title: Identification of the veratryl alcohol binding site in lignin peroxidase by site-directed mutagenesis.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, 16802, USA.
authors.forename: K
authors.initials: K
authors.lastname: Ambert-Balay
authors.forename: S M
authors.initials: SM
authors.lastname: Fuchs
authors.forename: M
authors.initials: M
authors.lastname: Tien
mesh_terms: D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001592:Benzyl Alcohols / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli; D018698:Glutamic Acid / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001592:Benzyl Alcohols; D006420:Hemeproteins; D011994:Recombinant Proteins; D018698:Glutamic Acid; D012694:Serine; D010544:Peroxidases; C042858:lignin peroxidase; C042197:veratryl alcohol; D000409:Alanine