Engineering of a manganese-binding site in lignin peroxidase isozyme H8 from Phanerochaete chrysosporium.
A Mn(2+)-binding site was created in the recombinant lignin peroxidase isozyme H8 from Phanerochaete chrysosporium. In fungal Mn peroxidase, the Mn-binding site is composed of Glu35, Glu39, and Asp179. We generated a similar site in lignin peroxidase by generating an anionic binding site. We generated three mutations: Asn182Asp, Asp183Lys, and Ala36Glu. Its activity, veratryl alcohol, and Mn(2+) oxidation were compared to those of native recombinant enzyme and to fungal Mn peroxidase isozyme H4, respectively. The mutated enzyme was able to oxidize Mn(2+) and still retain its ability to oxidize veratryl alcohol. Steady-state results indicate that the enzyme's ability to oxidize veratryl alcohol was lowered slightly. The K(m) for Mn(2+) was determined to be 1.57 mM and the k(cat) = 5.45 s(-1). These results indicate that the mutated lignin peroxidase is less effective in Mn(2+) oxidation that the wild type fungal enzyme. The pH optima of veratryl alcohol and Mn oxidation were altered by the mutation. They are one unit of pH value higher than those of recombinant H8 and wild type fungal Mn peroxidase isozyme H4.
Mester
T
T
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Tien
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M
D000595:Amino Acid Sequence; D001592:Benzyl Alcohols / Q000378:metabolism; D001665:Binding Sites; D005800:Genes, Fungal; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D020075:Phanerochaete / Q000201:enzymology*; D010802:Phylogeny; D016415:Sequence Alignment
D001592:Benzyl Alcohols; D008345:Manganese; D006861:Hydrogen Peroxide; D010544:Peroxidases; C042858:lignin peroxidase; C051129:manganese peroxidase; C042197:veratryl alcohol