A precise swaying map for how promiscuous cellobiose-2-epimerase operate bi-reaction

Area of Science:

• Biochemistry and Enzymology
• Structural Biology
• Computational Chemistry

Background:

• Promiscuous enzymes are vital for organism survival and discovering novel biochemical reactions.
• The intricate catalytic and regulatory mechanisms of promiscuous enzymes remain incompletely understood.
• Cellobiose 2-epimerase (CsCE) from Caldicellulosiruptor saccharolyticus exhibits complex epimerization and isomerization activities.

Purpose of the Study:

• To comprehensively investigate the promiscuous mechanisms of cellobiose 2-epimerase (CsCE).
• To elucidate the catalytic and regulatory strategies employed by CsCE.
• To provide a detailed structural and mechanistic understanding of enzyme promiscuity.

Main Methods:

• Determination of the crystal structure of CsCE.
• Determination of the structure of CsCE complexed with its isomerized product, glucopyranosyl-β1,4-fructose.
• Integration of X-ray crystallography with computational calculations.

Main Results:

• The catalytic framework involving ring-opening, cis-enediol intermediate formation, and ring-closing was elucidated.
• Structural analysis revealed a precise cooperation between double active sites, flexible loop rearrangements, and intermediate swaying.
• The flexible loop acts as a regulatory gear, controlling intermediate positioning and catalytic directionality.

Conclusions:

• Enzyme promiscuity in CsCE arises from the coordinated action of multiple structural elements.
• The protonation states of the cis-enediol intermediate, particularly involving H188, are critical for catalysis.
• Promiscuous enzymes exhibit a remarkable ability to leverage available structural and chemical features for diverse functions.