The Structure of Sucrose-Soaked Levansucrase Crystals from Erwinia tasmaniensis reveals a Binding Pocket for Levanbiose

Polsinelli, Ivan; Caliandro, Rosanna; Demitri, Nicola; Benini, Stefano

doi:10.3390/ijms21010083

Cited by 15 publications

(10 citation statements)

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“…The difference in the quantity and size of the FOS production may have been due to differences in a pocket, which has been shown to retain acceptor molecules. Polsinelli et al [38] determined that there was a levanbiose-binding site on LS from Erwinia tasmaniensis and E. amylovora, defined by Ala34, Phe35, Pro36, Val37, Arg73, Ile89, Trp371, Phe376, Arg377, and Ile378 (residues correspond to E. amylovora). The authors suggested that the binding of levanbiose or another small oligofructan to the enzymatic structure close to the active site would increase its likelihood to be used as an acceptor molecule, thereby increasing FOS production [38].…”

Section: Amino Acid Sequence Comparisonmentioning

confidence: 99%

“…Polsinelli et al [38] determined that there was a levanbiose-binding site on LS from Erwinia tasmaniensis and E. amylovora, defined by Ala34, Phe35, Pro36, Val37, Arg73, Ile89, Trp371, Phe376, Arg377, and Ile378 (residues correspond to E. amylovora). The authors suggested that the binding of levanbiose or another small oligofructan to the enzymatic structure close to the active site would increase its likelihood to be used as an acceptor molecule, thereby increasing FOS production [38]. There were substitutions in each of the five enzyme sequences, with LS from P. graminis containing smaller and less polar residues (Ala79Asp, Val37Leu, Arg73Pro, Ile89-, Trp371Pro, and Phe376-).…”

Section: Amino Acid Sequence Comparisonmentioning

confidence: 99%

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Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates

Hill

Karboune

Narwani

et al. 2020

IJMS

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The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via β-(2→6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans, and Paraburkolderia graminis. The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree.

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Section: Amino Acid Sequence Comparisonmentioning

confidence: 99%

Section: Amino Acid Sequence Comparisonmentioning

confidence: 99%

Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates

Hill

Karboune

Narwani

et al. 2020

IJMS

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show abstract

“…This secondary site supported the binding of a levanhexaose molecule mainly by lodging two of its fructosyl moieties. Similarly, a secondary OB site distant from the catalytic site was recently identified in the catalytic pocket periphery of E. tasmaniensis LS, in which levanbiose was observed ( 14 ). This disaccharide is located at 22.5 and 24.8 Å over the surface from the hexasaccharides bound to the OB1 and OB2 sites observed in the SacB–oligosaccharide complex ( Fig.…”

Section: Discussionmentioning

confidence: 70%

“…These observations indicate that SacB has the structural machinery for both elongation mechanisms. At present, the crystallographic structures of LSs from B. subtilis, Gluconacetobacter diazotrophicus , Bacillus megaterium , Erwinia amylovora , and Erwinia tasmaniensis have been determined ( 8 , 11 , 12 , 13 , 14 ). All of these structures display a single domain with a five-bladed β-propeller architecture, in which each β sheet adopts the classical “W” topology of four antiparallel β strands.…”

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confidence: 99%

The molecular basis of the nonprocessive elongation mechanism in levansucrases

Raga-Carbajal

Diaz-Vilchis

Rojas-Trejo

et al. 2021

Journal of Biological Chemistry

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Levansucrases (LSs) synthesize levan, a β2-6-linked fructose polymer, by successively transferring the fructosyl moiety from sucrose to a growing acceptor molecule. Elucidation of the levan polymerization mechanism is important for using LSs in the production of size-defined products for application in the food and pharmaceutical industries. For a deeper understanding of the levan synthesis reaction, we determined the crystallographic structure of Bacillus subtilis LS (SacB) in complex with a levan-type fructooligosaccharide and utilized site-directed mutagenesis to identify residues involved in substrate binding. The presence of a levanhexaose molecule in the central catalytic cavity allowed us to identify five substrate-binding subsites (−1, +1, +2, +3, and +4). Mutants affecting residues belonging to the identified acceptor subsites showed similar substrate affinity ( K m) values to the wildtype (WT) K m value but had a lower turnover number and transfructosylation/hydrolysis ratio. Of importance, compared with the WT, the variants progressively yielded smaller-sized low-molecular-weight levans, as the affected subsites that were closer to the catalytic site, but without affecting their ability to synthesized high-molecular-weight levans. Furthermore, an additional oligosaccharide-binding site 20 Å away from the catalytic pocket was identified, and its potential participation in the elongation mechanism is discussed. Our results clarify, for the first time, the interaction of the enzyme with an acceptor/product oligosaccharide and elucidate the molecular basis of the nonprocessive levan elongation mechanism of LSs.

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“…LBS is a di-fructose FOS intermediate formed during the synthesis of longer-chain FOSs. The binding pocket discovered in this crystal structure represents a starting point for planning mutagenesis studies in order to understand its biological relevance and role in FOS chain elongation [5].…”

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confidence: 99%

Carbohydrate-Active Enzymes: Structure, Activity, and Reaction Products

Benini¹

2020

IJMS

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Carbohydrate-active enzymes are responsible for both the biosynthesis and breakdown of carbohydrates and glycoconjugates. They are involved in many metabolic pathways, in the biosynthesis and degradation of various biomolecules, such as bacterial exopolysaccharides, starch, cellulose, and lignin, and in the glycosylation of proteins and lipids. Their interesting reactions have attracted the attention of researchers belonging to different scientific fields ranging from basic research to biotechnology. Interest in carbohydrate-active enzymes is due not only to their ability to build and degrade biopolymers, which is highly relevant in biotechnology, but also because they are involved in bacterial biofilm formation, and in the glycosylation of proteins and lipids, which has important health implications.This Special Issue features a collection of research papers and reviews representing an up-to-date state of the art in this growing field of research to broaden our understanding about carbohydrate-active enzymes, their mutants, and their reaction products at the molecular level.Tindara Venuto et al. wrote a paper about alpha2,8-sialyltransferases from ray-finned fish and showed that, in tetrapods, duplicated st8sia genes like st8sia7, st8sia8, and st8sia9 have disappeared while orthologues are maintained in teleosts [1]. They reconstructed the evolutionary history of st8sia genes in fish genomes and by bioinformatics analysis showed changes in the conserved polysialyltransferase domain of the fish sequences possibly accounting for variable enzymatic activities [1].PhNah20A, a β-N-acetylhexosaminidase from the marine bacterium Paraglaciecola hydrolytica S66T, was identified and characterized. By phylogenetic analysis, the authors discovered that PhNah20A is located outside clusters of other studied β-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. PhNah20A is the first characterized member of a distinct subgroup within GH20 β-N-acetylhexosaminidases. The recombinant PhNah20A showed optimum hydrolytic activity on GlcNAc β-1,4 and β-1,3 linkages in chitobiose (GlcNAc)2 and GlcNAc-1,3-β-Gal-1,4-β-Glc (LNT2), the core structure of a human milk oligosaccharide, at pH 6.0 and 50 • C. Interestingly PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide β-Gal-1,4-β-Glc-1,1-β-GlcNAc, and in low amounts the β-1,2or β-1,3-linked trisaccharide β-Gal-1,4(β-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor [2].The production of biofuels from lignocellulosic biomasses is hampered by the saccharification step of the biomasses requiring the concerted action of lignocellulose cellulases and hemicellulases.Zhang et al. characterized the secretome of Trichoderma harzianum EM0925 under induction of lignocellulose cellulases and hemicellulases [3]. Compared with the commercial enzyme preparations, the T. harzianum EM0925 enzyme cocktail presented significantly higher lignocellulolytic enzyme activi...

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The Structure of Sucrose-Soaked Levansucrase Crystals from Erwinia tasmaniensis reveals a Binding Pocket for Levanbiose

Cited by 15 publications

References 40 publications

Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates

Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates

The molecular basis of the nonprocessive elongation mechanism in levansucrases

Carbohydrate-Active Enzymes: Structure, Activity, and Reaction Products

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