Abstract
The metazoan cytosolic fatty acid synthase (FAS) contains all of the enzymes required for de novo fatty acid biosynthesis covalently linked around two reaction chambers. Although the three-dimensional architecture of FAS has been mostly defined, it is unclear how reaction intermediates can transfer between distant catalytic domains. Using single-particle EM, we have identified a near continuum of conformations consistent with a remarkable flexibility of FAS. The distribution of conformations was influenced by the presence of substrates and altered by different catalytic mutations, suggesting a direct correlation between conformation and specific enzymatic activities. We interpreted three-dimensional reconstructions by docking high-resolution structures of individual domains, and they show that the substrate-loading and condensation domains dramatically swing and swivel to access substrates within either reaction chamber. Concomitant rearrangement of the β-carbon–processing domains synchronizes acyl chain reduction in one chamber with acyl chain elongation in the other.
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Acknowledgements
We thank A. Witkowski for helpful discussions. We also acknowledge the National Resource for Automated Molecular Microscopy for assistance with data collection. The work was supported by a research fellowship F32 DK080622 (to E.J.B.) and grant RO1 DK16073 (to S.S.) from the US National Institutes of Health.
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E.J.B. performed all experiments and data analysis; S.S. provided purified FAS; all authors contributed to designing experiments, interpreting results and writing the manuscript.
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Supplementary Text and Figures
Supplementary Figures 1–5 (PDF 2633 kb)
Supplementary Video 1
2D analysis reveals a continuum of domain rearrangements within the β-carbon processing portion of FAS. (GIF 2693 kb)
Supplementary Video 2
An animation illustrates how FAS may interchange between the experimentally observed conformations to facilitate contacts between the ACP and active sites of the catalytic domains in a typical reaction cycle. (MOV 9889 kb)
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Brignole, E., Smith, S. & Asturias, F. Conformational flexibility of metazoan fatty acid synthase enables catalysis. Nat Struct Mol Biol 16, 190–197 (2009). https://doi.org/10.1038/nsmb.1532
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DOI: https://doi.org/10.1038/nsmb.1532
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