Abstract

The redesign of enzymes to produce catalysts for a predefined transformation remains a major challenge in protein engineering. Here, we describe the structure-based engineering of methylaspartate ammonia lyase (which in nature catalyses the conversion of 3-methylaspartate to ammonia and 2-methylfumarate) to accept a variety of substituted amines and fumarates and catalyse the asymmetric synthesis of aspartic acid derivatives. We obtained two single-active-site mutants, one exhibiting a wide nucleophile scope including structurally diverse linear and cyclic alkylamines and one with broad electrophile scope including fumarate derivatives with alkyl, aryl, alkoxy, aryloxy, alkylthio and arylthio substituents at the C2 position. Both mutants have an enlarged active site that accommodates the new substrates while retaining the high stereo- and regioselectivity of the wild-type enzyme. As an example, we demonstrate a highly enantio- and diastereoselective synthesis of threo-3-benzyloxyaspartate (an important inhibitor of neuronal excitatory glutamate transporters in the brain).

  • Compound C6H8O4

    2-Ethylfumaric acid

  • Compound C7H10O4

    2-Propylfumaric acid

  • Compound C8H12O4

    2-Butylfumaric acid

  • Compound C9H14O4

    2-Pentylfumaric acid

  • Compound C10H16O4

    2-Hexylfumaric acid

  • Compound C6H8O5

    2-Ethoxyfumaric acid

  • Compound C10H8O5

    2-Phenoxyfumaric acid

  • Compound C11H10O5

    2-(Benzyloxy)fumaric acid

  • Compound C6H8O4S

    2-Ethylthiofumaric acid

  • Compound C10H8O4S

    2-(Phenylthio)fumaric acid

  • Compound C11H10O4S

    2-(Benzylthio)fumaric acid

  • Compound C11H10O4

    2-Benzylfumaric acid

  • Compound C5H9NO4

    2-Amino-3-methylsuccinic acid

  • Compound C6H11NO4

    2-Methyl-3-(methylamino)succinic acid

  • Compound C7H13NO4

    2-(Ethylamino)-3-methylsuccinic acid

  • Compound C8H15NO4

    2-Methyl-3-(propylamino)succinic acid

  • Compound C9H17NO4

    2-(Butylamino)-3-methylsuccinic acid

  • Compound C10H19NO4

    2-Methyl-3-(pentylamino)succinic acid

  • Compound C11H21NO4

    2-(Hexylamino)-3-methylsuccinic acid

  • Compound C8H15NO4

    2-(Isopropylamino)-3-methylsuccinic acid

  • Compound C9H15NO4

    2-(Cyclobutylamino)-3-methylsuccinic acid

  • Compound C10H17NO4

    2-(Cyclopentylamino)-3-methylsuccinic acid

  • Compound C11H19NO4

    2-(Cyclohexylamino)-3-methylsuccinic acid

  • Compound C9H15NO4

    2-((Cyclopropylmethyl)amino)-3-methylsuccinic acid

  • Compound C12H15NO4

    2-(Benzylamino)-3-methylsuccinic acid

  • Compound C8H13NO4

    2-(Cyclopropylamino)-3-methylsuccinic acid

  • Compound C7H13NO5

    2-(Ethoxyamino)-3-methylsuccinic acid

  • Compound C7H13NO5

    2-((2-Hydroxyethyl)amino)-3-methylsuccinic acid

  • Compound C8H15NO5

    2-((3-Hydroxypropyl)amino)-3-methylsuccinic acid

  • Compound C8H15NO5

    2-((2-Methoxyethyl)amino)-3-methylsuccinic acid

  • Compound C8H16N2O4

    2-Methyl-3-((2-(methylamino)ethyl)amino)-succinic acid

  • Compound C7H14N2O4

    2-((2-Aminoethyl)amino)-3-methylsuccinic acid

  • Compound C8H16N2O4

    2-((3-Aminopropyl)amino)-3-methylsuccinic acid

  • Compound C4H7NO4

    2-Aminosuccinic acid

  • Compound C5H9NO4

    2-Amino-3-methylsuccinic acid

  • Compound C6H11NO4

    2-Amino-3-ethylsuccinic acid

  • Compound C7H13NO4

    2-Amino-3-propylsuccinic acid

  • Compound C8H15NO4

    2-Amino-3-butylsuccinic acid

  • Compound C9H17NO4

    2-Amino-3-pentylsuccinic acid

  • Compound C10H19NO4

    2-Amino-3-hexylsuccinic acid

  • Compound C6H11NO5

    2-Amino-3-ethoxysuccinic acid

  • Compound C10H11NO5

    2-Amino-3-phenoxysuccinic acid

  • Compound C11H13NO5

    2-Amino-3-(benzyloxy)succinic acid

  • Compound C6H11NO4S

    2-Amino-3-(ethylthio)succinic acid

  • Compound C10H11NO4S

    2-Amino-3-(phenylthio)succinic acid

  • Compound C11H13NO4S

    2-Amino-3-(benzylthio)succinic acid

  • Compound C11H13NO4

    2-Amino-3-benzylsuccinic acid

  • Compound C12H12O4S

    Dimethyl 2-(phenylthio)fumarate

  • Compound C8H12O7S

    (4R,5R)-Diethyl 1,3,2-dioxathiolane-4,5-dicarboxylate 2-oxide

  • Compound C8H13N3O5

    (2S,3R)-Diethyl 2-azido-3-hydroxysuccinate

  • Compound C8H13NO4

    (2S,3S)-Diethyl aziridine-2,3-dicarboxylate

  • Compound C10H15NO5

    (2S,3S)-Diethyl 1-acetylaziridine-2,3-dicarboxylate

  • Compound C12H21NO5S

    (2R,3R)-Diethyl 2-acetamido-3-(ethylthio)succinate

  • Compound C16H21NO5S

    (2R,3R)-Diethyl 2-acetamido-3-(phenylthio)succinate

  • Compound C17H23NO5S

    (2R,3R)-Diethyl 2-acetamido-3-(benzylthio)succinate

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Acknowledgements

The authors thank B. Wu, B. Kaptein and O. May for helpful discussions. This research was financially supported by VENI grant 700.54.401 and ECHO grant 700.59.042 (both to G.J.P.) from the Division of Chemical Sciences of the Netherlands Organisation of Scientific Research (NWO-CW), and by the Netherlands Ministry of Economic Affairs and the B-Basic partner organizations (www.b-basic.nl) through B-Basic, a public-private NWO–ACTS programme (ACTS, Advanced Chemical Technologies for Sustainability). Financial support from the Royal Netherlands Academy of Arts and Sciences (KNAW to W.S. and B.L.F.) is also gratefully acknowledged.

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Affiliations

  1. Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands

    • Hans Raj
    • , Jandré de Villiers
    • , Vinod Puthan Veetil
    • , Carlos R. Reis
    • , Marianne de Villiers
    • , Wim J. Quax
    •  & Gerrit J. Poelarends
  2. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

    • Wiktor Szymański
    •  & Dick B. Janssen
  3. Center for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

    • Wiktor Szymański
    •  & Ben L. Feringa
  4. Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands

    • Henriëtte J. Rozeboom
    •  & Andy-Mark W. H. Thunnissen
  5. Department of Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands

    • Frank J. Dekker
  6. DSM Pharmaceutical Products, PO Box 18, 6160 MD, Geleen, The Netherlands

    • Stefaan de Wildeman

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Contributions

H.R. performed mutagenesis, library screening and activity measurements. W.S. and J.d.V. synthesized starting substrates and reference compounds. H.R., W.S., M.d.V. and F.J.D. performed preparative biocatalysis. H.J.R. performed X-ray crystallography experiments. V.P.V. performed chiral HPLC. C.R.R. performed the molecular docking experiments. S.d.W., W.J.Q., A.M.W.H.T., B.L.F., D.B.J. and G.J.P. supervised scientific work. All authors contributed to writing the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Gerrit J. Poelarends.

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DOI

https://doi.org/10.1038/nchem.1338

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