The Complex GH32 Enzyme Orchestra from Priestia megaterium Holds the Key to Better Discriminate Sucrose-6-phosphate Hydrolases from Other ß-Fructofuranosidases in Bacteria.

Inulin is widely used as a prebiotic and emerging as a priming compound to counteract plant diseases. We isolated inulin-degrading strains from the lettuce phyllosphere, identified as Bacillus subtilis and Priestia megaterium, species hosting well-known biocontrol organisms. To better understand their varying inulin degradation strategies, three intracellular ß-fructofuranosidases from P. megaterium NBRC15308 were characterized after expression in Escherichia coli: a predicted sucrose-6-phosphate (Suc6P) hydrolase (SacAP1, supported by molecular docking), an exofructanase (SacAP2), and an invertase (SacAP3). Based on protein multiple sequence and structure alignments of bacterial glycoside hydrolase family 32 enzymes, we identified conserved residues predicted to be involved in binding phosphorylated (Suc6P hydrolases) or nonphosphorylated substrates (invertases and fructanases). Suc6P hydrolases feature positively charged residues near the structural catalytic pocket (histidine, arginine, or lysine), whereas other ß-fructofuranosidases contain tryptophans. This correlates with our phylogenetic tree, grouping all predicted Suc6P hydrolases in a clan associated with genomic regions coding for transporters involved in substrate phosphorylation. These results will help to discriminate between Suc6P hydrolases and other ß-fructofuranosidases in future studies and to better understand the interaction of B. subtilis and P. megaterium endophytes with sucrose and/or fructans, sugars naturally present in plants or exogenously applied in the context of defense priming.

Insights in inulin binding and inulin oligosaccharide formation by novel multi domain endo-inulinases from Botrytis cinerea.

World-wide, pathogenic fungi such as Botrytis cinerea cause tremendous yield losses in terms of food production and post-harvest food decay. Many fungi produce inulin-type oligosaccharides (IOSs) from inulin through endo-inulinases which typically show a two domain structure. B.cinerea lacks a two domain endo-inulinase but contains a three domain structure instead. Genome mining revealed three and four domain (d4) enzymes in the fungal kingdom. Here, three and two domain enzymes were compared in their capacity to produce IOSs from inulin. Hill kinetics were observed in three domain enzymes as compared to Michaelis-Menten kinetics in two domain enzymes, suggesting that the N-terminal extension functions as a carbohydrate binding module. Analysis of the IOS product profiles generated from purified GF6, GF12, GF16 and GF18 inulins and extensive sugar docking approaches led to enhanced insights in the active site functioning, revealing subtle differences between the endo-inulinases from Aspergillus niger and B. cinerea. Improved insights in structure-function relationships in fungal endo-inulinases offer opportunities to develop superior enzymes for the production of specific IOS formulations to improve plant and animal health (priming agents, prebiotics).

A novel chicory fructanase can degrade common microbial fructan product profiles and displays positive cooperativity.

Fructan metabolism in bacteria and plants relies on fructosyltransferases and fructanases. Plant fructanases (fructan exohydrolase, FEH) only hydrolyse terminal fructose residues. Levan (ß-2,6 linkages) is the most abundant fructan type in bacteria. Dicot fructan accumulators, such as chicory (Cichorium intybus), accumulate inulin (ß-2,1 linkages), harbouring several 1-FEH isoforms for their degradation. Here, a novel chicory fructanase with high affinity for levan was characterized, providing evidence that such enzymes widely occur in higher plants. It is adapted to common microbial fructan profiles, but has low affinity towards chicory inulin, in line with a function in trimming of microbial fructans in the extracellular environment. Docking experiments indicate the importance of an N-glycosylation site close to the active site for substrate specificity. Optimal pH and temperature for levan hydrolysis are 5.0 and 43.7 °C, respectively. Docking experiments suggested multiple substrate binding sites and levan-mediated enzyme dimerization, explaining the observed positive cooperativity. Alignments show a single amino acid shift in the position of a conserved DXX(R/K) couple, typical for sucrose binding in cell wall invertases. A possible involvement of plant fructanases in levan trimming is discussed, in line with the emerging 'fructan detour' concepts, suggesting that levan oligosaccharides act as signalling entities during plant-microbial interactions.

Functional and Molecular Characterization of the Halomicrobium sp. IBSBa Inulosucrase.

Fructans are fructose-based (poly)saccharides with inulin and levan being the best-known ones. Thanks to their health-related benefits, inulin-type fructans have been under the focus of scientific and industrial communities, though mostly represented by plant-based inulins, and rarely by microbial ones. Recently, it was discovered that some extremely halophilic Archaea are also able to synthesize fructans. Here, we describe the first in-depth functional and molecular characterization of an Archaeal inulosucrase from Halomicrobium sp. IBSBa (HmcIsc). The HmcIsc enzyme was recombinantly expressed and purified in Escherichia coli and shown to synthesize inulin as proven by nuclear magnetic resonance (NMR) analysis. In accordance with the halophilic lifestyle of its native host, the enzyme showed maximum activity at very high NaCl concentrations (3.5 M), with specific adaptations for that purpose. Phylogenetic analyses suggested that Archaeal inulosucrases have been acquired from halophilic bacilli through horizontal gene transfer, with a HX(H/F)T motif evolving further into a HXHT motif, together with a unique D residue creating the onset of a specific alternative acceptor binding groove. This work uncovers a novel area in fructan research, highlighting unexplored aspects of life in hypersaline habitats, and raising questions about the general physiological relevance of inulosucrases and their products in nature.

Implications of the mutation S164A on Bacillus subtilis levansucrase product specificity and insights into protein interactions acting upon levan synthesis.

Mutation S164A largely affects the transfructosylation properties of Bacillus subtilis levansucrase (SacB). The variant uses acceptors such as glucose and short levans with an average molecular weight of 7.6 kDa more efficiently than SacB, leading to the enhanced synthesis of medium and high molecular weight polymer and a blasto-oligosaccharide series with a polymerization degree of 2-10. A 3-fold increase in blasto-oligosaccharides yield is provoked by the modified interplay between the variant and glucose. Despite its modified product specificity, protein-carbohydrate and protein-protein interactions are still a major factor affecting size and distribution of levan molecular weight. This study highlights the importance of critical factors such as protein concentration in the analysis of wild-type and mutagenized levansucrases. Docking experiments with the crystal structures of SacB and variant S164A - the latter obtained at a 2.6 Å resolution - identified unreported potential binding subsites for fructosyl moieties on the surface of both enzymes.