JBEI - Structural control of regioselectivity in an unusual bacterial acyl-CoA dehydrogenase

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Mutants assays_WT-but-val_pub data_2019-08-11_07-39-25.sky.zip2019-08-14 14:10:541836669693
Malonate assays_pub data_2019-08-11_07-36-51.sky.zip2019-08-14 14:10:5491830607
WT assays_set 1_pub data_2019-08-11_07-41-51.sky.zip2019-08-14 14:10:5320407410417
Structural control of regioselectivity in an unusual bacterial acyl-CoA dehydrogenase
  • Organism: Escherichia coli
  • Instrument: 6460 Triple Quadrupole LC/MS
  • SpikeIn: No
  • Keywords: Ppant ejection assay
  • Lab head: Chris Petzold Submitter: Chris Petzold
Terminal alkenes are easily derivatized, making them desirable functional group targets for polyketide synthase (PKS) engineering. However, they are rarely encountered in natural PKS systems. One mechanism for terminal alkene formation in PKSs is through the activity of an acyl-CoA dehydrogenase (ACAD). Herein, we use biochemical and structural analysis to understand the mechanism of terminal alkene formation catalyzed by an ACAD from the biosynthesis of the polyketide natural product FK506, TcsD. We show that, while TcsD is homologous to canonical α,β-ACADs, it acts regioselectively at the γ,ẟ-position and only on α,β-unsaturated substrates. Furthermore, this regioselectivity is controlled by a combination of bulky residues in the active site and a lateral shift in the positioning of the FAD cofactor within the enzyme. Substrate modeling suggests that TcsD utilizes a novel set of hydrogen bond donors for substrate activation and positioning, preventing dehydrogenation at the α,β position of substrates. From the structural and biochemical characterization of TcsD, key residues that contribute to regioselectivity and are unique to the protein family were determined and used to identify other putative γ,ẟ-ACADs that belong to diverse natural product biosynthetic gene clusters. This work exemplifies a powerful approach to understanding unique enzymatic reactions and will facilitate future enzyme discovery, inform enzyme engineering, and aid natural product characterization efforts.
Experiment Description
Assays of TcsD activity on TcsAS98A-bound substrates were carried out as follows: First, purified TcsAS98A was loaded with acyl-Coenzyme A substrates via the action of the promiscuous phosphopantetheinyl transferase Sfp. Loading reactions (150 µL) contained 60 µM TcsAS98A, 10 µM Sfp, 250 µM acyl-Coenzyme A, 20 mM MgCl2, and 50 mM sodium phosphate, pH 7.2. Reactions were initiated by the addition of Sfp and allowed to proceed at 37 deg. C for 30 minutes. The loaded protein was then immediately used for TcsD assays, each of which contained: 45 µL of loading reaction, 0.5 mM FeHFP (dissolved in 10 mM HCl), and 2 µM TcsD mutant (or boiled TcsD mutant). FeHFP was prepared as described above. Reactions were allowed to proceed for 16 hours then quenched with and equal volume of HPLC methanol (Sigma Aldrich) and either stored at -20 deg. C or immediately processed for LC-MS analysis.
Created on 8/14/19, 2:33 PM

This data is available under the CC BY 4.0 license.