Enzymatic Properties and Substrate Specificity of the Trehalose Phosphorylase from Caldanaerobacter subterraneus

Abstract

A putative glycoside phosphorylase from Caldanaerobacter subterraneus subsp. pacificus was recombinantly expressed in Escherichia coli, after codon optimization and chemical synthesis of the encoding gene. The enzyme was purified by His tag chromatography and was found to be specifically active toward trehalose, with an optimal temperature of 80°C. In addition, no loss of activity could be detected after 1 h of incubation at 65°C, which means that it is the most stable trehalose phosphorylase reported so far. The substrate specificity was investigated in detail by measuring the relative activity on a range of alternative acceptors, applied in the reverse synthetic reaction, and determining the kinetic parameters for the best acceptors. These results were rationalized based on the enzyme-substrate interactions observed in a homology model with a docked ligand. The specificity for the orientation of the acceptor's hydroxyl groups was found to decrease in the following order: C-3 > C-2 > C-4. This results in a particularly high activity on the monosaccharides D-fucose, D-xylose, L-arabinose, and D-galactose, as well as on L-fucose. However, determination of the kinetic parameters revealed that these acceptors bind less tightly in the active site than the natural acceptor D-glucose, resulting in drastically increased K m values. Nevertheless, the enzyme's high thermostability and broad acceptor specificity make it a valuable candidate for industrial disaccharide synthesis.

Chemoenzymatic Synthesis of Trehalose Analogues: Rapid Access to Chemical Probes for Investigating Mycobacteria

“…Two enzymes are particularly fitting for this purpose because they employ simple and readily available glucose (Glc) analogues as substrates: trehalose phosphorylase (TreP), which catalyzes the cleavage of trehalose‐6‐phosphate to release Glc and β‐ D ‐Glc‐1‐phosphate; and trehalose synthase (TreT), which catalyzes the formation of trehalose directly from Glc and uridine diphosphate glucose (UDP‐Glc). TreP was recently explored for its ability to produce trehalose analogues for biopreservation applications by running the enzyme in the reverse “synthetic” direction, but substrate specificity tests were limited to natural sugars, and no yields or product characterization data were reported 13. Further, the donor in these reactions, β‐ D ‐Glc‐1‐phosphate, is prohibitively expensive and difficult to synthesize.…”

Role of Glycoside Phosphorylases in Mannose Foraging by Human Gut Bacteria

“…In GH reaction mechanisms, the nucleophilic attack of the C-1 of the glycoside is performed by a water molecule activated by the catalytic base. The natural structural and functional diversity of GPs thus appears to be highly restricted because of the following: (i) they are found in only 7 of the 226 GH and GT families listed in the CAZy database (March 2013); (ii) approximately only 15 EC entries are currently assigned to GPs (24); (iii) their specificity toward glycosyl phosphates is limited to ␣-and ␤-D-glucopyranose 1-phosphate (25)(26)(27)(28)(29), which are the most prevalent substrates; ␣-D-galactopyranose 1-phosphate (30); N-acetyl-␣-D-glucosamine 1-phosphate (31), and ␣-Dmannopyranose 1-phosphate (32). In July 2013, ␣-D-mannopyranose 1-phosphate specificity was described for just one enzyme produced by a human gut bacterium, the Bacteroides fragilis NCTC 9343 mannosylglucose phosphorylase (BfMP), FIGURE 1.…”

Identification and Characterization of an Archaeal Kojibiose Catabolic Pathway in the Hyperthermophilic Pyrococcus sp. Strain ST04

“…The optimal temperature of PsKP was 90°C, while those of kojibiose phosphorylase from C. saccharolyticus and trehalose phosphorylase from Ca. pacificum DSM 12653 were reported as 85°C and 80°C, respectively (28,30). The half-life of PsKP at 95°C was 72 h, which corresponds to the high optimal growth temperature of Pyrococcus sp.…”

“…brockii (30%) (29), Ca. pacificum (27%) (30), and C. saccharolyticus (28%) (28). In members of the Thermococcales, there are some PsKP homologs, such as maltose phosphorylase from T. barophilus (53%) and hypothetical protein PH0746 from P. horikoshii OT3 (55%) (Fig.…”