Thermostabilization of glutamate decarboxylase B from Escherichia coli by structure-guided design of its pH-responsive N-terminal interdomain

•Glutamate decarboxylase B from E. coli (GadB) showed pH-dependent thermostability.•N-terminal helical bundle formed at acidic pH contributed to the GadB thermostability.•GadB was thermostabilized by engineering its pH-responsive N-terminal interdomain.•Thermostable triple mutant showed 7.9 and 7.7 °C increases in Tm and T5010 values.•The triple mutant showed no reduction of catalytic activity in enzyme kinetics.

Glutamate decarboxylase B (GadB) from Escherichia coli is a highly active biocatalyst that can convert l-glutamate to γ-aminobutyrate (GABA), a precursor of 2-pyrrolidone (a monomer of Nylon 4). In contrast to vigorous studies of pH shifting of GadB, mesophilic GadB has not been stabilized by protein engineering. In this study, we improved the thermostability of GadB through structural optimization of its N-terminal interdomain. According to structural analysis, the N-terminal fourteen residues (1–14) of homo-hexameric GadB formed a triple-helix bundle interdomain at acidic pH and contributed to the thermostability of GadB in preliminary tests as the pH shifted from 7.6 to 4.6. GadB thermostabilization was achieved by optimization of hydrophobic and electrostatic interactions at the N-terminal interdomain. A triple mutant (GadB-TM: Gln5Asp/Val6Ile/Thr7Glu) showed higher thermostability than the wild-type (GadB-WT), i.e., 7.9 and 7.7 °C increases in the melting temperature (Tm) and the temperature at which 50% of the initial activity remained after 10 min incubation (T5010), respectively. The triple mutant showed no reduction of catalytic activity in enzyme kinetics. Molecular dynamics (MD) simulation at high temperature showed that reinforced interactions of the triple mutant rigidified the N-terminal interdomain compared to the wild-type, leading to GadB thermostabilization.