title: Site-directed mutagenesis of a tetrameric dandelion polyphenol oxidase (PPO-6) reveals the site of subunit interaction.
abstract: Polyphenol oxidases (PPOs) catalyze the oxidation of ortho-diphenols to the corresponding quinones (EC 1.10.3.1). In plants PPOs appear in gene families, and the corresponding isoenzymes are located to the thylakoid lumen of chloroplasts. Although plant PPOs are often discussed with regard to their role in defense reactions, a common physiological function has not yet been defined. We analyzed a tetrameric PPO isoenzyme (PPO-6) from dandelion (Taraxacum officinale) heterologously expressed in Escherichia coli, and found it to display cooperativity in catalysis, a phenomenon that has rarely been shown for plant PPOs previously. The identification of a surface-exposed cysteine (197) through molecular modeling followed by site-directed mutagenesis proved this amino acid residue to stabilize the tetramer via a disulfide linkage. The C197S-mutein still forms a tetrameric structure but shows impaired enzymatic efficiency and cooperativity and a reduction in stability. These findings indicate that oligomerization may be a physiological requirement for PPO-6 stability and function in vivo and raise new questions regarding distinct functions for specific PPO isoenzymes in plants.
authors: Department of Plant Biology and Biotechnology, Westphalian Wilhelms-University of Münster, Hindenburgplatz 55, 48143 Münster, Germany.
authors: Mareike E
authors: ME
authors: Dirks-Hofmeister
authors: Jennifer K
authors: JK
authors: Inlow
authors: Bruno M
authors: BM
authors: Moerschbacher
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D004156:Catechol Oxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D031205:Taraxacum / Q000201:enzymology / Q000235:genetics*
chemical_list: D050505:Mutant Proteins; D010940:Plant Proteins; D021122:Protein Subunits; D004156:Catechol Oxidase; D003545:Cysteine