Structural and Thermodynamic Signatures of Ligand Binding to the Enigmatic Chitinase D of Serratia proteamaculans.

The Gram-negative bacteria Serratia marcescens and Serratia proteamaculans have efficient chitinolytic machineries that degrade chitin into N-acetylglucosamine (GlcNAc), which is used as a carbon and energy source. The enzymatic degradation of chitin in these bacteria occurs through the synergistic action of glycoside hydrolases (GHs) that have complementary activities; an endo-acting GH (ChiC) making random scissions on the polysaccharide chains and two exo-acting GHs mainly targeting single reducing (ChiA) and nonreducing (ChiB) chain ends. Both bacteria produce low amounts of a fourth GH18 (ChiD) with an unclear role in chitin degradation. Here, we have determined the thermodynamic signatures for binding of (GlcNAc) and the inhibitor allosamidin to SpChiD as well as the crystal structure of SpChiD in complex with allosamidin. The binding free energies for the two ligands are similar (Δ G° = -8.9 ± 0.1 and -8.4 ± 0.1 kcal/mol, respectively) with clear enthalpic penalties (Δ H° = 3.2 ± 0.1 and 1.8 ± 0.1 kcal/mol, respectively). Binding of (GlcNAc) is dominated by solvation entropy change (- TΔ S° = -17.4 ± 0.4 kcal/mol) and the conformational entropy change dominates for allosamidin binding (- TΔ S° = -9.0 ± 0.2 kcal/mol). These signatures as well as the interactions with allosamidin are very similar to those of SmChiB suggesting that both enzymes are nonreducing end-specific.