title: Acyl-CoA subunit selectivity in the pikromycin polyketide synthase PikAIV: steady-state kinetics and active-site occupancy analysis by FTICR-MS.
abstract: Polyketide natural products generated by type I modular polyketide synthases (PKSs) are vital components in our drug repertoire. To reprogram these biosynthetic assembly lines, we must first understand the steps that occur within the modular "black boxes." Herein, key steps of acyl-CoA extender unit selection are explored by in vitro biochemical analysis of the PikAIV PKS model system. Two complementary approaches are employed: a fluorescent-probe assay for steady-state kinetic analysis, and Fourier Transform Ion Cyclotron Resonance-mass spectrometry (FTICR-MS) to monitor active-site occupancy. Findings from five enzyme variants and four model substrates have enabled a model to be proposed involving catalysis based upon acyl-CoA substrate loading followed by differential rates of hydrolysis. These efforts suggest a strategy for future pathway engineering efforts using unnatural extender units with slow rates of hydrolytic off-loading from the acyltransferase domain.
authors.affiliation: Department of Chemistry, Portland State University, Portland, OR 97201, USA.
authors.forename: Shilah A
authors.initials: SA
authors.lastname: Bonnett
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Rath
authors.forename: Abdur-Rafay
authors.initials: AR
authors.lastname: Shareef
authors.forename: Joanna R
authors.initials: JR
authors.lastname: Joels
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Chemler
authors.forename: Kristina
authors.initials: K
authors.lastname: Håkansson
authors.forename: Kevin
authors.initials: K
authors.lastname: Reynolds
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry; D020134:Catalytic Domain; D005583:Fourier Analysis; D006868:Hydrolysis; D007700:Kinetics; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D009154:Mutation; D048630:Polyketide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D000900:Anti-Bacterial Agents; D018942:Macrolides; D011993:Recombinant Fusion Proteins; D048630:Polyketide Synthases; C003145:picromycin