A GH115 α-glucuronidase structure reveals dimerization-mediated substrate binding and a proton wire potentially...

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Xylan, a plant cell wall component, is a source of biomaterials and prebiotic oligosaccharides.
• Substituents like α-D-glucuronic acid on xylan hinder enzymatic hydrolysis by xylanases.
• Glycoside hydrolase family 115 (GH115) α-glucuronidases specifically remove these substituents.

Purpose of the Study:

• To elucidate the molecular basis of bacterial GH115 member wtsAgu115A interaction with xylan.
• To investigate the indirect involvement of divalent ions in the enzyme-substrate complex formation.
• To characterize the structure and function of wtsAgu115A for potential biomaterial applications.

Main Methods:

• X-ray crystallography at 2.65 Å resolution to determine the structure of wtsAgu115A in complex with xylohexaose.
• Identification of active site residues (Asp303 as general acid) and observation of a proton wire.
• Analytical size-exclusion chromatography to confirm dimerization and kinetic analysis with aldouronic acids.

Main Results:

• The crystal structure revealed wtsAgu115A forms a dimer, with xylohexaose recognition occurring at the dimer interface.
• Kinetic analysis indicated cooperativity between the two binding sites (Hill coefficient > 2).
• Three Ca²⁺ ions were identified; one interacts with substrate-binding loops. Mg²⁺ or Mn²⁺ enhanced activity, while Ca²⁺ decreased it.

Conclusions:

• The dimeric structure of wtsAgu115A is crucial for substrate recognition and cooperative binding.
• Divalent metal ions, particularly Mg²⁺ and Mn²⁺, play a significant role in modulating enzyme activity.
• This structural and mechanistic understanding of wtsAgu115A provides insights for xylan valorization and biomaterial development.