequent religation to a different acceptor chain. These enzymes belong to a larger family of enzymes known as glycoside hydrolases, which catalyze fission of glycosidic bonds using general acid catalysis. XETs show a strong preference for xyloglucan polysaccharides as both substrate donor and acceptor molecules. XET activity is largely responsible for cutting and rejoining xyloglucan chains within the cell wall matrix, thereby controlling wall extensibility (Fry et al., 1992). In this issue of The Plant Cell, Johansson et al. (pages 874–886) report the crystal structure of XET16A from Populus tremula x tremuloides (PttXET16A) at 1.8-Å resolution. Because of the primary importance of XET activity to plant cell wall structure and dynamics, this represents one of the most long sought after structures in plant cell wall enzymology.

One of the best-characterized XET genes is Arabidopsis TCH4, transcription of which is strongly induced by a variety of stimuli, including touch, darkness, heat shock, and cold (Braam and Davis, 1990) and is influenced by brassinosteroids and auxin (Xu et al., 1995). Further studies (Campbell and Braam, 1999; Iliev et al., 2002) have led to the conclusion that TCH4 plays a role in cell wall modifications in response to environmental stress and during morphogenesis. The existence of a large XET gene family in Arabidopsis and the fundamental role these enzymes play in regulating cell wall architecture and mechanical strength prompt the hypothesis that there may be a wide range of tissue and developmental specificities within the encoded family of enzymes.

PttXET16A is one of the most abundant XET clones in a cDNA library constructed from hybrid aspen cambial tissue (Sterky et al., 1998). Bourquin et al. (2002) have shown that PttXET16A likely plays an important role in restructuring primary walls during the time that secondary walls are deposited, for example, by creating and reinforcing the connections between primary and secondary walls. These authors measured XET activity and confirmed localization of PttXET16A in xylem and phloem fibers at the stage of secondary wall formation.

Johansson et al. analyzed the crystal structure of native PttXET16A alone and in complex with a xyloglucan-derived nonasaccharide XLLG. Models for the apoenzyme and the enzyme in complex with XLLG were resolved to 2.1 and 1.8 Å, respectively. The enzyme was found to have an overall structure typical of glycoside hydrolase family 16, with the addition of a C-terminal linker that crosses the convex surface and forms a short, additional β-strand on the concave side of the molecule in the region of the acceptor binding site (Figure 1). This extension of the acceptor binding site helps to create an active site that is unique to XET enzymes.

Figure 1.

Schematic Diagram of the Structure of PttXET16A, Which Consists of Two Large β-Sheets Arranged in a Sandwich-Like Manner.

The active site is highlighted in blue and appears at the bottom of the image. The single, small, blue β-strand (bottom right of green β-strands on concave side) connected by a long loop to the blue β-barrel at the bottom represents a unique characteristic of XET enzymes among the larger family of glycoside hydrolases and likely imparts some of the unique substrate binding properties of XETs. (Figure courtesy of Alwyn Jones.)

Several observations were made on the conformation of the acceptor binding site and the loops that connect various β-strands, which help to explain the unique specificity properties of XETs; namely, the strong preference for xyloglucan as the donor substrate and for transfer to another xyloglucan polysaccharide as the acceptor molecule (transglycosylation). Certain other XET-like enzymes exhibit xyloglucan endohydrolase (XEH) activity, whereby the acceptor molecule is water, resulting in the cleavage of xyloglucan without religation to another oligosaccharide or polysaccharide molecule. PttXET16A, like many other XETs, is a strict transglycosylase and does not hydrolyze xyloglucan to a measurable extent.

Rose et al. (2002) recently proposed the name xyloglucan endotransglucosylase/hydrolase (XTH) to encompass XETs and XEHs and to recognize the apparent dual activities of these enzymes. These authors describe 33 members of the XTH gene family in Arabidopsis that potentially encode proteins having XET or XEH activity. These are further divided into three groups based on differences in gene structure, and the authors note that certain members of groups 1 and 2 have been demonstrated to catalyze exclusively endotransglycosylation reactions (Rose et al., 2002). The work of Johansson et al. supports the observation that PttXET16A is a strict endotransglycosylase, and these authors chose to retain the XET nomenclature, in part to avoid confusion with enzymes that exhibit hydrolase activity. In the Arabidopsis genome, PttXET16A is most similar to sequence At5g13870, which corresponds to a gene named ENDOXYLOGLUCAN TRANFERASE-A4 (EXGT-A4), according to terminology of Nishitani and Tominaga (1992), and renamed At-XTH5 (and positioned in group 1) by Rose et al. (2002). Readers are also referred to the Web site of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology for future updates on nomenclature of these enzymes. XET enzymes are currently designated as EC 2.4.1.207 (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/4/1/207.html) and XEH enzymes as EC 3.2.1.151 (http://www.chem.qmw.ac.uk/iubmb/enzyme/EC3/2/1/151.html).

The structure and analysis of PttXET16A offers some insight into why glycosyl transfer to another xyloglucan polysaccharide molecule predominates over the alternate hydrolase activity (transfer to water as the acceptor molecule). Johansson et al. made use of a synthetic XLLG molecule bearing a chromogenic aglycone, called XLLG-CNP, to distinguish between binding of the donor and acceptor polysaccharide molecules. If XLLG-CNP were bound as the donor molecule to be cleaved to form a glycosyl-enzyme intermediate, a burst of phenylate would be expected, which could easily be measured spectrophotometrically. The authors did not observe release of phenylate in these reactions, suggesting that XLLG-CNP could not serve as the donor molecule. However, XLLG-CNP could serve as the acceptor molecule, which was confirmed by determining the XET/XLLG-CNP complex structure. This structure, identical to that of the XET/XLLG complex, indicated several sugar residues in the acceptor subsites of the enzyme. Johansson et al. therefore proposed that the binding of sugars in the acceptor site is a prerequisite for catalysis and that the observed XLLG is well placed for the glycosyl transfer step.

The structure of PttXET16A thus confirms the unique characteristics of XETs that confer their singular ability to remodel the cell wall matrix during plant growth and development at the same time allowing for both wall extensibility and maintenance of wall strength.