Engineering of N-Acetyl-L-glutamate kinase from Corynebacterium glutamicum toward improved catalytic efficiency and thermostability

Abstract

N-Acetyl-L-glutamate kinase (NAGK) is a key enzyme in the synthesis of L-arginine. Although, solving the problem of L-arginine feedback inhibition for NAGK is a vital way for improving L-arginine production, an equally important mechanism is improving catalytic activity and thermostability of NAGK. We successfully improved thermostability and catalytic efficiency of NAGK from Corynebacterium glutamicum (CgNAGK) by site-saturated mutagenesis. Residues G71, I74 and F91 that located in the substrate-binding pocket were selected to be substituted according to sequences alignment and homologous modeling. Comparing with wild-type CgNAGK, mutants I74VNAGK, F91YNAGK and F91HNAGK displayed 38, 41 and 52% higher enzyme activities, respectively, and exhibited superior performance in thermostability, opening the way to potential increases in arginine production by C. glutamicum. Results indicated that the length and shape of residue, hydrophobic interaction, hydrogen bond interactions and conjugation effect had significant impact on catalytic efficiency and stability by analyzing the structure of mutants and CgNAGK. Substitution of Val for I74 and replacement of F91 with His or Tyr both resulted in small structure changes of substrate binding pocket, which contributed to improve protein stability and catalytic efficiency. Our strategy could provide a way of improving enzyme activity and stability. Simultaneously, it could provide an insight into the catalytic mechanism of CgNAGK and amino acid kinase family.