Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases

Abstract

Among carbohydrate active enzymes, glycoside phosphorylases (GPs) are valuable catalysts for white biotechnologies, due to their exquisite capacity to efficiently re-modulate oligo- and poly-saccharides, without the need for costly activated sugars as substrates. The reversibility of the phosphorolysis reaction, indeed, makes them attractive tools for glycodiversification. However, discovery of new GP functions is hindered by the difficulty in identifying them in sequence databases, and, rather, relies on extensive and tedious biochemical characterization studies. Nevertheless, recent advances in automated tools have led to major improvements in GP mining, activity predictions, and functional screening. Implementation of GPs into innovative in vitro and in cellulo bioproduction strategies has also made substantial advances. Herein, we propose to discuss the latest developments in the strategies employed to efficiently discover GPs and make the best use of their exceptional catalytic properties for glycoside bioproduction.

Substrate-Assisted Mechanism for the Degradation of N-Glycans by a Gut Bacterial Mannoside Phosphorylase

“…We provide the precise itinerary of the substrate during catalysis, which can serve in the design of not only more efficient enzymes as biosynthetic tools but also ligands able to inhibit N -glycan degradation by GH130 enzymes. The mechanism disclosed herein is expected to apply to other GH130 enzymes with phosphorolytic activity, as well as the recently reported dual-activity GT108 mannoside phosphorylases …”

“…UhgbMP belongs to the glycoside hydrolase family 130 (GH130), which contains both mannosidases and phosphorylases that cleave β-mannosides at the nonreducing ends of their substrates. 15,16 UhgbMP is a homohexamer, with each monomer consisting of a five-bladed-propeller fold (Figure 1A), with the catalytic center located in the central cleft.…”

“…The design of UhgbMP inhibitors, as well as the utilization of UhgbMP and more generally that of mannoside phosphorylases as biosynthetic tools, requires further insight into their substrate recognition and catalytic mechanism. UhgbMP belongs to the glycoside hydrolase family 130 (GH130), which contains both mannosidases and phosphorylases that cleave β-mannosides at the nonreducing ends of their substrates. , UhgbMP is a homohexamer, with each monomer consisting of a five-bladed-propeller fold (Figure A), with the catalytic center located in the central cleft. Structural analyses have shown that the active site can accommodate a phosphate ion (Pi), mannose, and N -acetylglucosamine in the central furrow (Figure B) …”

Cross-Feeding and Enzymatic Catabolism for Mannan-Oligosaccharide Utilization by the Butyrate-Producing Gut Bacterium Roseburia hominis A2-183

“…The activity, stability, and products of RhMOP130A were investigated in enzyme reactions set up in triplicates. The activity was determined by reverse phosphorolysis (synthesis) [49,50] by incubation (60 µL total reaction volume) of 0.14 mg/mL enzyme at 37 • C with α-D-mannose 1-phosphate (10 mM) and M4 (10 mM) in 100 mM sodium citrate buffer (pH 5.5, for pH-optimum pH 4.0-6.2). After 10 min, the reaction was stopped by boiling (5 min), and the released phosphate was quantified by using a colorimetric malachite green phosphate assay kit (MAK308, Sigma-Aldrich, St. Louis, MO, USA).…”

Energetics of the Glycosyl Transfer Reactions of Sucrose Phosphorylase

“…Glycoside hydrolases (GHs) promote the cleavage of glycosidic bonds with rate accelerations of up to ∼10 17 -fold. − GHs have drawn mechanistic interest to elucidate the interactions that lead to the impressive proficiency in catalysis. − GHs have additionally been studied for the importance and application potential that their transformations have in diverse fields of chemistry, biology, and medicine. − Glycoside phosphorylases (GPs) are a specialized group of enzymes within the large GH class. − Instead of water as in hydrolysis, the GPs use phosphate as nucleophile of the reaction. Glycosyl transfer to phosphate is called phosphorolysis.…”