title: Probing the selectivity of β-hydroxylation reactions in non-ribosomal peptide synthesis using analytical ultracentrifugation.
abstract: Bacteria and fungi use non-ribosomal peptide synthetases (NRPSs) to produce peptides of broad structural diversity and biological activity, many of which have proven to be of great importance for human health. The impressive diversity of non-ribosomal peptides originates in part from the action of tailoring enzymes that modify the structures of single amino acids and/or the mature peptide. Studying the interplay between tailoring enzymes and the peptidyl carrier proteins (PCPs) that anchor the substrates is challenging owing to the transient and complex nature of the protein-protein interactions. Using sedimentation velocity (SV) methods, we studied the collaboration between the PCPs and cytochrome P450 enzyme that results in the installation of β-hydroxylated amino acid precursors in the biosynthesis of the depsipeptide skyllamycin. We show that SV methods developed for the analytical ultracentrifuge are ideally suited for a quantitative exploration of PCP-enzyme equilibrium interactions. Our results suggest that the PCP itself and the presence of substrate covalently tethered to the PCP together facilitate productive PCP-P450 interactions, thereby revealing one of nature's intricate strategies for installing interesting functionalities using natural product synthetases.
authors.affiliation: Department of Biology, Haverford College, Haverford, PA 19041, USA.
authors.forename: Bashkim
authors.initials: B
authors.lastname: Kokona
authors.affiliation: Department of Chemistry, Haverford College, Haverford, PA 19041, USA.
authors.forename: Emily S
authors.initials: ES
authors.lastname: Winesett
authors.affiliation: Department of Chemistry, Haverford College, Haverford, PA 19041, USA.
authors.forename: A Nikolai
authors.initials: AN
authors.lastname: von Krusenstiern
authors.affiliation: Max Planck Institute for Medical Research, 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
authors.affiliation: Department of Biology, Haverford College, Haverford, PA 19041, USA. Electronic address: rfairman@haverford.edu.
authors.forename: Robert
authors.initials: R
authors.lastname: Fairman
authors.affiliation: Department of Chemistry, Haverford College, Haverford, PA 19041, USA. Electronic address: lcharkou@haverford.edu.
authors.forename: Louise K
authors.initials: LK
authors.lastname: Charkoudian
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D047630:Depsipeptides / Q000032:analysis* / Q000096:biosynthesis; D006900:Hydroxylation; D010453:Peptide Synthases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014461:Ultracentrifugation
chemical_list: D000596:Amino Acids; D047630:Depsipeptides; D003577:Cytochrome P-450 Enzyme System; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: Analytical ultracentrifugation; Cytochrome P450; Non-ribosomal peptide synthetase; Peptidyl carrier protein; Sedimentation velocity; Skyllamycin