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

Periplasmic chitodextrinase (Chdx) is involved in chitin metabolism in Vibrio species. Chdxs from Vibrio cholerae (VcChdx) and Vibrio harveyi consist of six domains: an N-terminal family 5/12 carbohydrate-binding module (N-CBM), two immunoglobulin-like domains (IgL-1 and IgL-2), a GH18 catalytic domain (CatD), a chitin-binding domain (ChBD), and a C-terminal family 5/12 carbohydrate-binding module (C-CBM). We produced recombinant VcChdx, which exhibited a single band at 116 kDa on SDS-PAGE. VcChdx fwas successfully crystallized in the ligand-free and bound state with the inhibitor allosamidin, providing insights into the catalytic center and substrate-binding groove of CatD, which tightly interacts with ChBD. However, no electron density was observed for two CBMs and two IgLs, probably due to their high conformational mobility. Size-exclusion chromatography coupled with multi-angle light scattering and small-angle X-ray scattering revealed the overall architecture of multi-modular VcChdx, in which the individual folded domains are extended in solution. Isothermal titration calorimetry analysis indicated that the interaction between VcChdx and allosamidin was enthalpy-driven with moderate affinity (Kd = 1.58 ± 0.48 μm). HPLC analysis of the reaction products from the substrates, chitooligosaccharides, (GlcNAc)n (n = 2-6), indicated that VcChdx hydrolyzes (GlcNAc)n in an endo-splitting manner. Among tested substrates, the specific activity was highest toward (GlcNAc)6. Taken together, VcChdx is a GH18 enzyme with an elongated, multi-modular structure and endo-splitting activity toward soluble (GlcNAc)n. It likely acts cooperatively with chitoporin localized to the outer membrane, which preferentially translocates (GlcNAc)6 into the periplasm.