Structural and biochemical studies of Patellamide biosynthesis
Final Report Abstract
Cyclic peptides have great potential as novel pharmaceuticals but to date their production has proven difficult for synthetic chemists. Several marine cyanobacteria have been found to produce diverse cyclic peptides, the cyanobactins, through ribosomal synthesis of precursor peptides and post-translational tailoring. The patellamides, a member of the cyanobactin family, are cyclic octapeptides containing D-stereo centers and heterocyclized amino acids. Patellamides are derived from a precursor peptide, PatE, which consists of an approximately 40-residue N-terminal leader sequence followed by two eight-residue core peptides. Each core peptide is flanked by N- and C-terminal protease recognition sites composed of five and three residues respectively. PatE undergoes several modifications, catalyzed by the enzymes PatA, B, C, D, F and G, to form the final cyclic peptide (macrocycle). These modifications include heterocyclization, proteolysis, oxidation, epimerization and macrocyclization with the definitive order of steps yet to be established. In this study all enzymes were expressed and purified for crystallography and biochemical studies. PatA and PatG were analyzed both structurally and biochemically. We determined the structure of the protease domain of PatA, which recognizes the protease recognition site N-terminal of the core peptide in PatE. The structure combined with modeling studies allowed us to understand the recognition requirements for the enzyme and led to the discovery of two new cyanobactins from the cyanobacterium Cyanothece PCC 7425. The molecular rationalization of the specificity of one of the processing enzymes is a significant step forward in harnessing the potential of these natural products and might aid the discovery of more cyanobactins in the future. We also determined the structure of the macrocyclase domain of PatG (PatGmac), which recognizes the C-terminal protease recognition site in PatE and catalyzes the macrocyclization of the linear peptide. In addition to the structure determination we also performed the biochemical characterization and initial engineering of the enzyme. PatGmac contains insertions in the subtilisin fold to allow it to recognize a three-residue signature, bind substrate in a preorganized and unusual conformation, shield an acyl-enzyme intermediate from water and catalyze peptide bond formation. The ability to macrocyclize a broad range of non-activated substrates has wide biotechnological applications.
Publications
- (2012): The discovery of new cyanobactins from Cyanothece PCC 7425 defines a new signature for processing of patellamides. ChemBioChem 13(18): pp2683‐9
Houssen, W.E., Koehnke, J., Zollman, D., Vendome, J., Raab, A., Smith, M.C., Naismith, J.H. and Jaspars, M.
- (2012): The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain. Nat. Struct. Mol. Biol. 19(8): pp767‐72.
Koehnke, J., Bent, A., Houssen, W.E., Zollman, D., Morawitz, F., Shirran, S., Vendome, J., Nneoyiegbe, A.F., Trembleau, L. Botting, C., Smith, M.C., Jaspars, M. and Naismith, J.H.
