Functional and cooperative stabilization of a two-metal (Ca, Zn) center in α-amylase derived from Flavobacteriaceae species

Abstract

Mesophilic α-amylase from Flavobacteriaceae (FSA) is evolutionary closely related to thermophilic archaeal Pyrococcus furiosus α-amylase (PWA), but lacks the high thermostability, despite the conservation of most residues involved in the two-metal (Ca, Zn) binding center of PWA. In this study, a disulfide bond was introduced near the two-metal binding center of FSA (designated mutant EH-CC) and this modification resulted in a slight improvement in thermostability. As expected, E204G mutations in FSA and EH-CC led to the recovery of Ca2+-binding site. Interestingly, both Ca2+- and Zn2+-dependent thermostability were significantly enhanced; 153.1% or 50.8% activities was retained after a 30-min incubation period at 50 °C, in the presence of Ca2+ or Zn2+. The C214S mutation, which affects Zn2+-binding, also remarkably enhanced Zn2+- and Ca2+- dependent thermostability, indicating that Ca2+- and Zn2+-binding sites function cooperatively to maintain protein stability. Furthermore, an isothermal titration calorimetry (ITC) analysis revealed a novel Zn2+-binding site in mutant EH-CC-E204G. This metal ion cooperation provides a possible method for the generation of α-amylases with desired thermal properties by in silico rational design and systems engineering, to generate a Zn2+-binding site adjacent to the conserved Ca2+-binding site.