Donor substrate recognition in the raffinose-bound E342A mutant of fructosyltransferase Bacillus subtilis levansucrase

Here, we report high-resolution structures of three levansucrase point mutants, D86A, D247A, and E342A, and that of raffinose-bound levansucrase-E342A. The D86A and D247A substitutions have little effect on the active site geometry. In marked contrast, the E342A mutant reveals conformational flexibility of functionally relevant side chains in the vicinity of the general acid Glu342, including Arg360, a residue required for levan polymerisation. The raffinose-complex reveals a conserved mode of donor substrate binding, involving minimal contacts with the raffinose galactosyl unit, which protrudes out of the active site, and specificity-determining contacts essentially restricted to the sucrosyl moiety.The present structures, in conjunction with prior biochemical data, lead us to hypothesise that the conformational flexibility of Arg360 is linked to it forming a transient docking site for the fructosyl-acceptor substrate, through an interaction network involving nearby Glu340 and Asn242 at the rim of a central pocket forming the active site.Oligo- and polyfructosyl-sucrose polymers, collectively known as fructans, are synthesized by a significant number of bacteria and an estimated 40,000 plant species [1] either replacing or supplementing starch as a carbohydrate reserve. Fructans are synthesised from sucrose as the sole substrate, sharing a single sucrose starter unit (αGlc(1–2)βFru) to which fructofuranose units can become attached at various positions of the fructosyl or glucosyl ring, resulting in highly branched or linear polymers in a species-dependent fashion [2]. The two prevailing forms of fructans are β (2 → 6)-linked levan (αGlc(1–2)βFru [(2–6)βFru]n) and β (2 → 1)-linked inulin (αGlc(1–2)βFru [(2-1)βFru]n), with the degree of polymerization of fructans varying between a few hundred and several thousands saccharide units. In plants, fructans are thought to contribute to drought and frost tolerance by preventing rupture of cell membranes [2], whereas in bac