Novel Molecular Insights into the Catalytic Mechanism of Marine Bacterial Alginate Lyase AlyGC from Polysaccharide Lyase Family 6.
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 bacteriumGlaciecola chathamensisS18K6T, 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 Tyr304in the active center are also important for the activity of AlyGC. In catalysis, Lys220and Arg241function 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.
From the State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Institute of Marine Science and Technology, Shandong University, Jinan 250100 and.
Fei
F
Xu
From the State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Institute of Marine Science and Technology, Shandong University, Jinan 250100 and.
Fang
F
Dong
From the State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Institute of Marine Science and Technology, Shandong University, Jinan 250100 and.
Peng
P
Wang
From the State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Institute of Marine Science and Technology, Shandong University, Jinan 250100 and.
Hai-Yan
HY
Cao
From the State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Institute of Marine Science and Technology, Shandong University, Jinan 250100 and.
Chun-Yang
CY
Li
From the State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Institute of Marine Science and Technology, Shandong University, Jinan 250100 and.
Ping-Yi
PY
Li
From the State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Institute of Marine Science and Technology, Shandong University, Jinan 250100 and.
Xiu-Hua
XH
Pang
From the State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Institute of Marine Science and Technology, Shandong University, Jinan 250100 and.|the Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266000, China.
Yu-Zhong
YZ
Zhang
From the State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Institute of Marine Science and Technology, Shandong University, Jinan 250100 and cxl0423@sdu.edu.cn.
Xiu-Lan
XL
Chen
D034241:Alteromonadaceae / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D016415:Sequence Alignment; D013379:Substrate Specificity
D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase
alginate lyase; bacteria; catalysis; polysaccharide; protein structure