Fructosyltransferase mutants specify a function for the ?-fructosidase motif of the sucrose-binding box in specifying the fructan type synthesized

Abstract

The onion fructosyltransferase fructan:fructan 6G-fructosyltransferase (6G-FFT) synthesizes fructans of the inulin neo-series using 1-kestose as a substrate. 6G-FFT couples a fructosyl residue to either the terminal glucose via a beta (2-6) linkage or a terminal fructose via a beta (2-1) linkage. The sucrose-binding box is present at the N-terminus of invertases and fructosyltransferases. We tested its function by producing swaps of the first 36 amino acids of 6G-FFT with that of onion sucrose:sucrose 1-fructosyltransferase (1-SST) (SST-GFT) and vacuolar invertase (INV-GFT). In contrast to 6G-FFT, invertase and 1-SST are able to utilize sucrose as their only substrate. The chimerical enzymes were unable to use sucrose, but were active when incubated with 1-kestose. INV-GFT synthesized a similar array of fructans as 6G-FFT, in contrast, SST-GFT showed a dramatic shift in activity towards synthesis of beta (2-1) linkages. Thus the region containing the sucrose-binding box is directing the fructan type synthesized. In invertases, the beta -fructosidase motif, which is part of the sucrose-binding box, consists of NDPNG/A. This motif is variable in fructosyltransferases and consists of NDPSG in 6G-FFT and ADPNA in 1-SST of onion. We studied the importance of the 6G-FFT beta -fructosidase motif using mutants S87N (NDPNG) and N84A;S87N (ADPNG). S87N has 6G-FFT activity, whereas N84A;S87N has a activity that was shifted towards synthesis of beta (2-1) linkages. This is in agreement with the observed activities of the chimerical proteins and indicates that the beta -fructosidase motif of the sucrose-binding box is specifying the fructan type synthesized.

Using Natural Variation to Investigate the Function of Individual Amino Acids in the Sucrose-Binding Box of Fructan:Fructan 6G-Fructosyltransferase (6G-FFT) in Product Formation

“…The profile of N84S;S87D is highly similar to that of the previously reported mutant N84A; S87N, which has the b-fructosidase motif ADPNG similar to that of onion 1-SST ( Figures 2D and 3, Table 3) (Ritsema et al, 2004). According to the results described above, mutation S87N might not influence product preference of 6G-FFT wild-type and mutants.…”

“…This b-fructosidase motif differs from the general consensus for invertases, NDPN[GA]. In a previous paper (Ritsema et al, 2004) we reported the 6G-FFT mutant S87N, with the b-fructosidase motif NDPNG, to have the same enzymatic specificity as wild-type 6G-FFT (NDPSG).…”

“…Previously, we showed that substituting the b-fructosidase motif of the sucrose-binding box of onion 6G-FFT for that of onion 1-SST resulted in a change in products synthesized from the substrate 1-kestose. Changing the b-fructosidase motif of 6G-FFT in that of invertase did not result in any change (Ritsema et al, 2004). Here we analyze the function of individual amino acids in the sucrose-binding box to obtain insight into the underlying principles of enzymatic specificity.…”

Fructan:fructan 1-fructosyltransferase, a key enzyme for biosynthesis of graminan oligomers in hardened wheat

“…Studies on microbial invertases and fructosyltransferases have revealed the catalytic mechanisms for sucrose hydrolysis and transfer of fructosyl units (Chambert and Petit-Glatron 1991;Reddy and Maley 1996). Recently, Ritsema et al (2004) studied the importance of the onion 6G-FFT ß-fructosidase motif (NDPSG) using mutants S87N (NDPNG) and N84A;S87N (ADPNG) and reported that the mutant S87N has 6F-FFT activity, whereas the mutant N84A;S87N had a activity that was shifted towards synthesis of ß-(2,1)-linkages. Wheat 1-SST has the same ß-fructosidase motif (NDPNG) as the mutant S87N (Fig.…”

Fructo-Oligosaccharide Synthesis by Mutant Versions of Saccharomyces cerevisiae Invertase

“…In this context, a challenging task is finding out the structural determinants of the docking of the fructosyl moiety and its acceptor in a particular orientation, which will finally result in a specific type of linkage. Several studies in which specific residues have been mutated by site-directed mutagenesis have reported changes in the pattern of products recovered (2,20,27), although the conclusions of these studies still do not offer a complete picture of the transfructosylation mechanism. One of the questions that remains unanswered is which residues determine the formation of either ␤(2,1) or ␤(2,6) linkages (34).…”