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

Fei Xu,Fang Dong,Peng Wang,Hai-Yan Cao,Chun-Yang Li,Ping-Yi Li,Xiu-Hua Pang,Yu-Zhong Zhang,Xiu-Lan Chen

doi:10.1074/jbc.m116.766030

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 Glaciecola chathamensis S18K6T, 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 Tyr130, Arg187, His242, Arg265, and Tyr304 in the active center are also important for the activity of AlyGC. In catalysis, Lys220 and Arg241 function as the Brønsted base and acid, respectively, and a Ca2+ 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.

Highlights

Alginate lyases that degrade alginate via a ␤-elimination reaction fall into seven polysaccharide lyase (PL) families
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
Structure and Catalytic Mechanism of a PL6 Alginate Lyase mechanism of the PL6 alginate lyases will broaden our understanding on alginate lyases

Summary

Results and Discussion

Two-domain Structure of AlyGC Predicted by Sequence Analysis—The gene alyGC predicted to encode a PL6 alginate lyase (AlyGC) was cloned from the genome of G. chathamensis S18K6T. alyGC is 2265 bp in length and encodes a protein of 754 amino acid residues containing a predicted 28-residue signal peptide. Sequence analysis indicates that the precursor of alginate lyase OalS6 has a similar CTD with unidentified function (Fig. 1). These two CTDs may represent an uncharacterized protein domain. Sequence analysis indicates that AlyGC is a PL6 alginate lyase with a two-domain structure. PM and PG, AlyGC displayed the highest activity toward PG, indicating its preference to PG (Fig. 2A). A carbonate ion, a phosphate ion, and a glycerol molecule, which are most likely from the crystallization buffer, are bound near the Ca2ϩ (Fig. 3A) Another two phosphate ions are found in AlyGC structure. The catalytic cleft of ChonB is more L-like compared with that of AlyGC (Fig. 4, B and C)

Conformational Change in AlyGC When Binding an Alginate

Structure and Catalytic Mechanism Comparison of Alginate

Experimental Procedures