title: Cloning and characterisation of chlorophyll synthase from Avena sativa.
abstract: The chlorophyll synthase gene from oat (Avena sativa) was cloned and expressed in Escherichia coli. The deduced amino acid sequence consists of 378 amino acids including a presequence of 46 amino acids. Deletion mutants show that a core protein comprising amino acid residues 88 to 377 is enzymatically active. The sequence of the mature protein shows 85% identity with the chlorophyll synthase of Arabidopsis thaliana and 62% identity with the chlorophyll synthase of Synechocystis PCC 6803. The gene is constitutively expressed as the same transcript level is found in dark-grown and in light-grown seedlings. The enzyme requires magnesium ions for activity; manganese ions can reconstitute only part of the activity. Diacetyl and N-phenylmaleimide (NPM) inhibit the enzyme activity. Site-directed mutagenesis reveals that, out of the 4 Arg residues present in the active core protein, Arg-91 and Arg-161 are essential for the activity. Five cysteine residues are present in the core protein, of which only Cys-109 is essential for the enzyme activity. Since the wild-type and all other Cys-mutants with the exception of the mutant C304A are inhibited by N-phenylmaleimide, we conclude that the inhibitor binds to a non-essential Cys residue to abolish activity. The role of the various Arg and Cys residues is discussed.
authors.affiliation: Botanisches Institut der Ludwig-Maximilians-Universität München, Germany.
authors.forename: H C
authors.initials: HC
authors.lastname: Schmid
authors.forename: U
authors.initials: U
authors.lastname: Oster
authors.forename: J
authors.initials: J
authors.lastname: Kögel
authors.forename: S
authors.initials: S
authors.lastname: Lenz
authors.forename: W
authors.initials: W
authors.lastname: Rüdiger
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D018554:Avena / Q000201:enzymology*; D019729:Carbon-Oxygen Ligases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D010940:Plant Proteins; D019729:Carbon-Oxygen Ligases; C026901:chlorophyll synthetase