Novel Molecular Insights into the Catalytic Mechanism of Marine Bacterial Alginate Lyase AlyGC from Polysaccharide Lyase Family 6

Abstract

Alginate lyases that degrade alginate via a β-elimination reaction fall into seven polysaccharide lyase (PL) families. Although the structures and catalytic mechanisms of alginate lyases in the other PL families have been clarified, those in family PL6 have yet to be revealed. Here, the crystal structure of AlyGC, a PL6 alginate lyase from marine bacterium S18K6, was solved, and its catalytic mechanism was illustrated. AlyGC is a homodimeric enzyme and adopts a structure distinct from other alginate lyases. Each monomer contains a catalytic N-terminal domain and a functionally unknown C-terminal domain. A combined structural and mutational analysis using the structures of AlyGC and of an inactive mutant R241A in complex with an alginate tetrasaccharide indicates that conformational changes occur in AlyGC when a substrate is bound and that the two active centers in AlyGC may not bind substrates simultaneously. The C-terminal domain is shown to be essential for the dimerization and the catalytic activity of AlyGC. Residues Tyr, Arg, His, Arg, and Tyr in the active center are also important for the activity of AlyGC. In catalysis, Lys and Arg function as the Brønsted base and acid, respectively, and a Ca in the active center neutralizes the negative charge of the C5 carboxyl group of the substrate. Finally, based on our data, we propose a metal ion-assisted catalytic mechanism of AlyGC for alginate cleavage with a state change mode, which provides a better understanding for polysaccharide lyases and alginate degradation.

Cloning, expression, and characterization of a new pH‐ and heat‐stable alginate lyase from Pseudoalteromonas carrageenovora ASY5

“…Most endolytic bacterial alginate lyases have been assigned to PL‐5 and PL‐7 families (Hashimoto, Miyake, Ochiai, & Murata, ; Ogura, Yamasaki, Mikami, Hashimoto, & Murata, ; Yamasaki, Ogura, Hashimoto, Mikami, & Murata, ). Few reports have been provided on PL‐6 alginate lyases, such as AlyGC from Glaciecola chathamensis S18K6 T (Xu et al, ), TsAly6A from Thalassomonas sp. LD5 (Gao et al, ), OalS6 from Shewanella sp.…”

“…The activity of Alg823 could be recovered by adding Ca 2+ which was completely depleted by EDTA conforming to the results of AlyGC. When the metal chelator EDTA was added to the reaction mixture, the enzyme activity of AlyGC decreased as the EDTA concentration increased, and 0.2 mM EDTA completely abolished the enzyme activity and could be recovered by adding Ca 2+ ; the enzyme activity almost fully recovered by adding 0.2 mM Ca 2+ (Xu et al, ). Therefore, Ca 2+ in Alg823 is a biological metal ion involved in catalysis.…”

“…The radical scavenging activity is mainly attributed to the existence of the conjugated alkene acid structure formed during enzymatic depolymerization(Falkeborg et al, ). Alginate oligosaccharides produced by enzymatic methods have a better DPPH scavenging ability than that of alginate oligosaccharides produced by chemical methods possibly because of the formation of double bonds during enzymatic hydrolysis (Xu et al, ); thus, producing alginate oligosaccharides via enzymatic methods has a greater application. Oligosaccharides may exert hydroxyl radical scavenging activity by transferring anomeric hydrogen in the intrinsic monosaccharide group through the hydrogen transfer mechanism (Chen et al, ).…”

Characterization and Application of an Alginate Lyase, Aly1281 from Marine Bacterium Pseudoalteromonas carrageenovora ASY5

“…Falkeborg et al [40] purposed that radical scavenging activity of AOS mainly originates from a conjugated alkene acid structure. In addition, Xu et al [45] purposed the catalytic mechanism of alginate lyase for the formation of the alkene acid structure based on structural and mutational analysis. In summary, the AOS obtained from Aly1281 presents good scavenging activities of up to >88%, >81%, and >69% toward hydroxyl, ABTS, and DPPH radicals, respectively.…”

Alginate Oligosaccharides: Production, Biological Activities, and Potential Applications

“…A1 (PDB code 5GKD) is the only structure solved alginate lyase, which adopts right‐handed β‐helix fold. As an exotype polyG‐specific alginate lyase from PL6, AlyGC forms dimers in solution and domains of each monomer adopt β‐helix fold (Xu et al., ).…”