Control of Substrate Conformation by Hydrogen Bonding in a Retaining β-Endoglycosidase

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Bacterial β-glycosidases are crucial hydrolytic enzymes with broad industrial and biomedical applications.
• Enzyme catalysis necessitates a conformational shift in the polysaccharide substrate, yet the stabilizing factors remain unclear.

Purpose of the Study:

• To investigate the molecular mechanisms stabilizing substrate distortion during bacterial β-glycosidase catalysis.
• To elucidate the role of the interaction between the reactive sugar and the catalytic nucleophile.

Main Methods:

• Utilized quantum mechanics/molecular mechanics (QM/MM) simulations.
• Employed molecular dynamics to analyze substrate-enzyme interactions at subsite -1.

Main Results:

• Identified a critical hydrogen bond between the C2 exocyclic group of the sugar and the catalytic nucleophile (glutamate).
• Demonstrated that this hydrogen bond stabilizes the required distorted sugar conformation for catalysis.
• Confirmed that 2-deoxy-2-fluoro derivatives accurately mimic the hydrolysable substrate conformation.

Conclusions:

• The 2-OH⋅⋅⋅nucleophile interaction is vital for substrate recognition and catalysis in endo-glycosidases.
• Findings provide insights for engineering more efficient β-glycosidases through targeted mutagenesis.