Purification, cDNA cloning and homology modeling of endo-1,3-beta-D-glucanase from scallop Mizuhopecten yessoensis.
The retaining endo-1,3-beta-D-glucanase (LV) with molecular mass of 36 kDa was purified to homogeneity from the crystalline styles of scallop Mizuhopecten yessoensis. The purified enzyme catalyzed hydrolysis of laminaran as endo-enzyme forming glucose, laminaribiose and higher oligosaccharides as products (Km approximately 600 microg/mL). The 1,3-beta-D-glucanase effectively catalyzed transglycosylation reaction that is typical of endo-enzymes too. Optima of pH and temperature were at 4.5 and 45 degrees C, respectively. cDNA encoding the endo-1,3-beta-D-glucanase was cloned by PCR-based methods. It contained an open reading frame that encoded 339-amino acids protein. The predicted endo-1,3-beta-D-glucanase amino acid sequence included a characteristic domain of the glycosyl hydrolases family 16 and revealed closest homology with 1,3-beta-D-glucanases from bivalve Pseudocardium sachalinensis, sea urchin Strongylocentrotus purpuratus and invertebrates lipopolysaccharide and beta-1,3-glucan-binding proteins. The fold of the LV was more closely related to kappa-carrageenase, agarase and 1,3;1,4-beta-D-glucanase from glycosyl hydrolases family 16. Homology model of the endo-1,3-beta-D-glucanase from M. yessoensis was obtained with MOE on the base of the crystal structure of kappa-carrageenase from P. carrageonovora as template. Putative three-dimensional structures of the LV complexes with substrate laminarihexaose or glucanase inhibitor halistanol sulfate showed that the binding sites of the halistanol sulfate and laminarihexaose are located in the enzyme catalytic site and overlapped.
Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, 159 pr.100 let Vladivostoku, Vladivostok, 690022, Russia. kovalchuk@piboc.dvo.ru
Svetlana N
SN
Kovalchuk
Elena V
EV
Sundukova
Mikhail I
MI
Kusaykin
Konstantin V
KV
Guzev
Stanislav D
SD
Anastiuk
Galina N
GN
Likhatskaya
Evgeni V
EV
Trifonov
Evgeni A
EA
Nurminski
Valeri B
VB
Kozhemyako
Tatyana N
TN
Zvyagintseva
Valeri A
VA
Rasskazov
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D004187:Disaccharides / Q000737:chemistry; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D049897:Pectinidae / Q000201:enzymology / Q000235:genetics*; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D013379:Substrate Specificity
D018076:DNA, Complementary; D004187:Disaccharides; C014637:laminaribiose; D044644:Endo-1,3(4)-beta-Glucanase