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.

Multifunctional fructans and raffinose family oligosaccharides

“…These residues are essential to stabilize the Glc part of Suc in the active site of GH32 Suc splitting enzymes (CWINV, VI, 1-SST, 6-SFT; Van den Ende et al, 2009), and they are absent in enzymes that use fructans as donor substrates (FEH, 1-FFT, 6G-FFT). This was confirmed by site directed mutagenesis experiments on invertase, defective invertase, FEH and 6G-FFT (Le Roy et al, 2007, 2008, 2013; Lasseur et al, 2009). However, the presence of an Asp/Lys or Asp/Arg is not sufficient; this couple needs to be in the right 3D configuration as well (Schroeven et al, 2009).…”

“…Intriguingly, defective invertases are never affected in their catalytic triad, but rather in a neighboring “Asp/Lys” or “Asp/Arg couple” (present in a flexible loop in the proximity of the acid/base catalyst) and in some Trp residues (Le Roy et al, 2007, 2013). These residues are essential to stabilize the Glc part of Suc in the active site of GH32 Suc splitting enzymes (CWINV, VI, 1-SST, 6-SFT; Van den Ende et al, 2009), and they are absent in enzymes that use fructans as donor substrates (FEH, 1-FFT, 6G-FFT).…”

“…Within the basal eudicots, a type I VI developed into a pFT in Pachysandra terminalis ( Figure 2 ) and this is considered as a rather “recent” evolutionary event (Van den Ende et al, 2011a). On the contrary, defective invertases and FEHs evolved from CWIs within GH32 (Le Roy et al, 2007, 2013). It can be speculated that the loss or alteration of the above-mentioned “couple” is an early evolutionary event that led to the formation of defective invertases with cell wall localization and a high iso-electric point (pI) for interaction with the cell wall.…”

Molecular and Functional Characterization of Novel Fructosyltransferases and Invertases from Agave tequilana

“…Conserved motifs shown in previous reports to be important for the determination of the activity of each enzyme were also identified in the deduced protein sequences from A. tequilana [21], [27], [38]–[40]. Specific changes within the WMNDPNG motif (DPNG) correlate well with the proposed activity of the enzyme: DPNA in Atq1-SST2, DPCG/DPSG in Atq6G-FFT-1 and -2 respectively and the conservation of the DPNG motif in both forms of invertase as described previously for other species [38], [41].…”

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

“…Both dandelion 1‐FEHs showed lower levels of identity with the chicory enzymes Ci1‐FEHI (CAC19366) with 52% identity, and an invertase (CAA72009) with 59% identity. The hydrolase‐specific W‐A/S/G‐W motif and the three conserved regions common to GH32 enzymes, including the three highly active amino acids mentioned above, were also found in Tb1‐FEH and Tk1‐FEH (Altenbach et al ., 2005; Lasseur et al ., 2009; Le Roy et al ., 2007, 2008) (Figure S1).…”