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A conserved threonine prevents self-intoxication of enoyl-thioester reductases

Nature Chemical Biology volume 13, pages 745–749 (2017)Cite this article

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Abstract

Enzymes are highly specific biocatalysts, yet they can promote unwanted side reactions. Here we investigated the factors that direct catalysis in the enoyl-thioester reductase Etr1p. We show that a single conserved threonine is essential to suppress the formation of a side product that would otherwise act as a high-affinity inhibitor of the enzyme. Substitution of this threonine with isosteric valine increases side-product formation by more than six orders of magnitude, while decreasing turnover frequency by only one order of magnitude. Our results show that the promotion of wanted reactions and the suppression of unwanted side reactions operate independently at the active site of Etr1p, and that the active suppression of side reactions is highly conserved in the family of medium-chain dehydrogenases/reductases (MDRs). Our discovery emphasizes the fact that the active destabilization of competing transition states is an important factor during catalysis that has implications for the understanding and the de novo design of enzymes.

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Figure 1: Reaction schemes of wild-type Etr1p and variants, and their effect on a hypothetical catalytic landscape.
Figure 2: Detailed characterization of the reactions catalyzed by wild-type Etr1p and variants.
Figure 3: Crystal structures of wild-type Etr1p and the T175V variant.
Figure 4: Cross-inhibition between wild-type Etr1p and Ccr T195V.

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Acknowledgements

The authors thank R. Lill and U. Mühlenhoff at the Core Facility for Protein Spectroscopy and Protein Biochemistry at the University of Marburg for providing access to the stopped-flow instrument, and N.S. Cortina for analysis of high-resolution mass spectrometric analyses. This work was supported by the Max Planck Society and the European Research Council (ERC 637675 “SYBORG” to T.J.E.).

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Author notes

  1. Raoul G Rosenthal

    Present address: John A. Paulson School of Engineering and Applied Sciences (SEAS), Harvard University, Cambridge, Massachusetts, USA

Authors and Affiliations

  1. Biochemistry and Synthetic Biology of Microbial Metabolism Group, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

    Raoul G Rosenthal, Bastian Vögeli & Tobias J Erb

  2. Microbial Protein Structure Group, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

    Tristan Wagner & Seigo Shima

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Contributions

R.G.R., B.V. and T.J.E. conceived and designed all experiments, with the exception of crystallography experiments, which they designed with T.W. and S.S. Enzyme kinetic assays and stopped-flow measurements were performed by R.G.R. and B.V. MS experiments were carried out and analyzed by B.V. and R.G.R. Crystallography experiments were carried out by B.V. and T.W. T.W. and B.V. collected the diffraction data, and T.W. interpreted the results. R.G.R., B.V. and T.J.E. wrote the paper.

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Correspondence to Tobias J Erb.

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Rosenthal, R., Vögeli, B., Wagner, T. et al. A conserved threonine prevents self-intoxication of enoyl-thioester reductases. Nat Chem Biol 13, 745–749 (2017). https://doi.org/10.1038/nchembio.2375

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