Functional characterization of three Azotobacter chroococcum alginate-modifying enzymes related to the Azotobacter vinelandii AlgE mannuronan C-5-epimerase family

Abstract

Bacterial alginate initially consists of 1–4-linked β-D-mannuronic acid residues (M) which can be later epimerized to α- L -guluronic acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum . The three A. chroococcum genes, here designated AcalgE1 , AcalgE2 and AcalgE3 , were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions − 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.

Mechanistic basis for understanding the dual activities of the bifunctionalAzotobacter vinelandiimannuronan C-5 epimerase and alginate lyase AlgE7

“…The product pattern changed somewhat, especially on polyMG where NaCl enhances epimerization (Figure S4 and Table S1). 13 C NMRexperiments on polyM without NaCl showed the same trends as discussed here, except for a total lack of MGG-signals during the reaction course. Lyase activity seems to be more dominating in the absence of salt, and this was investigated in more detail (see later section).…”

“…The epimerization pattern and preferred cleavage sites of AlgE7 have been studied previously (11,23), but the mode of action is not understood. To verify the end products and further elucidate the mode of action, we have used 1 H NMR at different reaction time points, timeresolved 13 C NMR, and 13 C HSQC (Heteronuclear single quantum coherence spectroscopy) spectra (Figure 2, Figure S1, and Table S1). The lyase and epimerase activities are closely linked in how they affect each other in the creation of the product pattern.…”

“…The decline in G-residues, seen as a decreasing F(GG) during the reaction course, implies that X could be a G-residue and that AlgE7 is able to abstract a proton from G-residues. From the time-resolved 13 C NMR-spectra (Figure 2B), we can infer that AlgE7 likely is able to abstract H-5 from M-residues, as ΔM is created from the start. The ΔMpeak is much larger than the ΔMG-peak, suggesting that there is more ΔMM created, and this is supported by the HSQC-spectrum recorded at the end of the reaction (Figure S1).…”

“…Fractions of residues calculated from integration of the 1 H-spectra at different time points are plotted to the right and shown in Table S1 (calculation of sequential parameters explained in Figure S3). B: Time resolved 13 C-spectra of the reaction from 0-1000 min (16.7 h), recorded at 25 °C with 10 mM MOPS, 75 mM NaCl, 2.5 mM CaCl2 and pH 6.9. 11 mg/ml of substrate and 2.9 µM of enzyme were used (1:42 enzyme:substrate, w/w).…”

“…and some strains of Pseudomonas. The AlgE epimerases comprise seven well-studied enzymes from A. vinelandii (AlgE1-7) (10, 11), PsmE from P. syringae (12), and three recently identified A. chroococcum enzymes (AcAlgE1-3) (13). Fourteen polysaccharide lyase (PL) families in the Carbohydrate-Active enZYmes (CAZy) database have so far been described to include alginate lyases, where they are characterized based on their overall structure, catalytic amino acids residues, and mode of action (14,15).…”