Exploring novel non-Leloir β-glucosyltransferases from proteobacteria for modifying linear (β1→3)-linked gluco-oligosaccharide chains

Abstract

Over the years several β-glucan transferases from yeast and fungi have been reported, but enzymes with such an activity from bacteria have not been characterized so far. In this work, we describe the cloning and expression of genes encoding β-glucosyltransferase domains of glycosyl hydrolase family GH17 from three species of proteobacteria: Pseudomonas aeruginosa PAO1, P. putida KT2440 and Azotobacter vinelandii ATCC BAA-1303. The encoded enzymes of these GH17 domains turned out to have a non-Leloir trans-β-glucosylation activity, as they do not use activated nucleotide sugar as donor, but transfer a glycosyl group from a β-glucan donor to a β-glucan acceptor. More particularly, the activity of the three recombinant enzymes on linear (β1 → 3)-linked gluco-oligosaccharides (Lam-Glc(4-9)) and their corresponding alditols (Lam-Glc(4-9)-ol) was studied. Detailed structural analysis, based on thin-layer chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry, and 1D/2D (1)H and (13)C nuclear magnetic resonance data, revealed diverse product spectra. Depending on the enzyme used, besides (β1 → 3)-elongation activity, (β1 → 4)- or (β1 → 6)-elongation, or (β1 → 6)-branching activities were also detected.

Structural investigation of water-soluble polysaccharides extracted from the fruit bodies of Coprinus comatus

“…The TOCSY pattern for the F H-1 signal at ı 4.532 (=residue A in Table 5), combined with the F C-6 signal at ı 69.5, is typical for -(1→6)-␤-d-Glcp-(1→6)-residues and strongly suggest the additional presence of a (1→6)-␤-d-glucan (minor component) (Hreggvidsson et al, 2011), also found, but more abundant, in fraction F4-3. Comparison of the NMR data of the fractions F3-2 and F4-3 (see below), including the TOCSY, HSQC, and NOESY data, showed that the Glc residues present in fraction F3-2 had no correlations with the Gal and Fuc residues, supporting separate components.…”

“…The presence of a reducing Glc unit is reflected by the R␣ and R␤ H-1 signals at ı 5.225 and 4.645, respectively. Starting from the anomeric signals of A, B, R␣, and R␤ in the 2D TOCSY spectrum, the chemical shifts of all non-anomeric protons for each residue could be determined (Table 5) (Hreggvidsson et al, 2011). Although the anomeric signals of A and B strongly overlap, their specific H-2,3,4,5,6a,6b signal sets could be found making use of the A H-6a (ı 4.226) track.…”

The first crystal structure of a glycoside hydrolase family 17 β-1,3-glucanosyltransferase displays a unique catalytic cleft

“…All clade I members are from plants. It is worth noting that all five structurally characterized enzymes of GH family 17 were placed into clade I. Clade II contains ten sequences, including all known GH family 17 -1,3-glucanosyltransferases from fungi, yeast and bacteria (Klebl & Tanner, 1989;Mouyna et al, 1998;Hreggvidsson et al, 2011). Alignment of sequences from clade II indicated that all of the proteins lack the subdomain around helix 6, which plays a key role in substrate binding at subsites +3 to +5.…”

“…Moreover, clade II includes four distinct -1,3-glucanosyltransglucosidase sequences from bacteria. These members appear to code for two protein domains: a region encoding a glycoside hydrolase domain of GH family 17 and a region encoding a domain belonging to the Leloir glycosyltransferase of family GT2 (Hreggvidsson et al, 2011).…”

Biochemical characterization of Aspergillus niger CfcI, a glycoside hydrolase family 18 chitinase that releases monomers during substrate hydrolysis

“…In addition, degradation of a (GlcNAc) 5 alditol was analysed. (GlcNAc) 5 (1 mg) was reduced (Hreggvidsson et al, 2011) and desalted using a Carbograph Ultra-Clean solid phase extraction column (Grace). Complete reduction was confirmed with MALDI-TOF-MS. (GlcNAc) 5 alditol (500 mM) was incubated with 4 mU CfcI-MBP in 60 ml containing 10 mM sodium acetate buffer pH 5 for 40 min, or 50 mM (GlcNAc) 5 alditol was incubated with 20 mU CfcI-MBP in 100 ml for 15 h. Reactions were stopped by heating and analysed by HPAEC.…”