The Azotobacter vinelandii Mannuronan C-5-Epimerase AlgE1 Consists of Two Separate Catalytic Domains

Abstract

The Azotobacter vinelandii enzyme AlgE1 is a member of a family of secreted mannuronan C-5-epimerases. These enzymes convert ␤-D-mannuronic acid residues (M) to ␣-L-guluronic acid residues (G) at the polymer level in the industrially important polysaccharide alginate, leading to altered physical and immunological properties of the polymer. The reaction product of AlgE1 was found to be a mixture of blocks of continuous G residues (G-blocks) and blocks containing alternating M and G residues (MG-blocks). The enzyme is dependent on Ca 2؉ for activity, and only Sr 2؉ of those tested was able to replace Ca 2؉ . Zn 2؉ blocked the activity even at low concentrations. algE1 has been divided into two parts based on the modular type of structure previously reported to be a characteristic of the secreted epimerases, and each part has been expressed in Escherichia coli. These experiments showed that AlgE1 contains two catalytic domains, AlgE1-1, which introduces both Gblocks and MG-blocks, and AlgE1-2, which only introduces MG-blocks. AlgE1-1 has a much lower specific activity than both AlgE1-2 and AlgE1. However, the two halves of AlgE1 seem to cooperate in such a way that they contribute approximately equally to the overall epimerization reaction.

The Catalytic Activities of the Bifunctional Azotobacter vinelandii Mannuronan C-5-Epimerase and Alginate Lyase AlgE7 Probably Originate from the Same Active Site in the Enzyme

“…Mg 2ϩ on the other hand had a weak stimulatory effect. The detrimental effect of Zn 2ϩ was also observed for AlgE1 (47), AlgE4 (54), and for the periplasmic epimerase AlgG (28). In conclusion, the responses of the AlgE7 lyase to divalent cations are very similar to that of the epimerases, strengthening the hypothesis that the same catalytic site is involved in both enzymatic activities.…”

“…The pH optimum was found to be between 6.9 and 7.3, with about 85 and 74% activity at pH 6.5 and pH 7.9, respectively (results not shown). The lyase activity of AlgE7 was inhibited by high NaCl concentrations, as also observed earlier for AlgE epimerases (47,54,55). At 300 mM only 11% of the activity was retained (Fig.…”

“…The band representing AlgE7 in the SDS-polyacrylamide electrophoresis gel corresponded to a higher molecular mass than expected based on the deduced amino acid sequence of the enzyme (105 kDa compared to 90.4 kDa, respectively). Such aberrant migration rates, possibly A-module-associated (30,53), have been observed also for other AlgE proteins (45,47,54).…”

“…Plasmid Constructions and Site-specific Mutagenesis-AlgE1 was previously shown to consist of two separate catalytic domains, of which the N-terminal part, AlgE1-1, introduces both G-and MG-blocks into the alginate substrates (encoded by pHE37) (47). AlgE1-1 has the same modular structure as AlgE7 (AR1-3), and its epimerization products have been extensively characterized (47).…”

“…AlgE1-1 has the same modular structure as AlgE7 (AR1-3), and its epimerization products have been extensively characterized (47). The expression vectors pHE37 and pBG27 (encodes AlgE7 (29)) were digested by NcoI and BglII, and the 5Ј-798 bp of algE1-1 were substituted with the same A-module encoding part of algE7 and vice versa.…”