TY - JOUR
T1 - Increasing the storage and oxidation stabilities of N-acyl-d-amino acid amidohydrolase by site-directed mutagenesis of critical methionine residues
AU - Peng, I. Chen
AU - Lo, Kai Yin
AU - Hsu, Chun Hua
AU - Lee, Chia Yin
N1 - Funding Information:
This work was supported by Grant NSC-92-2317-B-002-010 from the National Science Council, Taipei, Taiwan. The authors would like to thank professors SH Liaw for the valuable discussion regarding protein homology modeling, TC Chang for assistance with obtaining the CD spectra, and YM Pong for the calculation of enzyme half-lives.
PY - 2012/12
Y1 - 2012/12
N2 - 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.
AB - 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.
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U2 - 10.1016/j.procbio.2012.06.003
DO - 10.1016/j.procbio.2012.06.003
M3 - Article
AN - SCOPUS:84870831753
SN - 1359-5113
VL - 47
SP - 1785
EP - 1790
JO - Process Biochemistry
JF - Process Biochemistry
IS - 12
ER -