Structure-based functionality of a multi-domain periplasmic chitodextrinase from marine bacterium Vibrio cholerae

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Periplasmic chitodextrinase (Chdx) plays a role in chitin metabolism in Vibrio species.
• Chdx enzymes are composed of multiple domains, including catalytic and carbohydrate-binding modules.

Purpose of the Study:

• To characterize the structure and function of Vibrio cholerae chitodextrinase (VcChdx).
• To elucidate the catalytic mechanism and substrate specificity of VcChdx.

Main Methods:

• Recombinant protein expression and purification of VcChdx.
• Crystallography for structural analysis of the catalytic domain.
• Size-exclusion chromatography, multi-angle light scattering, and small-angle X-ray scattering for structural architecture.
• Isothermal titration calorimetry for binding affinity studies.
• High-performance liquid chromatography (HPLC) for enzymatic activity assays.

Main Results:

• VcChdx is a 116 kDa protein with a multi-modular, elongated structure in solution.
• Crystallography provided insights into the catalytic domain (CatD) and its interaction with the chitin-binding domain (ChBD).
• VcChdx exhibits enthalpy-driven binding to allosamidin with moderate affinity (Kd = 1.58 μM).
• The enzyme hydrolyzes chitooligosaccharides ((GlcNAc)n) in an endo-splitting manner, with highest activity towards (GlcNAc)6.

Conclusions:

• VcChdx is a GH18 enzyme with a unique extended architecture.
• Its endo-splitting activity on soluble chitooligosaccharides is important for chitin degradation.
• VcChdx likely collaborates with chitoporin for efficient translocation of substrates into the periplasm.