Structural Superimposition

Abstact

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 (K(d) = 1.58 +/- 0.48 mum). 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.