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Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

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Abstract

FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is thus of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl-CoA, we predicted amino acid positions within the effector binding pocket that would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.

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Abbreviations

TR:

Transcriptional regulator

LCACoAs:

Long chain acyl-coenzyme A thioesters

WT:

Wild-type

GST:

Glutathione S-Transferase

SDS:

Sodium dodecyl sulfate

EMSA:

Electrophoretic mobility shift assay

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Authors and Affiliations

  1. Bioenergy and Biome Sciences Group, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, 87544, USA

    John-Paul Bacik, Chris M. Yeager, Scott N. Twary & Ricardo Martí-Arbona

  2. Bioscience Division, Group B-11, Los Alamos National Laboratory, MS E529, PO Box 1663, Los Alamos, NM, 87545, USA

    Ricardo Martí-Arbona

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  1. John-Paul Bacik
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  2. Chris M. Yeager
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  4. Ricardo Martí-Arbona
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Correspondence to Ricardo Martí-Arbona.

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Bacik, JP., Yeager, C.M., Twary, S.N. et al. Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis. Protein J 34, 359–366 (2015). https://doi.org/10.1007/s10930-015-9630-1

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