title: cDNA cloning and bacterial expression of a PL-14 alginate lyase from a herbivorous marine snail Littorina brevicula.
abstract: Herbivorous marine snails like Littorina species are known to possess alginate lyases in their digestive tracts. The Littorina enzymes have been identified as endolytic polymannuronate (poly(M)) lyases (EC 4.2.2.3); however, it is still unclear which polysaccharide-lyase family (PL) the Littorina enzymes belong to, since no complete primary structure of Littorina enzymes has been determined. Thus, in the present study, we analyzed the primary structure of LbAly28, a 28kDa alginate lyase isozyme of Littorina brevicula, by the cDNA method. LbAly28 cDNAs were amplified by PCR followed by 5'- and 3'-RACE PCRs from the L. brevicula hepatopancreas cDNA. A cDNA covering entire coding region of LbAly28 consisted of 1129bp and encoded an amino-acid sequence of 291 residues. The deduced amino-acid sequence comprised an initiation methionine, a putative signal peptide of 14 residues, a propeptide-like region of 16 residues, and a mature LbAly28 domain of 260 residues. The mature LbAly28 domain showed 43-53% amino-acid identities with other molluscan PL-14 enzymes. The catalytically important residues in PL-14 enzymes, which were identified in the Chlorella virus glucuronate-specific lyase vAL-1 and Aplysia poly(M) lyase AkAly30, were also conserved in LbAly28. Site-directed mutagenesis regarding these residues, that is, replacements of Lys94, Lys97, Thr121, Arg 123, Tyr135, and Tyr137 to Ala, decreased the activity of recombinant LbAly28 to various degrees. From these results we concluded that LbAly28 is a member of PL-14 alginate lyases. Besides the effects of above mutations, we noticed that the replacement of T121 by Ala changed the substrate preference of LbAly28. Namely, the activities toward sodium alginate and poly(MG)-block substrate increased and became comparable with the activity toward poly(M)-block substrate. This suggests that the region including T121 of LbAly28 closely relates to the recognition of poly(MG) region of alginate.
authors.affiliation: Laboratory of Marine Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, Minato-cho 3-1-1, Hakodate 041-8611, Japan.
authors.forename: Mohammad Matiur
authors.initials: MM
authors.lastname: Rahman
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.forename: Akira
authors.initials: A
authors.lastname: Inoue
authors.forename: Takao
authors.initials: T
authors.lastname: Ojima
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000096:biosynthesis* / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D012908:Snails / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase