Oligosaccharide substrate binding in Escherichia coli maltodextrin phosphorylase

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Alpha-glucan phosphorylases are enzymes that catalyze the phosphorolysis of alpha-linked glucans.
• Understanding substrate binding and catalytic mechanisms is key to enzyme function.
• The crystal structure of E. coli maltodextrin phosphorylase complexed with an oligosaccharide has not been previously determined.

Purpose of the Study:

• To elucidate the structural basis of oligosaccharide binding to E. coli maltodextrin phosphorylase.
• To investigate the catalytic mechanism of alpha-glucan phosphorylase through structural analysis.
• To identify key residues involved in substrate recognition and binding.

Main Methods:

• X-ray crystallography was used to solve the crystal structure of E. coli maltodextrin phosphorylase co-crystallized with an oligosaccharide at 3.0 A resolution.
• Site-directed mutagenesis was employed to study the role of specific residues in enzyme activity.
• Kinetic assays (Kcat/Km) were performed to quantify the effect of mutations on enzyme efficiency.

Main Results:

• The study provides the first structure of an oligosaccharide bound at the catalytic site of an alpha-glucan phosphorylase.
• An induced fit mechanism was observed, involving the repositioning of two domains to accommodate the substrate within the catalytic site tunnel.
• A critical stacking interaction between the glucosyl residue and a remote tyrosine residue was identified as essential for substrate recognition, with mutation to alanine reducing catalytic efficiency (Kcat/Km) by 10^4-fold.
• Analysis suggests that substrate binding may induce alterations in glycosidic torsion angles, which are important for the phosphorolysis mechanism.

Conclusions:

• The crystal structure reveals a novel induced fit mechanism for oligosaccharide binding in E. coli maltodextrin phosphorylase.
• A remote tyrosine residue plays a critical role in substrate recognition through a stacking interaction.
• These findings provide insights into the catalytic mechanism of alpha-glucan phosphorylases and substrate binding, with implications for enzyme engineering and drug design.