The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain

• Published in: Nature structural & molecular biology Vol. 19; no. 8; pp. 767 - 772
• Main Authors: Koehnke, Jesko, Bent, Andrew, Houssen, Wael E, Zollman, David, Morawitz, Falk, Shirran, Sally, Vendome, Jeremie, Nneoyiegbe, Ada F, Trembleau, Laurent, Botting, Catherine H, Smith, Margaret C M, Jaspars, Marcel, Naismith, James H
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.08.2012; Nature Publishing Group
• Subjects: 631/45/2783; 631/45/535; Amino Acid Sequence; Amino acids; Animals; Bacterial genetics; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biochemistry; Biological Microscopy; Biomedical and Life Sciences; Biotechnology; Chemical synthesis; Cyanobacteria; Genetic aspects; Life Sciences; Macrocycles; Membrane Biology; Models, Molecular; Oligopeptides - chemistry; Oligopeptides - metabolism; Peptides; Peptides, Cyclic - chemistry; Peptides, Cyclic - genetics; Peptides, Cyclic - metabolism; Physiological aspects; Prochloron - genetics; Prochloron - metabolism; Protein Conformation; Protein Structure; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Substrate Specificity; Substrates; Subtilisins - chemistry; Subtilisins - genetics; Subtilisins - metabolism; Symbiosis; Urochordata - microbiology; United States; United Kingdom
• ISSN: 1545-9993; 1545-9985
• DOI: 10.1038/nsmb.2340

The subtilisin-like domain of PatG can catalyze the macrocyclization of linear peptide. The crystal structure of the PatG macrocyclase domain, along with mutagenesis and functional analyses, reveals how the enzyme recognizes its substrate and enforces macrocyclization over hydrolysis of the acyl-enzyme intermediate. This information is also used to engineer variants with altered specificity and enhanced catalytic activity. Peptide macrocycles are found in many biologically active natural products. Their versatility, resistance to proteolysis and ability to traverse membranes has made them desirable molecules. Although technologies exist to synthesize such compounds, the full extent of diversity found among natural macrocycles has yet to be achieved synthetically. Cyanobactins are ribosomal peptide macrocycles encompassing an extraordinarily diverse range of ring sizes, amino acids and chemical modifications. We report the structure, biochemical characterization and initial engineering of the PatG macrocyclase domain of Prochloron sp. from the patellamide pathway that catalyzes the macrocyclization of linear peptides. The enzyme 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 nonactivated substrates has wide biotechnology applications.