Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetobacter diazotrophicus

Abstract

The endophytic Gram-negative bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10), which converts sucrose into fructooligosaccharides and levan. The enzyme is included in GH (glycoside hydrolase) family 68 of the sequence-based classification of glycosidases. The three-dimensional structure of LsdA has been determined by X-ray crystallography at a resolution of 2.5 A (1 A=0.1 nm). The structure was solved by molecular replacement using the homologous Bacillus subtilis (Bs) levansucrase (Protein Data Bank accession code 1OYG) as a search model. LsdA displays a five-bladed beta-propeller architecture, where the catalytic residues that are responsible for sucrose hydrolysis are perfectly superimposable with the equivalent residues of the Bs homologue. The comparison of both structures, the mutagenesis data and the analysis of GH68 family multiple sequences alignment show a strong conservation of the sucrose hydrolytic machinery among levansucrases and also a structural equivalence of the Bs levansucrase Ca2+-binding site to the LsdA Cys339-Cys395 disulphide bridge, suggesting similar fold-stabilizing roles. Despite the strong conservation of the sucrose-recognition site observed in LsdA, Bs levansucrase and GH32 family Thermotoga maritima invertase, structural differences appear around residues involved in the transfructosylation reaction.

The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions

“…In contrast, the enzymes of Gluconacetobacter diazotrophicus, Zymomonas mobilis and Lactobacillus sanfranciscensis have been found to synthesize mainly short fructooligosaccharides (FOSs; kestose and nystose) from sucrose (Doelle et al, 1993;Hernández et al, 1995;Korakli et al, 2001Korakli et al, , 2003; thus, these enzymes may employ a nonprocessive type of reaction, involving release of the fructan chain after (virtually) each fructosyl transfer. The available three-dimensional structures of the B. subtilis SacB levansucrase, which synthesizes mainly large polymers, and the FOS-synthesizing levansucrase from G. diazotrophicus (Martinez-Fleites et al, 2005;Meng & Futterer, 2003), show that the active-site architecture of both levansucrase enzymes is identical (Martinez-Fleites et al, 2005;Meng & Futterer, 2003). Therefore, it is unclear which structural features determine the polymerization versus oligosaccharide synthesis ratio, and the use of a processive or a non-processive mechanism for fructan-chain growth in FTF enzymes.…”

Structural and Functional Insights into Intramolecular Fructosyl Transfer by Inulin Fructotransferase

“…Because both fructosyl units in DFA-IV remain in the ␤-configuration, GH32 levan fructotransferases appear to carry out catalysis in a manner similar to that of the members of GH32, employing a mechanism that results in retention of the anomeric configuration. To date, structural analyses for enzymes involved in fructan degradation and biosynthesis in the GH32 (8 -11) and GH68 (3,5) families have resulted exclusively in five-bladed ␤-propeller structures. In contrast, the present study demonstrates that the structurally unrelated ␤-helix carries out an intramolecular fructosyl transfer in a way analogous to intermolecular glycosyl transfer by glycosyltransferases.…”

“…By contrast, in bacteria, the multifunctional enzymes levansucrase (3) and inulosucrase (4) catalyze fructan biosynthesis, producing inulin and levan, the predominant bacterial fructans, respectively. Details of the levan biosynthetic mechanism in bacteria were recently revealed by structural studies of levansucrase (3,5).…”

Inulin and levan synthesis by probiotic Lactobacillus gasseri strains: characterization of three novel fructansucrase enzymes and their fructan products

“…Various groups have attempted to elucidate structure-function relationships of FTF enzymes, following site-directed mutagenesis and FTF protein 3D structure analysis. At present, high-resolution 3D structures are only available for the levansucrase proteins of Bacillus subtilis, also with sucrose and raffinose bound in the active site (Meng & Futterer, 2003, and Gluconacetobacter diazotrophicus (Martinez-Fleites et al, 2005). It has been proven by site-directed mutagenesis that the amino acid residues located at the 21 (donor) and +2 (acceptor) sugar-binding subsites are important in determining the size of the products synthesized and polymer versus oligosaccharide product ratio (Homann et al, 2007;Ozimek et al, 2006a).…”