title: Regulation of the P450 Oxygenation Cascade Involved in Glycopeptide Antibiotic Biosynthesis.
abstract: Glycopeptide antibiotics (GPAs) are nonribosomal peptides rich in modifications introduced by external enzymes. These enzymes act on the free peptide aglycone or intermediates bound to the nonribosomal peptide synthetase (NRPS) assembly line. In this process the terminal module of the NRPS plays a crucial role as it contains a unique recruitment platform (X-domain) interacting with three to four modifying Cytochrome P450 (P450) enzymes that are responsible for cyclizing bound peptides. However, whether these enzymes share the same binding site on the X-domain and how the order of the cyclization steps is orchestrated has remained elusive. In this study we investigate the first two reactions in teicoplanin aglycone maturation catalyzed by the enzymes OxyBtei and OxyAtei. We demonstrate that both enzymes interact with the X-domain via the identical interaction site with similar affinities, irrespective of the peptide modification stage, while their catalytic activity is restricted to the correctly cross-linked peptide. On the basis of steady state kinetics of the OxyBtei-catalyzed reaction, we propose a model for P450 recruitment and peptide modification that involves continuous association/dissociation of the P450 enzymes with the NRPS, followed by specific recognition of the peptide cyclization state by the P450 (scanning). This leads to an induced conformational change that enhances the affinity of the enzyme/substrate complex and initiates catalysis; product release then occurs, with the product itself becoming the substrate for the second enzyme in the pathway. This model rationalizes our experimental findings for this complex enzyme cascade and provides insights into the orchestration of the sequential peptide tailoring reactions on the terminal NRPS module in GPA biosynthesis.
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research , Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Madeleine
authors.initials: M
authors.lastname: Peschke
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research , Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Haslinger
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research , Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Clara
authors.initials: C
authors.lastname: Brieke
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research , Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Reinstein
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research , Jahnstrasse 29, 69120 Heidelberg, Germany.|EMBL Australia, Monash University , Clayton, Victoria 3800, Australia.|The Department of Biochemistry and Molecular Biology and ARC Centre of Excellence in Advanced Molecular Imaging, Monash University , Clayton, Victoria 3800, Australia.
authors.forename: Max J
authors.initials: MJ
authors.lastname: Cryle
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001665:Binding Sites; D003500:Cyclization; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D006020:Glycopeptides / Q000096:biosynthesis*; D010100:Oxygen / Q000737:chemistry*; D010453:Peptide Synthases / Q000737:chemistry*; D011485:Protein Binding
chemical_list: D000900:Anti-Bacterial Agents; D006020:Glycopeptides; D003577:Cytochrome P-450 Enzyme System; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D010100:Oxygen