Insights into the role of the (α + β) insertion in the TIM-barrel catalytic domain, regarding the stability and the enzymatic activity of Chitinase A from Serratia marcescens

Chitinase A (ChiA) from Serratia marcescens is a mesophilic enzyme with high catalytic activity and high stability. The crystal structure of ChiA has revealed a TIM-barrel fold of the catalytic domain, an (α + β) insertion between the B7 β-strand and A7 α-helix of the TIM-barrel, an FnIII domain at the N-terminus of the molecule and a hinge region that connects the latter to the catalytic domain. In this study, the role of the (α + β) domain on the stability, catalytic activity and specificity of the enzyme was investigated by deleting this domain and studying the enzymatic and structural properties of the resulting truncated enzyme. The obtained data clearly show that by removing the (α + β) domain, the thermal stability of the enzyme is substantially reduced, with an apparent Tm of 42.0 ± 1.0 °C, compared to the apparent Tm of 58.1 ± 1.0 °C of ChiA at pH 9.0. The specific activity of ChiAΔ(α + β) was substantially decreased, the pH optimum was shifted from 6.5 to 5.0 and the substrate and product specificities were altered.