Cyanophycin (multi-L-arginyl-poly-L-aspartate), a water-insoluble reserve polymer of cyanobacteria, is a product of nonribosomal peptide synthesis. The purification of cyanophycin synthetase of the cyanobacterium Anabaena variabilis is described. In sodium dodecylsulfate/polyacrylamide gel electrophoresis, the enzyme preparation shows one band with an apparent molecular mass of 100 kDa. The native enzyme has an apparent molecular mass of approximately 230 kDa, as determined by size-exclusion chromatography, suggesting that the active form is a homodimer. During catalysis, ATP is converted to ADP. The gene coding for cyanophycin synthetase has been identified in the sequenced genome of Synechocystis sp. PCC 6803. The C-terminal 60% of the deduced amino acid sequence of cyanophycin synthetase show sequence similarity to enzymes of the superfamily of ligases involved in the biosynthesis of murein and of folyl-poly(γ-glutamate). Cells of Escherichia coli harbouring the gene on a plasmid express active synthetase and accumulate cyanophycin-like material. The results prove that a single enzyme catalyzes the de novo synthesis of cyanophycin.Keywords : non-ribosomal peptide synthesis; cyanobacteria ; cyanophycin synthetase; purification; heterologous expression.Most cyanobacteria contain the nitrogen-rich reserve mateFrom the cyanobacterium Anabaena cylindrica, Simon [9] has enriched an enzyme that synthesizes cyanophycin in vitro rial multi-L-arginyl-poly (L-aspartic acid) (trivial name, cyanoand has studied the basic properties of the biosynthetic reaction. phycin), which is deposited in the cytoplasm in the form of gran-The substrates of the reaction are the constituent amino acids Lules [1Ϫ4]. In this polymer, nearly all β-carboxy groups of a arginine and L-aspartic acid, Mg-ATP, and cyanophycin as poly(aspartate) backbone are linked to A-amino groups of argiprimer [9]. In a strict sense, the assay measures an elongation nine residues by iso-peptide bonds, resulting in an approximate reaction. A thiol reagent such as 2-mercaptoethanol and K ϩ were 1:1 stoichiometry of aspartate/arginine [1,5,6]. The polymer is found to be required for full activity [9]. The latter properties the product of nonribosomal peptide synthesis [7]. Its molecular are, in addition to the formation of iso-peptide bonds, reminismass, as estimated by SDS/PAGE, in a given cyanobacterial specent of the requirements of the biosynthesis of glutathione [10] cies is not uniform, but ranges over about 25Ϫ100 kDa [1]. Cyaand of poly(γ-D-glutamate) [11]. nophycin is also present in filamentous cyanobacteria such asIn this communication, we describe the purification of cyaAnabaena which differentiate, in a semiregular pattern, so-called nophycin synthetase from the cyanobacterium Anabaena varheterocysts, cells specialized for fixation of N 2 under aerobic iabilis American Type Culture Collection (ATCC) 29413. It is conditions. In these species, cyanophycin may not only serve as demonstrated that a homodimer of a 100-kDa protein incorpoa reserve material,...