Increasing the storage and oxidation stabilities of N-acyl-d-amino acid amidohydrolase by site-directed mutagenesis of critical methionine residues

• Published in: Process biochemistry (1991) 2012-12, Vol.47 (12), p.1785-1790
• Main Authors: Peng, I-Chen, Lo, Kai-Yin, Hsu, Chun-Hua, Lee, Chia-Yin
• Format: Article
• Language: English
• Subjects: Alcaligenes faecalis; Enzyme stability; Enzymes; Inactivation; Leucine; Methionine; Methionine oxidation; N-acyl-d-amino acid amidohydrolase; N-d-AAase; Oxidation; Recombinant; Residues; Site-directed mutagenesis; Stability; Variovorax paradoxus; Variovorax paradoxus Iso1
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2012.06.003

The recombinant N-acyl-d-amino acid amidohydrolase (N-d-AAase) of Variovorax paradoxus Iso1 was unstable during protein purification and storage at 4°C. Since the methionine oxidation might be the artificial factor leading to the inactivation of N-d-AAase, eight potential oxidation sensitive methionine residues of the enzyme were individually substituted with leucine utilizing site-directed mutagenesis. Among them, five mutants, M39L, M56L, M221L, M254L, and M352L remained at least 70% of wild-type specific activity. The enzyme kinetic parameters of M221L revealed a 44% decrease in Km, and finally reflected a 2.4-fold increase in kcat/Km. Moreover, its half-life at 4°C increased up to 6-fold longer than that of the wild-type. Structural analysis of each methionine substitution was carried out based on the crystal structure of N-d-AAase from Alcaligenes faecalis DA1. Met221 spatial closeness to the zinc-assistant catalytic center is highly potential as the primary site for oxidative inactivation. We conclude that the replacement of methionine M221 with leucine in N-d-AAase successfully enhances the oxidative resistance, half-life, and enzyme activity. This finding provides a promising basis for the engineering the stability and activity of N-d-AAase.