Identification of the veratryl alcohol binding site in lignin peroxidase by site-directed mutagenesis

Katia Ambert-Balay, Stephen M. Fuchs, Ming Tien

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Site-directed mutagenesis was used to identify the veratryl alcohol binding site of lignin peroxidase. The cDNA encoding isozyme H8 was mutated at Glu146 to both an Ala and a Ser residue. The H8 polypeptide was produced by E. coli as inclusion bodies and refolded to yield active enzyme. The wild type recombinant enzyme and the mutants were purified to homogeneity and characterized by steady state kinetics. The kcat is decreased for both mutants of Glu146. The reactivity of mutants (kcat/Km) toward H2O2 were not affected. In contrast, the kcat/Km of the mutants for veratryl alcohol were decreased by at least half. The oxidation of guaiacol by these mutants were more significantly affected. These results collectively suggest that E146 plays a central role in the binding of veratryl alcohol by lignin peroxidase.

Original languageEnglish (US)
Pages (from-to)283-286
Number of pages4
JournalBiochemical and Biophysical Research Communications
Volume251
Issue number1
DOIs
StatePublished - Oct 9 1998

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Mutagenesis
Site-Directed Mutagenesis
Binding Sites
Guaiacol
Inclusion Bodies
Enzymes
Escherichia coli
Isoenzymes
Complementary DNA
Oxidation
Peptides
Kinetics
veratryl alcohol
lignin peroxidase

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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abstract = "Site-directed mutagenesis was used to identify the veratryl alcohol binding site of lignin peroxidase. The cDNA encoding isozyme H8 was mutated at Glu146 to both an Ala and a Ser residue. The H8 polypeptide was produced by E. coli as inclusion bodies and refolded to yield active enzyme. The wild type recombinant enzyme and the mutants were purified to homogeneity and characterized by steady state kinetics. The kcat is decreased for both mutants of Glu146. The reactivity of mutants (kcat/Km) toward H2O2 were not affected. In contrast, the kcat/Km of the mutants for veratryl alcohol were decreased by at least half. The oxidation of guaiacol by these mutants were more significantly affected. These results collectively suggest that E146 plays a central role in the binding of veratryl alcohol by lignin peroxidase.",
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Identification of the veratryl alcohol binding site in lignin peroxidase by site-directed mutagenesis. / Ambert-Balay, Katia; Fuchs, Stephen M.; Tien, Ming.

In: Biochemical and Biophysical Research Communications, Vol. 251, No. 1, 09.10.1998, p. 283-286.

Research output: Contribution to journalArticle

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