Characterization of Properties and Transglycosylation Abilities of Recombinant α-Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants
Irina Bakunina,
Lubov Slepchenko,
Stanislav Anastyuk,
Vladimir Isakov,
Galina Likhatskaya,
Natalya Kim,
Liudmila Tekutyeva,
Oksana Son,
Larissa Balabanova
Affiliations
Irina Bakunina
Laboratory of Enzyme Chemistry, Laboratory of Marine Biochemistry, Laboratory of Bioassays and Mechanism of action of Biologically Active Substances, Laboratory of Instrumental and Radioisotope Testing Methods, Group of NMR-Spectroscopy of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok 690022, Russia
Lubov Slepchenko
Laboratory of Enzyme Chemistry, Laboratory of Marine Biochemistry, Laboratory of Bioassays and Mechanism of action of Biologically Active Substances, Laboratory of Instrumental and Radioisotope Testing Methods, Group of NMR-Spectroscopy of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok 690022, Russia
Stanislav Anastyuk
Laboratory of Enzyme Chemistry, Laboratory of Marine Biochemistry, Laboratory of Bioassays and Mechanism of action of Biologically Active Substances, Laboratory of Instrumental and Radioisotope Testing Methods, Group of NMR-Spectroscopy of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok 690022, Russia
Vladimir Isakov
Laboratory of Enzyme Chemistry, Laboratory of Marine Biochemistry, Laboratory of Bioassays and Mechanism of action of Biologically Active Substances, Laboratory of Instrumental and Radioisotope Testing Methods, Group of NMR-Spectroscopy of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok 690022, Russia
Galina Likhatskaya
Laboratory of Enzyme Chemistry, Laboratory of Marine Biochemistry, Laboratory of Bioassays and Mechanism of action of Biologically Active Substances, Laboratory of Instrumental and Radioisotope Testing Methods, Group of NMR-Spectroscopy of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok 690022, Russia
Natalya Kim
Laboratory of Enzyme Chemistry, Laboratory of Marine Biochemistry, Laboratory of Bioassays and Mechanism of action of Biologically Active Substances, Laboratory of Instrumental and Radioisotope Testing Methods, Group of NMR-Spectroscopy of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok 690022, Russia
Liudmila Tekutyeva
School of Economics and Management, School of Natural Sciences of Far Eastern Federal University, Russky Island, Vladivostok 690022, Russia
Oksana Son
School of Economics and Management, School of Natural Sciences of Far Eastern Federal University, Russky Island, Vladivostok 690022, Russia
Larissa Balabanova
Laboratory of Enzyme Chemistry, Laboratory of Marine Biochemistry, Laboratory of Bioassays and Mechanism of action of Biologically Active Substances, Laboratory of Instrumental and Radioisotope Testing Methods, Group of NMR-Spectroscopy of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok 690022, Russia
A novel wild-type recombinant cold-active α-d-galactosidase (α-PsGal) from the cold-adapted marine bacterium Pseudoalteromonas sp. KMM 701, and its mutants D451A and C494N, were studied in terms of their structural, physicochemical, and catalytic properties. Homology models of the three-dimensional α-PsGal structure, its active center, and complexes with D-galactose were constructed for identification of functionally important amino acid residues in the active site of the enzyme, using the crystal structure of the α-galactosidase from Lactobacillus acidophilus as a template. The circular dichroism spectra of the wild α-PsGal and mutant C494N were approximately identical. The C494N mutation decreased the efficiency of retaining the affinity of the enzyme to standard p-nitrophenyl-α-galactopiranoside (pNP-α-Gal). Thin-layer chromatography, matrix-assisted laser desorption/ionization mass spectrometry, and nuclear magnetic resonance spectroscopy methods were used to identify transglycosylation products in reaction mixtures. α-PsGal possessed a narrow acceptor specificity. Fructose, xylose, fucose, and glucose were inactive as acceptors in the transglycosylation reaction. α-PsGal synthesized -α(1→6)- and -α(1→4)-linked galactobiosides from melibiose as well as -α(1→6)- and -α(1→3)-linked p-nitrophenyl-digalactosides (Gal2-pNP) from pNP-α-Gal. The D451A mutation in the active center completely inactivated the enzyme. However, the substitution of C494N discontinued the Gal-α(1→3)-Gal-pNP synthesis and increased the Gal-α(1→4)-Gal yield compared to Gal-α(1→6)-Gal-pNP.
