Cloning and characterisation of chlorophyll synthase from Avena sativa

The chlorophyll synthase gene from oat (Avena sativa) was cloned and expres sed in Escherichia coli. The deduced amino acid sequence consists of 378 am ino acids including a presequence, of 46 amino acids. Deletion mutants show that a core protein comprising amino acid residues 88 to 377 is enzymatica lly active. The sequence of the mature protein shows 85% identity with the chlorophyll synthase of Arabidopsis thaliana and 62% identity with the chlo rophyll synthase of Synechocystis PCC 6803. The gene is constitutively expr essed as the same transcript level is found in dark-grown and in light-grow n seedlings. The enzyme requires magnesium ions for activity; manganese ion s can reconstitute only part of the activity. Diacetyl and N-phenylmaleimid e (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 acti vity. Since the wild-type and all other Cys-mutants with the exception of t he mutant C304A are inhibited by N-phenylmaleimide, we conclude that the in hibitor binds to a non-essential Cys residue to abolish activity. The role of the various Arg and Cys residues is discussed.