Cloning and characterisation of chlorophyll synthase from Avena sativa.
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.
Schmid
H C
HC
Botanisches Institut der Ludwig-Maximilians-Universität München, Germany.
Oster
U
U
Kögel
J
J
Lenz
S
S
Rüdiger
W
W
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
D004791:Enzyme Inhibitors; D010940:Plant Proteins; D019729:Carbon-Oxygen Ligases; C026901:chlorophyll synthetase