Structure-function analysis of a broad specificity Populus trichocarpa endo-β-glucanase reveals an evolutionary link between bacterial licheninases and plant XTH gene products

Abstract

The large xyloglucan endotransglycosylase/hydrolase (XTH) gene family continues to be the focus of much attention in studies of plant cell wall morphogenesis due to the unique catalytic functions of the enzymes it encodes. The XTH gene products compose a subfamily of glycoside hydrolase family 16 (GH16), which also comprises a broad range of microbial endoglucanases and endogalactanases, as well as yeast cell wall chitin/β-glucan transglycosylases. Previous whole-family phylogenetic analyses have suggested that the closest relatives to the XTH gene products are the bacterial licheninases (EC 3.2.1.73), which specifically hydrolyze linear mixed linkage β(1→3)/β(1→4)-glucans. In addition to their specificity for the highly branched xyloglucan polysaccharide, XTH gene products are distinguished from the licheninases and other GH16 enzyme subfamilies by significant active site loop alterations and a large C-terminal extension. Given these differences, the molecular evolution of the XTH gene products in GH16 has remained enigmatic. Here, we present the biochemical and structural analysis of a unique, mixed function endoglucanase from black cottonwood (Populus trichocarpa), which reveals a small, newly recognized subfamily of GH16 members intermediate between the bacterial licheninases and plant XTH gene products. We postulate that this clade comprises an important link in the evolution of the large plant XTH gene families from a putative microbial ancestor. As such, this analysis provides new insights into the diversification of GH16 and further unites the apparently disparate members of this important family of proteins.

Keywords: Carbohydrate Processing; Cellulase; Enzyme Kinetics; Enzyme Structure; Glycoside Hydrolases; Licheninase; Plant Cell Wall; Polysaccharide; Xyloglucan; Xyloglucan Endotransglycosylase/Hydrolase (XTH).

FIGURE 1.

Amino acid sequence alignment of Paenibacillus macerans licheninase (PDB code 1u0a), P. trichocarpa endoglucanase 16 (PtEG16, POPTR_0002s15460), P. tremula x tremuloides xyloglucan endotransglycosylase 16-34 (PttXET16-34, PDB code 1umz), and Tropaeolum majus xyloglucan endotransglycosylase (TmNXG1, PDB code 2uwa) of GH16. The alignment was performed with MUSCLE (25), and secondary structure elements (strands (arrows), helices (spirals), and turns (T)) from the corresponding crystal structures were added with ESPript (83) (protein names are in italic type). Conserved catalytic residues are marked with asterisks.

FIGURE 2.

Phylogenetic relationship of PtEG16-like proteins in GH16 and census among plant proteomes. A, phylogenetic relationship of EG16s to XTH gene products and licheninases. XTH gene product groups are according to Ref. . The confidence at each node in the Bayesian tree is given in posterior probabilities from the Bayesian analysis multiplied by 100. At selected nodes, the bootstrap values from maximum likelihood calculations are indicated in parentheses. A. thaliana, O. sativa, and P. patens identifiers are according to Refs. and 82). Specific EG16 sequence identifiers can be found in supplemental Table 2, and gymnosperm transcripts in Group III-A are denoted by their accession number at TIGR (24). B, occurrence of XTH Group, Group II, Group III-A, Group III-B, and PtEG16-like gene products (hatched) in selected plant in silico proteomes. The number of PtEG16-like members in charophycean green algal proteomes is presently unknown in the absence of genome data; however, corresponding transcripts have been tentatively identified (see “Results”).

FIGURE 3.

Time-dependent endohydrolytic cleavage of polysaccharides by PtEG16. The action of PtEG16 on barley β-glucan (A), Icelandic moss lichenin (B), and tamarind seed galactoxyloglucan (C) was analyzed using high performance size exclusion chromatography with evaporative light-scattering detection (ELS). The t = 0 trace corresponds to that of the corresponding polysaccharide before the addition of the enzyme. Compositional analyses of the oligosaccharide mixtures resulting from overnight incubation are shown in supplemental Figs. S3–S5. Peaks marked with an asterisk arise from low components the enzyme preparation (e.g. buffer and salts).

FIGURE 4.

Initial rate kinetic data for the PtEG16-catalyzed hydrolysis of oligosaccharides. A, rates of XXXG production from XXXGXXXG (XXXG₂). B, rates of production of Glc₄/Glc₂ (inverted triangles), Glc₃ (triangles), and Glc₅/Glc (hexagons) from cellohexaose (C6; Glc₆); circles represent the sum of rates for all cleavage modes. Error bars, S.E. from duplicate measurements. Lines represent the non-linear least-squares fits of the Michaelis-Menten equation to the data.

FIGURE 5.

Modeling and experimental validation of the PtEG16 tertiary structure. A, superimposition of the M4T (marine) and Phyre2 (magenta) homology models and the chemical shift-derived CS23D (orange) model of PtEG16 in a wall-eyed stereo representation. Models are oriented with the positive enzyme subsites on the right, relative to the conserved active-site residues Glu-88, Asp-90, and Glu-92 (bottom to top) of PtEG16 (cf. Fig. 1). B, assigned ¹⁵N TROSY-HSQC spectrum of carbon-perdeuterated and ¹³C,¹⁵N-labeled PtEG16 in H₂O buffer at pH 7.5 and 25 °C.

FIGURE 6.

Proposed evolution of EG16 and XTH gene products in GH16 from a licheninase-like ancestor. With reference to Fig. 1, the gold coloring represents the licheninase loop extension, the XET C-terminal extension, and the XEH YNIIG loop insertion in the respective proteins.