Bacterial polysaccharide lyase family 33: Specificity from an evolutionarily conserved binding tunnel

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Acidic glycans are crucial for eukaryotic cell biology.
• Microbial polysaccharide lyases (PL) cleave glycosidic linkages in glycans.
• PL family 33 (PL33) enzymes exhibit broad substrate specificity, including glycosaminoglycans (GAGs) and gellan gum.

Purpose of the Study:

• To investigate the biochemical and biophysical properties of PL33 enzymes.
• To understand how substrate specificity is determined in PL33 enzymes.
• To identify key amino acids and structural features governing PL33 activity.

Main Methods:

• Recombinant expression of 10 PL33 members.
• Biochemical and biophysical characterization of five PL33 enzymes.
• Structural analysis and bioinformatic comparisons.
• Molecular modeling to elucidate substrate-induced structural changes.

Main Results:

• Substrate specificity is dictated by variations in the enzyme's substrate-binding tunnel length and topography.
• Key amino acids responsible for catalysis and substrate interaction were identified.
• Tunnel topography is conformationally induced upon glycan substrate binding.
• Conserved structural features were observed across multiple lyase families and mammalian GAG epimerases.

Conclusions:

• PL33 enzyme activity and specificity are modulated by substrate-induced conformational changes in the binding tunnel.
• The identified structural and catalytic mechanisms offer insights into glycan processing by lyases.
• Conserved features suggest a common evolutionary basis for glycan-modifying enzymes.