beta-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro; RDP) which is absent from alpha-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp-309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in beta-fructofuranosidases.
Abstract:The three-dimensional~3D! structure of fructan biosynthetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence-structure compatibility search using TOPITS, SDP, 3D-PSSM, and SAM-T98 programs identified a b-propeller fold with scores above the confidence threshold that indicate a structurally conserved catalytic domain in fructosyltransferases~FTFs! of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase~Protein Data Bank file: 5nn9! as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases~Batista et al., 1999; Song & Jacques, 1999!. The sequence-sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern $H-x~2!-@PTV#-x~4!-@LIVMA#-@NSCAYG#-@DE#-P-@NDSC#-@GA#% for this family.Keywords: fold recognition; glycoside hydrolases; levansucrase; sequence analysis Fructans are commercially used in both food and nonfood applications. In nature, fructan biosynthesis occurs from sucrose by several microbial species and by about 15% of higher plants~Hen-dry & Wallace, 1993!. Bacterial levansucrases~EC 2.4.1.10! are multifunctional enzymes capable of synthesizing high-molecularmass levans directly from sucrose; however, plant fructans are synthesized by the concerted action of at least two fructosyltransferases~FTFs! exhibiting a distinct fructosyl-donor and fructosyl-acceptor specificities. Sucrose:sucrose 1-fructosyltransferasẽ 1-SST! generally initiates fructan synthesis in plants by catalyzing the transfer of the fructosyl residue from one sucrose to another sucrose molecule, resulting in the formation of the trisaccharide 1-kestose. Then, structurally different fructans are formed by the action of fructan:fructan 1-fructosyltransferase~1-FFT!, fructan: fructan 6G-fructosyltransferase~6G-FFT! or sucrose:fructan 6-fructosyltransferase~6-SFT!~for review see Vijn & Smeekens, 1999!.b-Fructofuranosidases are considered to function by a double displacement mechanism with an overall retention of the anomeric configuration of the fructosyl residue. These enzymes are grouped in the glycoside hydrolase family 32~invertases, levanases, inulinases, sucrose-6-phosphate hydrolases, and fungal and plant FTFs!, and glycoside hydrolase family 68~bacterial FTFs and invertases from Zymomonas mobilis and Bacillus sp.!~http:00afmb.cnrs-mrs.fr0 ;pedro0CAZY0ghf.html!.The three-dimensional~3D! structures and key residues at active sites of enzymes are generally better conserved than amino acid sequences. Consequently, structural studies combined with sequence comparisons have allowed many glycoside hydrolase families to be grouped accord...
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