Crystal structure of 6-SST/6-SFT from Pachysandra terminalis, a plant fructan biosynthesizing enzyme in complex with its acceptor substrate 6-kestose.

Fructans play important roles as reserve carbohydrates and stress protectants in plants, and additionally serve as prebiotics with emerging antioxidant properties. Various fructan types are synthesized by an array of plant fructosyltransferases belonging to family 32 of the glycoside hydrolases (GH32), clustering together with GH68 in Clan-J. Here, the 3D structure of a plant fructosyltransferase from a native source, the Pachysandra terminalis 6-SST/6-SFT (Pt6-SST/6-SFT), is reported. In addition to its 1-SST (1-kestose-forming) and hydrolytic side activities, the enzyme uses sucrose to create graminan- and levan-type fructans, which are probably associated with cold tolerance in this species. Furthermore, a Pt6-SST/6-SFT complex with 6-kestose was generated, representing a genuine acceptor binding modus at the +1, +2 and +3 subsites in the active site. The enzyme shows a unique configuration in the vicinity of its active site, including a unique D/Q couple located at the +1 subsite that plays a dual role in donor and acceptor substrate binding. Furthermore, it shows a unique orientation of some hydrophobic residues, probably contributing to its specific functionality. A model is presented showing formation of a ß(2-6) fructosyl linkage on 6-kestose to create 6,6-nystose, a mechanism that differs from the creation of a ß(2-1) fructosyl linkage on sucrose to produce 1-kestose. The structures shed light on the evolution of plant fructosyltransferases from their vacuolar invertase ancestors, and contribute to further understanding of the complex structure-function relationships within plant GH32 members.

Unexpected presence of graminan- and levan-type fructans in the evergreen frost-hardy eudicot Pachysandra terminalis (Buxaceae): purification, cloning, and functional analysis of a 6-SST/6-SFT enzyme.

About 15% of flowering plants accumulate fructans. Inulin-type fructans with ß(2,1) fructosyl linkages typically accumulate in the core eudicot families (e.g. Asteraceae), while levan-type fructans with ß(2,6) linkages and branched, graminan-type fructans with mixed linkages predominate in monocot families. Here, we describe the unexpected finding that graminan- and levan-type fructans, as typically occurring in wheat (Triticum aestivum) and barley (Hordeum vulgare), also accumulate in Pachysandra terminalis, an evergreen, frost-hardy basal eudicot species. Part of the complex graminan- and levan-type fructans as accumulating in vivo can be produced in vitro by a sucrose:fructan 6-fructosyltransferase (6-SFT) enzyme with inherent sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan 6-exohydrolase side activities. This enzyme produces a series of cereal-like graminan- and levan-type fructans from sucrose as a single substrate. The 6-SST/6-SFT enzyme was fully purified by classic column chromatography. In-gel trypsin digestion led to reverse transcription-polymerase chain reaction-based cDNA cloning. The functionality of the 6-SST/6-SFT cDNA was demonstrated after heterologous expression in Pichia pastoris. Both the recombinant and native enzymes showed rather similar substrate specificity characteristics, including peculiar temperature-dependent inherent 1-SST and fructan 6-exohydrolase side activities. The finding that cereal-type fructans accumulate in a basal eudicot species further confirms the polyphyletic origin of fructan biosynthesis in nature. Our data suggest that the fructan syndrome in P. terminalis can be considered as a recent evolutionary event. Putative connections between abiotic stress and fructans are discussed.