title: Purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3.
abstract: ι-Carrageenases belong to family 82 of glycoside hydrolases that degrade sulfated galactans in the red algae known as ι-carrageenans. The catalytic mechanism and some substrate-binding residues of family GH82 have been studied but the substrate recognition and binding mechanism of this family have not been fully elucidated. We report here the purification, cloning and characterization of a new ι-carrageenase CgiA_Ce from the marine bacterium Cellulophaga sp. QY3. CgiA_Ce was the most thermostable carrageenase described so far. It was most active at 50°C and pH 7.0 and retained more than 70% of the original activity after incubation at 50°C for 1 h at pH 7.0 or at pH 5.0-10.6 for 24 h. CgiA_Ce was an endo-type ι-carrageenase; it cleaved ι-carrageenan yielding neo-ι-carrabiose and neo-ι-carratetraose as the main end products, and neo-ι-carrahexaose was the minimum substrate. Sequence analysis and structure modeling showed that CgiA_Ce is indeed a new member of family GH82. Moreover, sequence analysis of ι-carrageenases revealed that the amino acid residues at subsites -1 and +1 were more conserved than those at other subsites. Site-directed mutagenesis followed by kinetic analysis identified three strictly conserved residues at subsites -1 and +1 of ι-carrageenases, G228, Y229 and R254 in CgiA_Ce, which played important roles for substrate binding. Furthermore, our results suggested that Y229 and R254 in CgiA_Ce interacted specifically with the sulfate groups of the sugar moieties located at subsites -1 and +1, shedding light on the mechanism of ι-carrageenan recognition in the family GH82.
authors.affiliation: Key Laboratory of Marine Drugs, Chinese Ministry of Education, Ocean University of China, Qingdao, China.
authors.forename: Su
authors.initials: S
authors.lastname: Ma
authors.forename: Gaofei
authors.initials: G
authors.lastname: Duan
authors.forename: Wengang
authors.initials: W
authors.lastname: Chai
authors.forename: Cunliang
authors.initials: C
authors.lastname: Geng
authors.forename: Yulong
authors.initials: Y
authors.lastname: Tan
authors.forename: Lushan
authors.initials: L
authors.lastname: Wang
authors.forename: Frédéric
authors.initials: F
authors.lastname: Le Sourd
authors.forename: Gurvan
authors.initials: G
authors.lastname: Michel
authors.forename: Wengong
authors.initials: W
authors.lastname: Yu
authors.forename: Feng
authors.initials: F
authors.lastname: Han
mesh_terms: D020418:Algal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002351:Carrageenan / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D000461:Rhodophyta / Q000737:chemistry / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D020418:Algal Proteins; D002351:Carrageenan; D006026:Glycoside Hydrolases