원문정보
초록
영어
Glutamate decarboxylase (GAD, EC 4.1.1.15) is a pyridoxal 5'-phosphate (PLP) dependent enzyme, which catalyses the α-decarboxylation of L-glutamate to produce γ-aminobutyrate (GABA). Glutamate decarboxylase B (GadB) from E. coli shows multimeric structure (hexamer) and its N-terminal residues 1-15 form the triple helix bundle at acidic pH. In this study, we showed that the thermostability of GadB is considerably affected by the N-terminal structure, which is changed dramatically at different pH conditions. And the thermostability of GadB was improved through structural optimization of the N-terminal helical bundle interdomains. The amino acid residues (Gln5, Val6, Thr7, Ser11) located at N-terminal helical bundle of GadB were redesigned considering the electrostatic interaction (Gln5-Lys4, Thr7-Lys3, Ser11-Lys341) and hydrophobic interaction (Val6-Val6) between the N-terminal α-helix residues. The GadB-WT and its N-terminal mutants were expressed successfully in E. coli expression host. The T50 10 (the temperature at which 50% of initial enzymatic activity remains after 10 min heat treatment) and Tm of mutants were increased compared to that of GadB-WT. The T50 10 value of GadB TM7 [Q5D:V6I:T7E] was especially 8.5oC higher than that of GadB-WT and the Tm value was also 7.9oC higher than that of GadB-WT.