The structure and function of cyanophycin synthetase Itai Sharon 1 , Sharon Pinus 2 , Marcel Grogg 3 , Nicolas Moitessier 2 , Donald Hilvert 3 and T. Martin Schmeing 1 1 Department of Biochemistry and Centre de recherche en biologie structurale, McGill University, Canada, 2 Department of Chemistry, McGill University, Canada, 3 ETH Zürich, Switzerland. Cyanophycin is a natural biopolymer consisting of a chain of poly-L-Asp residues with L-Arg residues attached to the b-carboxylate side chains by isopeptide bonds 1 . First discovered in cyanobacteria in 1886, cyanophycin is produced by a wide range of bacteria and is important for cellular nitrogen storage 2 . Cyanophycin is synthesized from ATP, aspartic acid, arginine and a suitable primer by a homooligomeric enzyme called cyanophycin synthetase (CphA1) 3 . CphA1 has domains that are homologous to glutathione synthetases and muramyl ligases, but no other structural information has been available, and many questions regarding its structure and mechanism of action were outstanding. We used cryo-electron microscopy to determine structures of CphA1 from the cyanobacterium synechocystic sp. UTEX2470,including co-complex structures of CphA1 with ATP and cyanophycin polymer analogs at 2.6 Å resolution 4 . These structures show the configuration of active sites and polymer-binding regions, indicate dynamic conformational changes, and afford insight into the enzyme's catalytic mechanism. We also make the discovery that CphA1 can make minute quantities of cyanophycin without primer, and an unexpected, cryptic metallopeptidase-like active site in the N-terminal domain of many CphA1s digests these into primers, solving the problem of primer availability 5 . These results show howevolution combined three different enzymes into one elegant macromolecular machine. References 1. Simon, R.D. & Weathers, P. Determination of the structure of the novel polypeptide containing aspartic acid and arginine which is found in cyanobacteria. Biochim Biophys Acta 420 , 165–76 (1976). 2. Liang, B. et al. Cyanophycin mediates the accumulation and storage of fixed carbon in non-heterocystous filamentous cyanobacteria from coniform mats. PLoS One 9, e88142 (2014). 3. Ziegler, K. et al. Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartate (cyanophycin). European journal of biochemistry 254, 154-9 (1998). 4. Sharon, I. et al. Structures and function of the amino acid polymerase cyanophycin synthetase. Nat Chem Biol 17, 1101–1110 (2021). 5. Sharon, I. et al. A cryptic third active site in cyanophycin synthetase creates primers for polymerization. Nat comm , under review.
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