Unraveling the difference between invertases and fructan exohydrolases: a single amino acid (Asp-239) substitution transforms Arabidopsis cell wall invertase1 into a fructan 1-exohydrolase

Abstract

Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs.

Figure 1.

Schematic structure of Suc (A) and 1-kestose (B).

Figure 2.

Multiple sequence alignment (using EMBL-EBI ClustalW) of three conserved regions of some plant GH32 enzymes. The following sequences were used for the alignment: AtcwINV1, chicory vacuolar invertase, 1-FEH IIa, 1-SST, 1-FFT, and an invertase from yeast. The three crucial catalytic residues are shown in bold. Arrows indicate the residues subjected to mutagenesis. Numbering refers to AtcwINV1.

Figure 3.

Overview of the 3-D structure of the active site of AtcwINV1 (PDB code 2AC1; A) and chicory 1-FEH IIa (PDB code 1ST8; B). Figures were prepared with PyMol (Delano, 2002). The sequence motifs containing the indicated residues are shown in Figures 2, 4, and 7: Asp-23/Asp-22 (DPN region), Asp-149/Asp-147 (RDP region), Glu-203/Glu-201 (EC region), Asp-239/Glu-234, Lys-242 (LDDTKH/FEG--H region), Trp-19/Trp-20 (WMN region), Trp-47/Phe-46 (WGN/FGD region), and Trp-82/Trp-82 (WSGSAT region). [See online article for color version of this figure.]

Figure 4.

Multiple sequence alignment of the Asp-239/Lys-242 region (shown in bold) in cell wall invertases and all known plant FEHs. The following sequences were used for the alignment: Arabidopsis cell wall invertase 1, Solanum tuberosum cell wall invertase 1, Nicotiana tabacum cell wall invertase, S. tuberosum cell wall invertase 2, S. tuberosum cell wall invertase 3, tomato cell wall invertase Lin5, Vicia faba cell wall invertase, Pisum sativum cell wall invertase, V. faba cell wall invertase 2, Chenopodium rubrum cell wall invertase, Arabidopsis cell wall invertase 2, Arabidopsis cell wall invertase 4, Daucus carota cell wall invertase 1, Oryza sativa cell wall invertase, wheat cell wall invertase, Zea mays cell wall invertase 1, Z. mays cell wall invertase 2, Z. mays cell wall invertase 3, Arabidopsis cell wall invertase 5, Arabidopsis 6-FEH (ancient cell wall invertase 3), Beta vulgaris 6-FEH, chicory 1-FEH I, chicory 1-FEH IIa, chicory 1-FEH IIb, Campanula rapunculoides 1-FEH, Arabidopsis 6&1-FEH (ancient cell wall invertase 6), wheat 1-FEH w1, wheat 1-FEH w2, wheat 6&1-FEH, Hordeum vulgare 1-FEH, wheat 6-KEH w1, wheat 6-KEH w2, Lolium perenne 1-FEH, and wheat 6-FEH. Functionally characterized enzymes are marked with an asterisk.

Figure 5.

A, Chromatographic pattern of AtcwINV1 wild-type and Asp-239 mutant enzymatic activities. Reaction conditions were as follows: incubation of 50 ng purified enzyme with 10 mm Suc in 50 mm acetate buffer, pH 5.0, during 30 min at 30°C. B, Comparison of specific enzymatic activities [specific (enzymatic) activity in (mol Fru) × (mol enzyme)⁻¹ (s)⁻¹ and (nkat) × (mg protein)⁻¹] of AtcwINV1 wild-type and Asp-239 mutant purified proteins in function of increasing substrate concentrations.

Figure 6.

A, Superposition of the active sites of AtcwINV1 (turquoise) and B. subtilis levansucrase (green; PDB code 10YG) showing similarities in the catalytic region. B, Details of Suc (orange) in the active site of B. subtilis levansucrase Glu-342A mutant (PDB code 1PT2) in which Ala-342 was replaced in silico by Glu-342 (wild-type orientation; PDB code 1OYG). C, Illustration of the putative position of Suc in the active site of AtcwINV1 based on the known structural complex of B. subtilis levansucrase with Suc. Putative Suc-protein interactions are indicated with dashed lines. Figures were prepared with PyMol (Delano, 2002).

Figure 7.

Multiple sequence alignment of the three conserved hydrophobic residues (shown in bold) of some cell wall invertases: Arabidopsis cell wall invertase 1, S. tuberosum cell wall invertase 1, N. tabacum cell wall invertase, S. tuberosum cell wall invertase 2, S. tuberosum cell wall invertase 3, V. faba cell wall invertase, P. sativum cell wall invertase, V. faba cell wall invertase 2, O. sativa cell wall invertase, wheat cell wall invertase, Z. mays cell wall invertase 3, Z. mays cell wall invertase 1, and Z. mays cell wall invertase 2.