title: Rapid and flexible biochemical assays for evaluating 4'-phosphopantetheinyl transferase activity.
abstract: PPTases (phosphopantetheinyl transferases) are of great interest owing to their essential roles in activating fatty acid, polyketide and non-ribosomal peptide synthetase enzymes for both primary and secondary metabolism, as well as an increasing number of biotechnological applications. However, existing techniques for PPTase characterization and development are cumbersome and technically challenging. To address this, we have developed the indigoidine-synthesizing non-ribosomal peptide synthetase BpsA as a reporter for PPTase activity. Simple co-transformation allows rapid assessment of the ability of a PPTase candidate to activate BpsA in vivo. Kinetic parameters with respect to either CoA or BpsA as variable substrate can then be derived in vitro by continuously measuring the rate of indigoidine synthesis as the PPTase progressively converts BpsA from its apo into holo form. Subsequently, a competition assay, in which BpsA and purified carrier proteins compete for a limited pool of CoA, enables elucidation of kinetic parameters for a PPTase with those carrier proteins. We used this system to conduct a rapid characterization of three different PPTase enzymes: Sfp of Bacillus subtilis A.T.C.C.6633, PcpS of Pseudomonas aeruginosa PAO1, and the putative PPTase PP1183 of Ps. putida KT2440. We also demonstrate the utility of this system for discovery and characterization of PPTase inhibitors.
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington 6140, New Zealand.
authors.forename: Jeremy G
authors.initials: JG
authors.lastname: Owen
authors.forename: Janine N
authors.initials: JN
authors.lastname: Copp
authors.forename: David F
authors.initials: DF
authors.lastname: Ackerley
mesh_terms: D001051:Apoenzymes / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D003374:Coumarins / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D010453:Peptide Synthases / Q000378:metabolism*; D010881:Piperidones / Q000378:metabolism; D011550:Pseudomonas aeruginosa; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D001051:Apoenzymes; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003374:Coumarins; D010881:Piperidones; C082662:phosphopantetheinyl transferase; D005973:Glutamine; C071196:6-nitroso-1,2-benzopyrone; C446045:indigoidine; D017855:Transferases (Other Substituted Phosphate Groups); D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase