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Engineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acids


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).

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Figure 1: Crystal structure of wild-type MAL in complex with the natural substrate threo-(2S,3S)-3-methylaspartate.
Figure 2: Enzyme-catalysed transformations under optimized reaction conditions.
Figure 3: Structural comparison of wild-type MAL and the Gln73Ala and Leu384Ala mutants.
Figure 4: Molecular docking of substrates in the active sites of wild-type MAL and the two MAL mutants.


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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 ( 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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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.

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Correspondence to Gerrit J. Poelarends.

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The authors declare no competing financial interests.

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Raj, H., Szymański, W., de Villiers, J. et al. Engineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acids. Nature Chem 4, 478–484 (2012).

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