Abstract
Objective
To develop a model for binding and catalysis associated with the stimulation of 4-fluorophenol (4-FP) oxidation in the presence of long chain aldehydes by the enzymatic catalyst, cytochrome P450BM3-F87G.
Results
A variation of the Michaeli–Menten kinetic model was employed to describe interactions at the active site of the enzyme, along with computer aided modeling approaches. In addition to the hydroquinone product arising from de-fluorination of 4-FP, a second product (p-fluorocatechol) was also observed and, like the hydroquinone, its rate of formation increased in the presence of the aldehyde. When only aldehyde was present with the enzyme, BM3-F87G catalyzed its oxidation to the corresponding carboxylic acid; however, this activity was inhibited when 4-FP was added to the reaction. A 3D computer model of the active site containing both aldehyde and 4-FP was generated, guided by these kinetic observations. Finally, partitioning between the two phenolic products was examined with an emphasis on the conditions directing the initial epoxidation at either the 2,3- or 3,4-positions on the substrate. Temperature, reaction time, substrate concentration, and the structure of the aldehyde had no substantial effect on the overall product ratios, however the NADPH coupling efficiency decreased when unsaturated aldehydes were included, or when the temperature of the reaction was reduced.
Conclusions
The unsaturated aldehyde, trans-2-decenal, stimulates BM3-F87G catalyzed oxidation of 4-fluorophenol through a cooperative active site binding mode that doesn’t influence product distributions or coupling efficiencies, while 4-fluorophenol acts as a competitive inhibitor of aldehyde oxidation.
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Acknowledgements
Funding for this research was provided by The National Science Foundation (#0414301), Research Corporation (CC4924) and The American Chemical Society Petroleum Research Fund (41094-UFS and 37796-B4) to G.M.R.
Supporting information
Supplementary Fig. 1—(A) Representative titration for determining the Ks for trans-2-decenal binding to BM3-F87G. (B) Resulting titration curve used to determine the Ks for binding.
Supplementary Fig. 2—HPLC of reactions containing BM3-F87G and 15 mM 4-FP in the absence of NADPH, in the presence of 1 mM NADPH, and in the presence of 1 mM NADPH and 2 mM glutathione.
Supplementary Fig. 3—Raw data collected for calculating the NADPH coupling efficiency for the reaction of BM3-F87G with 4-FP at 27 °C for 20 min.
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Ledford, C., McMahon, M., Whitesell, A. et al. A dual substrate kinetic model for cytochrome P450BM3-F87G catalysis: simultaneous binding of long chain aldehydes and 4-fluorophenol. Biotechnol Lett 39, 311–321 (2017). https://doi.org/10.1007/s10529-016-2252-7
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DOI: https://doi.org/10.1007/s10529-016-2252-7