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

The Azotobacter vinelandii genome encodes a family of seven secreted Ca2+-d ependent epimerases (AlgE1-7) catalyzing the polymer level epimerization of beta -D-mannuronic. acid. (Al) to alpha -L-guluronic acid (G) in the comme rcially important polysaccharide alginate. AlgE1-7 are composed of two type s of protein modules, A and R, and the A-modules have previously been found to be sufficient for epimerization. AlgE7 is both an epimerase and an algi nase, and here we show that the lyase activity is Ca2+-dependent and also r esponds similarly to the epimerases in the presence of other divalent catio ns. The AlgE7 lyase degraded M-rich alginates and a relatively G-rich algin ate from the brown algae Macrocystis pyrifera most effectively, producing o ligomers of 4 (mannuronan) to 7 units. The sequences cleaved were mainly G down arrow MM and/or G down arrow GM. Since G-moieties dominated at the red ucing ends even when mannuronan was used as substrate, the AlgE7 epimerase probably stimulates the lyase pathway, indicating a complex interplay betwe en the two activities. A truncated form of AlgE1 (AlgE1-1) was converted to a combined epimerase and lyase by replacing the 5 ' -798 base pairs in the algE1-1 gene with the corresponding A-module-encoding DNA sequence from al gE7. Furthermore, substitution of an aspartic acid residue at position 152 with glycine in AlgE7A eliminated almost all of both the lyase and epimeras e activities. Epimerization and lyase activity are believed to be mechanist ically related, and the results reported here strongly support this hypothe sis by suggesting that the same enzymatic site can catalyze both reactions.