Discovery of a Kojibiose Phosphorylase in Escherichia coli K-12

Abstract

The substrate profiles for three uncharacterized enzymes (YcjM, YcjT, and YcjU) that are expressed from a cluster of 12 genes ( ycjM-W and ompG) of unknown function in Escherichia coli K-12 were determined. Through a comprehensive bioinformatic and steady-state kinetic analysis, the catalytic function of YcjT was determined to be kojibiose phosphorylase. In the presence of saturating phosphate and kojibiose (α-(1,2)-d-glucose-d-glucose), this enzyme catalyzes the formation of d-glucose and β-d-glucose-1-phosphate ( k_cat = 1.1 s^-1, K_m = 1.05 mM, and k_cat/ K_m = 1.12 × 10³ M^-1 s^-1). Additionally, it was also shown that in the presence of β-d-glucose-1-phosphate, YcjT can catalyze the formation of other disaccharides using 1,5-anhydro-d-glucitol, l-sorbose, d-sorbitol, or l-iditol as a substitute for d-glucose. Kojibiose is a component of cell wall lipoteichoic acids in Gram-positive bacteria and is of interest as a potential low-calorie sweetener and prebiotic. YcjU was determined to be a β-phosphoglucomutase that catalyzes the isomerization of β-d-glucose-1-phosphate ( k_cat = 21 s^-1, K_m = 18 μM, and k_cat/ K_m = 1.1 × 10⁶ M^-1 s^-1) to d-glucose-6-phosphate. YcjU was also shown to exhibit catalytic activity with β-d-allose-1-phosphate, β-d-mannose-1-phosphate, and β-d-galactose-1-phosphate. YcjM catalyzes the phosphorolysis of α-(1,2)-d-glucose-d-glycerate with a k_cat = 2.1 s^-1, K_m = 69 μM, and k_cat/ K_m = 3.1 × 10⁴ M^-1 s^-1.

Figure 1

Organization of the ycj gene cluster in E. coli. These genes encode proteins that are predicted to include five sugar transporters (YcjN, YcjO, YcjP, YcjV, and OmpG), two NAD⁺-dependent dehydrogenases (YcjS and YcjQ), two polysaccharide hydrolases/phosphorylases (YcjM and YcjT), an epimerase/isomerase (YcjR), a β-phosphoglucomutase (YcjU), and a LacI-type repressor (YcjW).

Figure 2

Sequence similarity network of cog0366 at an E-value cutoff of 1 x 10⁻⁵⁰. The network was created using Cytoscape (www.cytoscape.org). Each node represents a non-redundant protein sequence and each edge (or connecting line) represents a BLAST E-value between two sequences that is better than the arbitrary value of 1 x 10⁻⁵⁰. The lengths of the edges are not significant; in tight clusters the sequences are more closely related as compared to the clusters that contain fewer connections. YcjM is shown as a yellow triangle. Within the same cluster α-(1,2)-glucose-D-glycerate phosphorylase from Meiothermus silvanus is indicated with a diamond (dark blue). Diamonds in Groups 1, 2, 3, and 4 contain enzymes that have been experimentally verified. Group 1: sucrose phosphorylase from Bifidobacterium adolescentis (green); Group 2: amylosucrase from Neisseria polysaccharea (cyan); Group 3: oligo-1,6-glucosidase from Bacillus subtilis (light blue); and Group 4: neopullulanase from Geobacillus stearothermophillus (pink).

Figure 3

Sequence similarity network of cog1554 at an E-value cutoff of 1 x 10⁻¹⁵⁰. The network was generated using Cytoscape (www.cytoscape.org). Groups 1, 2, 3, 4, 5, 6, and 7 contain enzymes whose functions are experimentally verified (yellow arrowheads or red triangles). Group 1: α,α-trehalose phosphorylase (Bacillus stearothermophillus); Group 2: maltose phosphorylase (Lactobacillus brevis); Group 3: trehalose-6-phosphate phosphorylase (Lactococcus lactis); Group 4: nigerose phosphorylase (Lachnoclostridium phytofermantans); Group 5: α-(1,2)-D-glucose-glycerol phosphorylase (Bacillus selenitireducens); Group 6: kojibiose phosphorylase from Thermoanaerobacter brockii and Caldicellulosiruptor saccharolyticus; Group 7: kojibiose phosphorylase from Pyrococcus sp.

Figure 4

Sequence similarity network of Interpro family IPR010972 at an E-value cutoff of 1 x 10⁻⁸⁵. The network was created using Cytoscape (www.cytoscape.org). Diamonds (orange and yellow) represent two experimentally verified β-D-phosphoglucomutase enzymes: PgmB from Lactococcus lactis and YvdM from Bacillus subtilis. The blue triangle represents YcjU from Escherichia coli K-12 MG1655.

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