title: Functional Characterization of the Unique Terminal Thioesterase Domain from Polymyxin Synthetase.
abstract: Polymyxins remain one of the few antibiotics available for treating antibiotic resistant bacteria. Here we describe polymyxin B thioesterase which performs the final step in polymyxin B biosynthesis. Isolated thioesterase catalyzed cyclization of an N-acetylcystamine polymyxin B analogue to form polymyxin B. The thioesterase contained a catalytic cysteine unlike most thioesterases which possess a serine. Supporting this, incubation of polymyxin B thioesterase with reducing agents abolished enzymatic activity, while mutation of the catalytic cysteine to serine significantly decreased activity. NMR spectroscopy demonstrated that uncyclized polymyxin B was disordered in solution, unlike other thioesterase substrates which adopt a transient structure similar to their product. Modeling showed the thioesterase substrate-binding cleft was highly negatively charged, suggesting a mechanism for the cyclization of the substrate. These studies provide new insights into the role of polymyxin thioesterase in polymyxin biosynthesis and highlight its potential use for the chemoenzymatic synthesis of polymyxin lipopeptides.
authors.forename: Charles A
authors.identifier: 0000-0003-2730-1105
authors.initials: CA
authors.lastname: Galea
authors.forename: Kade D
authors.initials: KD
authors.lastname: Roberts
authors.affiliation: Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University , Parkville, Victoria 3800, Australia.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhu
authors.forename: Philip E
authors.initials: PE
authors.lastname: Thompson
authors.affiliation: Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University , Parkville, Victoria 3800, Australia.
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.forename: Tony
authors.identifier: 0000-0002-0017-7952
authors.initials: T
authors.lastname: Velkov
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003500:Cyclization; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011112:Polymyxin B / Q000096:biosynthesis* / Q000737:chemistry; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D013869:Thiolester Hydrolases; C044320:thioesterase B; D008025:Ligases; D011112:Polymyxin B; D003545:Cysteine