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

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.

Keywords: GH 36 family; Pseudoalteromonas sp. KMM 701; homology model; marine bacteria; mutation; transglycosylation; α-d-galactosidase.

Figure 1

Homology model of α-PsGal three-dimensional (3D) structure generated using X-ray structure of the α-galactosidase of Lactobacillus acidophilus (PDB ID 2XN2) as a template: (a) 3D-model of α-PsGal structure in a ribbon diagram representation: α-helixes (red), β-strands (yellow), coils (white), and turns (blue); (b) superimposition of the α-PsGal homology model (orange) with template active sites (turquoise); D-galactose is shown by sticks (green); and (c) the binding site of D-galactose in the active center of α-PsGal homology model. Hydrogen-bond contacts were determined using the Protein Contacts module of Molecular Operating Environment version 2018.01 (MOE) program (Chemical Computing Group ULC: 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2018) and are shown with a dashed line.

Figure 2

SDS-PAGE (12.5%) of the recombinant wild α-PsGal (lanes 1 and 2 before and after final purification stage, respectively) and its D451A and C494N mutants after the final stages of purification (lanes 3 and 4, respectively); molecular weight markers are shown in lane 5.

Figure 3

Circular dichroism spectra of wild α-PsGal (1) and C494N mutant (2) with 0.1 M sodium phosphate buffer (pH 7.0), 25 °C, and 0.1 cm cell.

Figure 4

Effect of pH on the activity of enzymes: (a) wild-type α-PsGal and (b) mutant C494N. Fragments of curves correspond to 0.1 M sodium citrate buffer (1), 0.1 M sodium phosphate buffer (2), and 0.1 M Tris HCl buffer (3).

Figure 5

Effect of temperature on activity of enzymes: (a) the dependence of relative activity on temperature of wild α-PsGal (1) and C494N mutant (2) and (b) thermal stability of wild α-PsGal (1) and C494N mutant (2). The solid line (3) indicates 100% activity and the dashed line (4) indicates 50% activity.

Figure 6

Two-dimensional (2D) diagrams of the D-Gal binding sites in (a) wild α-PsGal and (b) mutant C494N.

Figure 7

Thin layer chromatography (TLC) profiles of the hydrolysis and transglycosylation products produced by recombinant α-PsGal. Lanes 1 and 8—α-PsGal with Gal-α(1→6)-Glcα,β at 20 °C and 8 °C, respectively. Lanes 2 and 4—α-PsGal with pNP-α-Gal at 20 °C and 8 °C, respectively. Lane 3—α-PsGal with mixture of Gal-α(1→6)-Glcα,β/pNP-α-Gal at 20 °C. Lane 5—D451A mutant with Gal-α(1→6)-Glcα,β/NaN₃. Lane 6—D451A mutant with pNP-α-Gal/NaN₃. Lane 7—D451A mutant with pNP-α-Gal. Blank mixtures: lane 9—Gal-α(1→6)-Glcα,β, lane 10—pNP-α-Gal, lane 11—Gal, and lane 12—Glc.

Figure 8

Mass spectrometry monitoring of the reaction of melibiose hydrolysis and transglycosylation catalyzed by wild α-PsGal in the buffered heavy-oxygen water at 20 °C. (a) Experimental time-dependent changes in integral intensity of matrix-assisted laser desorption ionization-mass spectroscopy (MALDI-MS) signals of the Hex₂ ions at 365 m/z (1) and 367 m/z (2), Hex at 203 m/z (3) and 205 m/z (4), and Hex₃ at 527 m/z (5) and 529 m/z; (b) time dependences of the MALDI-MS signals intensity of Hex₃ ions at 527 m/z (1) and 529 m/z (2) in an expanded scale.