Prediction of a common β‐propeller catalytic domain for fructosyltransferases of different origin and substrate specificity

Abstract

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...

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

“…Although bacterial and plant fructosyltransferases share limited homology on the amino acid level, some similarities can be noted and a similar structure is predicted (Pons et al, 2000). The putative sucrose-binding box in bacterial fructosyltransferases is not similar to those from plants.…”

“…The NDPNG/A pattern of the sucrose-binding box is called the b-fructosidase motif, it contains the essential Asp residue that probably acts as a nucleophile. In fructosyltransferases the sucrosebinding box is also present although some variations occur, especially in the b-fructosidase motif, leading to a different consensus: H-X-X-[PTV]-X-X-X-X- (Pons et al, 2000). In 6G-FFT the sucrose-binding box is formed by amino acids 75-88, located at the N-terminus of the mature protein.…”

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 sucrose-binding box consensus of GH32 was determined to consist of H-X-X- Table 2) (Pons et al, 2000).…”

Structural and functional features of fructansucrases present in Leuconostoc mesenteroides ATCC 8293

“…The structural and functional properties of bacterial fructansucrases have been the subject of intensive research: among gram positive bacteria, those produced by Bacillus, Streptococcus, Lactobacillus and Leuconostoc species are classified in family 68 of glycoside hydrolases (Pons et al 2000). Levansucrases produced by Bacillus spp.…”