Role of ionic interactions and linker in the domain interaction and modulation of functional activity of hyaluronate lyases.

Hyaluronate lyases from Streptococcus pneumoniae (SpnHL) and Streptococcus agalactiae (SagHL) are composed of four domains; N-terminal domain, spacer domain, alpha-domain and C-terminal domain, which are connected through peptide linkers. We have earlier shown that the recombinant alpha- and C-terminal domains of SpnHL/SagHL interact with each other even in absence of the linker and form a functional complex with enhanced enzymatic activity. Here, we looked into the role of ionic interactions in the enzyme stability and also the role of C-terminal domain and linker in the functional regulation. Domain swapping studies showed that the C-terminal domain does not bind directly to the substrate; instead the domain contributes to the interaction with the polymeric hyaluronan for catalysis. Furthermore, the substrate specificity exchanges with the size of catalytic cleft. The role of linker connecting alpha-domain to C-terminal domain was found to hold the C-terminal domain in a conformation suitable for achieving maximum activity.