Structure and polymannuronate specificity of a eukaryotic member of the polysaccharide lyase family 14.

Research paper by Hui-Min HM Qin, Takuya T Miyakawa, Akira A Inoue, Ryuji R Nishiyama, Akira A Nakamura, Atsuko A Asano, Yoriko Y Sawano, Takao T Ojima, Masaru M Tanokura

Indexed on: 25 Dec '16Published on: 25 Dec '16Published in: Journal of Biological Chemistry


Alginate is an abundant algal polysaccharide, composed of β-D-mannuronate and its C5 epimer α-L-guluronate, that is a useful biomaterial in cell biology and tissue engineering, with applications in cancer and aging research. The alginate lyase (EC from Aplysia kurodai, AkAly30, is a eukaryotic member of the polysaccharide lyase 14 (PL-14) family and degrades alginate by cleaving the glycosidic bond through a β-elimination reaction. Here, we present the structural basis for the substrate specificity, with a preference for polymannuronate, of AkAly30. The crystal structure of AkAly30 at a 1.77 Å resolution and the putative substrate-binding model show that the enzyme adopts a β-jelly roll fold at the core of the structure and that Lys99, Tyr140, and Tyr142 form catalytic residues in the active site. Their arrangements allow the carboxy group of mannuronate residues at subsite +1 to form ionic bonds with Lys99. The coupled tyrosine forms a hydrogen-bond network with the glycosidic bond, and the hydroxy group of Tyr140 is located near the C5 atom of the mannuronate residue. These interactions could promote the β-elimination of the mannuronate residue at subsite +1. More interestingly, Gly118 and the disulfide bond formed by Cys115 and Cys124 control the conformation of an active-site loop, which makes the space suitable for substrate entry into subsite -1. The cleavage efficiency of AkAly30 is enhanced relative to that of mutants lacking either Gly118 or the Cys115-Cys124 disulfide bond. The putative binding model and mutagenesis studies provide a novel substrate-recognition mode explaining the polymannuronate specificity of PL-14 alginate lyases.