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

I-Chen Peng, Kai Yin Lo, Chun Hua Hsu, Chia Yin Lee

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1785-1790
Number of pages6
JournalProcess Biochemistry
Volume47
Issue number12
DOIs
Publication statusPublished - 2012 Dec 1

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Amidohydrolases
Mutagenesis
Site-Directed Mutagenesis
Methionine
Amino acids
Amino Acids
Oxidation
Leucine
Half-Life
Enzymes
Alcaligenes faecalis
Enzyme kinetics
Enzyme activity
Kinetic parameters
Structural analysis
Purification
Zinc
Substitution reactions
Crystal structure
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Bioengineering

Cite this

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title = "Increasing the storage and oxidation stabilities of N-acyl-d-amino acid amidohydrolase by site-directed mutagenesis of critical methionine residues",
abstract = "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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Increasing the storage and oxidation stabilities of N-acyl-d-amino acid amidohydrolase by site-directed mutagenesis of critical methionine residues. / Peng, I-Chen; Lo, Kai Yin; Hsu, Chun Hua; Lee, Chia Yin.

In: Process Biochemistry, Vol. 47, No. 12, 01.12.2012, p. 1785-1790.

Research output: Contribution to journalArticle

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