Acyl-CoA Subunit Selectivity in the Pikromycin Polyketide Synthase PikAIV: Steady-State Kinetics and Active-Site Occupancy Analysis by FTICR-MS

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• 作者：Shilah  ; A. Bonnett¹ ;   ; ⁵ ; Christopher  ; M. Rath² ;   ; ⁵ ; Abdur-Rafay Shareef² ; Joanna  ; R. Joels² ; Joseph  ; A. Chemler² ; Kristina Hå ; kansson³ ; Kevin Reynolds¹ ;   ; ^{reynoldsk@pdx.edu} ; David  ; H. Sherman² ;   ; ³ ;   ; ⁴ ;   ; ^{davidhs@umich.edu}
• 刊名：Chemistry & Biology
• 出版年：2011
• 出版时间：23 September 2011
• 年：2011
• 卷：18
• 期：9
• 页码：1075-1081
• 全文大小：372 K

文摘

Summary

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.