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Structures of carboxylic acid reductase reveal domain dynamics underlying catalysis

Nature Chemical Biology volume 13, pages 975–981 (2017)Cite this article

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Abstract

Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the nonribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation–PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP–reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that further reduction of the aldehyde product does not occur. Combining crystallography with small-angle X-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This theory is supported by the fact that (R)-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.

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Figure 1: Carboxylic acid reductase (CAR) is a modular enzyme.
Figure 2: Structure of the CARsr A–PCP didomain reveals a dynamic entity.
Figure 3: Structure of CAR reductase and PCP–R regions.
Figure 4: Modeling of the full-length CAR structure.
Figure 5: A dynamic model for CAR.

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Acknowledgements

This work was supported by BBSRC grant (BB/K00199X/1 to N.T., N.S.S. and D.L.). We thank the BBSRC/EPSRC SYNBIOCHEM Centre (grant BB/M017702/1 to N.T., N.S.S. and D.L.) for access to analytical equipment. We thank the Diamond Light Source for access to beam lines for macromolecular crystallography and bio-SAXS (proposal number MX12788). D.L. and N.T. are Royal Society Wolfson Merit Award holders. N.S.S. is an EPSRC Established Career Fellow. We thank the CoEBio3 Affiliates programme for funding to A.H.

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Author notes

  1. Daniela Quaglia

    Present address: Département de chimie, Université de Montréal, Montréal, Québec, Canada

  2. Deepankar Gahloth and Mark S Dunstan: These authors contributed equally to this work.

Authors and Affiliations

  1. Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester, UK

    Deepankar Gahloth, Mark S Dunstan, Daniela Quaglia, Evaldas Klumbys, Andrew M Hill, Sasha R Derrington, Nigel S Scrutton, Nicholas J Turner & David Leys

  2. Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, University of Manchester, Manchester, UK

    Michael P Lockhart-Cairns

  3. Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK

    Michael P Lockhart-Cairns

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Contributions

M.S.D. cloned, expressed and purified various CAR enzymes (both truncations and full-length as well as hybrid forms) and obtained crystal structures for both A and R domains. D.G. cloned, expressed and purified various CAR enzymes and obtained crystals structures for the PCP-didomain constructs in addition to isolated R domain structures. D.G. and M.P.L.-C. performed SAXS data collection and modeling. E.K., D.Q., A.M.H. and S.R.D. performed kinetic data analysis. All authors discussed the results and participated in writing of the manuscript. N.J.T., N.S.S. and D.L. initiated and directed this research.

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Correspondence to David Leys.

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Gahloth, D., Dunstan, M., Quaglia, D. et al. Structures of carboxylic acid reductase reveal domain dynamics underlying catalysis. Nat Chem Biol 13, 975–981 (2017). https://doi.org/10.1038/nchembio.2434

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