title: cDNA cloning of an alginate lyase from a marine gastropod Aplysia kurodai and assessment of catalytically important residues of this enzyme.
abstract: Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5'- and 3'-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed ∼40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg; however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1.
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: Akira
authors.initials: A
authors.lastname: Inoue
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Takao
authors.initials: T
authors.lastname: Ojima
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001048:Aplysia / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase