Characterization and structural analysis of a leucine aminopeptidase using site-directed mutagenesis.

Abstract

Amp0279 (EC 3.4.11.24, GenBank: CP000817.1) is a Co²⁺-dependent leucine aminopeptidase from the Lysinibacillus sphaericus C3-41 genome. After analyses using molecular docking and spatial structure analysis, site-directed mutagenesis mutants were performed as Amp0279-R131E, Amp0279-R131H, Amp0279-R131A and Amp0279-E349D. The optimum pH of Amp0279-R131E was shifted from the original 8.5 to 7.5, and the overall electrostatic potential was shifted towards acidic. Compared with the original enzyme, the mutant proteins all gained better structural stability, especially the apparent melting temperature (T_m) of Amp0279-R131H increased from 41.8 to 45.5 °C. Morever, when protein was bound to the substrate, the T_m of Amp0279-R131E was increased by 7.3 °C and Amp0279-R131H increased by 5.4 °C, compared to the original enzyme. This is consistent with the results that the mutants acquired higher binding energies to the substrates, and an increase the hydrogen bonding force. In addition, the molecular docking of mutant and substrate revealed that the truncation of R131 contributes to the increase in the binding capacity of the substrate molecules to the active centre. In contrast, the presence of π-Cation interactions generated by R131 with the substrate has an important effect on the ability of Amp0279 to hydrolyse the substrate. This study demostrated that R131 is a key site for activity and stability, which is important in the future exploration of the functional structure of Amp0279.