An alternative broad-specificity pathway for glycan breakdown in bacteria.

Area of Science:

• Microbiology
• Biochemistry
• Enzymology

Background:

• Most characterized glycosidases utilize variations of Koshland mechanisms for glycosidic bond hydrolysis via substitution reactions.
• The human gut microbiome harbors a vast diversity of enzymes with largely uncharacterized functions.

Purpose of the Study:

• To identify and characterize novel glycosidases with non-Koshland mechanisms from a human gut metagenomic library.
• To investigate the substrate specificity and catalytic mechanisms of these newly discovered enzymes.

Main Methods:

• Large-scale screening of a human gut metagenomic library using a selective assay for non-Koshland glycosidase activity.
• Mechanistic and structural characterization of identified enzyme clusters.
• Analysis of enzyme homolog distribution across bacterial species and environments.

Main Results:

• Identification of a cluster of enzymes with exceptionally broad substrate specificities, including thioglycosides and pseudoglycosidic bonds.
• Elucidation of a distinct hydrolysis mechanism involving oxidation/reduction and elimination/hydration steps.
• Demonstration of interchangeable enzyme modules catalyzing these steps, found in diverse bacterial species and environments.

Conclusions:

• These findings reveal a significant, previously unrecognized class of bacterial carbohydrate metabolism pathways.
• The discovered enzymes and mechanisms expand our understanding of glycan degradation beyond canonical glycosidases.
• The broad substrate range and modular nature of these enzymes suggest widespread ecological and biotechnological relevance.