Impaired coordination of nucleophile and increased hydrophobicity in the +1 subsite shift levansucrase activity towards transfructosylation.

Bacterial levansucrases produce ß(2,6)-linked levan-type polysaccharides using sucrose or sucrose analogs as donor/acceptor substrates. However, the dominant reaction of Bacillus megaterium levansucrase (Bm-LS) is hydrolysis. Single domain levansucrases from Gram-positive bacteria display a wide substrate-binding pocket with open access to water, challenging engineering for transfructosylation-efficient enzymes. We pursued a shift in reaction specificity by either modifying the water distribution in the active site or the coordination of the catalytic acid/base (E352) and the nucleophile (D95), thus affecting the fructosyl-transfer rate and allowing acceptors other than water to occupy the active site. Two serine (173/422) and two water-binding tyrosine (421/439) residues located in the first shell of the catalytic pocket were modified. Library variants of S173, Y421 and S422, which coordinate the position of D95 and E352, show increased transfructosylation (30-200%) and modified product spectra. Substitutions at position 422 have a higher impact on sucrose affinity, while changes at position 173 and 421 have a strong effect on the overall catalytic rate. As most retaining glycoside hydrolases (GHs) Bm-LS catalyzes hydrolysis and transglycosylation via a double displacement reaction involving two-transition states (TS1 and TS2). Hydrogen bonds of D95 with the side chains of S173 and S422 contribute a total of 2.4 kcal mol-1 to TS1 stabilization, while hydrogen bonds between invariant Y421, E352 and the glucosyl C-2 hydroxyl-group of sucrose contribute 2.15 kcal mol-1 stabilization. Changes at Y439 render predominantly hydrolytic variants synthesizing shorter oligosaccharides.

Extending the Scope of GTFR Glucosylation Reactions with Tosylated Substrates for Rare Sugars Synthesis.

Functionalized rare sugars were synthesized with 2-, 3-, and 6-tosylated glucose derivatives as acceptor substrates by transglucosylation with sucrose and the glucansucrase GTFR from Streptococcus oralis. The 2- and 3-tosylated glucose derivatives yielded the corresponding 1,6-linked disaccharides (isomaltose analogues), whereas the 6-tosylated glucose derivatives resulted in 1,3-linked disaccharides (nigerose analogue) with high regioselectivity in up to 95 % yield. Docking studies provided insight into the binding mode of the acceptors and suggested two different orientations that were responsible for the change in regioselectivity.

Abnormal Tin-Boron Exchange in the Attempted Synthesis of a Borylated Borole.

Boroles are important motifs within functional materials. With the aim to prepare a pinacolboryl-substituted derivative, the metallacycle transfer from corresponding zirconium and tin precursors has been explored. We show that the reaction of 1,1-dimethyl-2,3,4,5-tetrapinacolborylstannole with dichloro(phenyl)borane does not provide the desired borole, but instead a stannyl-substituted 1-chloroboracyclopent-3-ene. Spectroscopic and structural details of this highly functionalized boracycle indicate that intramolecular interactions between the tin and oxygen atoms of the boryl substituents may account for the unexpected outcome of the tin-boron exchange reaction.

Exploring the sequence variability of polymerization-involved residues in the production of levan- and inulin-type fructooligosaccharides with a levansucrase.

The connection between the gut microbiome composition and human health has long been recognized, such that the host-microbiome interplay is at present the subject of the so-called "precision medicine". Non-digestible fructooligosaccharides (FOS) can modulate the microbial composition and therefore their consumption occupies a central place in a strategy seeking to reverse microbiome-linked diseases. We created a small library of Bacillus megaterium levansucrase variants with focus on the synthesis of levan- and inulin-type FOS. Modifications were introduced at positions R370, K373 and F419, which are either part of the oligosaccharide elongation pathway or are located in the vicinity of residues that modulate polymerization. These amino acids were exchanged by residues of different characteristics, some of them being extremely low- or non-represented in enzymes of the levansucrase family (Glycoside Hydrolase 68, GH68). F419 seemed to play a minor role in FOS binding. However, changes at R370 abated the levansucrase capacity to synthesize levan-type oligosaccharides, with some mutations turning the product specificity towards neo-FOS and the inulin-like sugar 1-kestose. Although variants retaining the native R370 produced efficiently levan-type tri-, tetra- and pentasaccharides, their capacity to elongate these FOS was hampered by including the mutation K373H or K373L. Mutant K373H, for instance, generated 37- and 5.6-fold higher yields of 6-kestose and 6-nystose, respectively, than the wild-type enzyme, while maintaining a similar catalytic activity. The effect of mutations on the levansucrase product specificity is discussed.