Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi- L-arginyl-poly- L-aspartate (cyanophycin)

Authors


  • Correspondence to W. Lockau, Institut für Biologie, Humboldt-Universität zu Berlin, Chausseestr. 117, D-10115 Berlin, Germany

  • Fax: +49 30 2093 8164.

  • Abbreviations. ATCC, American Type Culture Collection; ORF, open reading frame; cphA, gene coding for cyanophycin synthetase; PCC, Pasteur Collection of Cyanobacteria.

  • Enzymes. UDP-N-acetylmuramate : L-alanine ligase ( EC6.3.2.8); UDP-N-acetylmuramoylalanine : D-glutamate ligase ( EC6.3.2.9); UDP-N-acetylmuramoylalanyl- D-glutamate : 2,6-diaminopimelate ligase ( EC6.3.2.13); UDP-N-acetylmuramoylalanyl- D-glutamyl-2,6-diaminopimelate : D-alanyl- D-alanine ligase ( EC6.3.2.15); folyl-poly(γ-glutamate) synthetase ( EC6.3.2.17).

  • Note. The cyanophycin synthetase gene sequence of Synechocystis sp. strain PCC 6803 is available via the Internet (CyanoBase; http://www.kazusa.or.jp/cyano/cyano.html).

Abstract

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.

Ancillary