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

• Published in: Scientific reports 2017-12, Vol.7 (1), p.17933-8, Article 17933
• Main Authors: Yin, Huijia, Yang, Zhou, Nie, Xinyu, Li, Shannan, Sun, Xuyang, Gao, Chao, Wang, Zenghang, Zhou, Guangming, Xu, Ping, Yang, Chunyu
• Format: Article
• Language: English
• Subjects: 38/70; 631/61; 631/61/318; 82/80; alpha-Amylases - genetics; alpha-Amylases - metabolism; Binding Sites; Calcium; Calcium - metabolism; Calcium-binding protein; Calorimetry; Flavobacteriaceae - enzymology; Flavobacteriaceae - genetics; Humanities and Social Sciences; Metals; multidisciplinary; Mutation; Science; Science (multidisciplinary); Sequence Alignment; Systems engineering; Thermal properties; Thermal stability; Thermotolerance; Titration; Zinc; Zinc - metabolism; α-Amylase
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-18085-4

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 Ca 2+ -binding site. Interestingly, both Ca 2+ - and Zn 2+ -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 Ca 2+ or Zn 2+ . The C214S mutation, which affects Zn 2+ -binding, also remarkably enhanced Zn 2+ - and Ca 2+ - dependent thermostability, indicating that Ca 2+ - and Zn 2+ -binding sites function cooperatively to maintain protein stability. Furthermore, an isothermal titration calorimetry (ITC) analysis revealed a novel Zn 2+ -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 Zn 2+ -binding site adjacent to the conserved Ca 2+ -binding site.