Site-Directed Mutagenesis-Based Functional Analysis and Characterization of Endolytic Lyase Activity of N- and C-Terminal Domains of a Novel Oligoalginate Lyase from Sphingomonas sp. MJ-3 Possessing Exolytic Lyase Activity in the Intact Enzyme.

A novel oligoalginate lyase from a marine bacterium, Sphingomonas sp. strain MJ-3, exhibited a unique alginate degradation activity that completely depolymerizes alginate to monomers through the formation of oligomers. In order to reveal the reason why MJ-3 oligoalginate can exhibit both endolytic and exolytic alginate lyase activities, ten mutants were developed and characterized on the basis of homology modeling. When the recombinant cell lysates containing the mutated proteins of MJ-3 oligoalginate lyase were allowed to react with alginate, the Asn177Ala, His178Ala, Tyr234Phe, His389Ala, and Tyr426Phe mutants showed reduced oligoalginate lyase activity, whereas the Arg236Ala mutant exhibited endolytic activity. Interestingly, the overexpressed Arg236Ala protein (79.6 kDa) was proteolytically cleaved into two fragments, i.e., the N-terminal 32.0-kDa and the C-terminal 47.6-kDa fragments. Both the purified N-terminal and C-terminal fragments showed endolytic lyase activity. They preferentially degraded a heteropolymeric (polyMG) block than poly-β-D-mannuronate (polyM) or poly-α-L-guluronate (polyG) blocks. These results suggest that the oligoalginate lyase activity of MJ-3 enzyme is derived from the cooperative interaction between the N- and C-terminal endolytic alginate lyase domains in the intact enzyme.