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.

Structure and function of the abasic site specificity pocket of an AP endonuclease from Archaeoglobus fulgidus.

The major AP endonuclease in Escherichia coli Exonuclease III (ExoIII) is frequently used in gene technology due to its strong exonucleolytic activity. A thermostabilized variant of ExoIII or a homologous enzyme from thermophilic organisms could be most useful for further applications. For this purpose we characterized a nuclease from the hyperthermophilic archaeon Archaeoglobus fulgidus (Af_Exo), which shares 33% overall sequence identity and 55% similarity to ExoIII. The gene coding for this thermostable enzyme was cloned and expressed in E. coli. The purified protein shows a strong Mg(2+)-dependent nicking activity at AP-sites, nicking of undamaged double-stranded (ds) DNA and a weak exonucleolytic activity. A V217G variant of the enzyme was crystallized with decamer ds-DNA molecule, and the three-dimensional structure was determined to 1.7A resolution. Besides our goal to find or produce a thermostable exonuclease, the structural and catalytic data of Af_Exo and a series of mutant proteins, based on the crystal structure, provide new insight into the mechanism of abasic site recognition and repair. Each of the hydrophobic residues Phe 200, Trp 215 and Val 217, forming a binding pocket for the abasic deoxyribose in Af_Exo, were mutated to glycine or serine. By expanding the size of the binding pocket the unspecific endonucleolytic activity is increased. Thus, size and flexibility of the mostly hydrophobic binding pocket have a significant influence on AP-site specificity. We suggest that its tight fitting to the flipped-out deoxyribose allows for a preferred competent binding of abasic sites. In a larger or more flexible pocket however, intact nucleotides more easily bind in a catalytically competent conformation, resulting in loss of specificity. Moreover, with mutations of Phe 200 and Trp 215 we induced a strong exonucleolytic activity on undamaged DNA.

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.