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Mar. Drugs 2018, 16(10), 349; https://doi.org/10.3390/md16100349

Characterization of Properties and Transglycosylation Abilities of Recombinant α-Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants

1
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
2
School of Economics and Management, School of Natural Sciences of Far Eastern Federal University, Russky Island, Vladivostok 690022, Russia
*
Author to whom correspondence should be addressed.
Received: 16 August 2018 / Revised: 20 September 2018 / Accepted: 21 September 2018 / Published: 24 September 2018
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Abstract

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. View Full-Text
Keywords: α-d-galactosidase; homology model; GH 36 family; mutation; transglycosylation; marine bacteria; Pseudoalteromonas sp. KMM 701 α-d-galactosidase; homology model; GH 36 family; mutation; transglycosylation; marine bacteria; Pseudoalteromonas sp. KMM 701
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Bakunina, I.; Slepchenko, L.; Anastyuk, S.; Isakov, V.; Likhatskaya, G.; Kim, N.; Tekutyeva, L.; Son, O.; Balabanova, L. Characterization of Properties and Transglycosylation Abilities of Recombinant α-Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants. Mar. Drugs 2018, 16, 349.

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