Engineering the substrate acceptance of l-amine dehydrogenase enables the collective biocatalytic synthesis of N-heterocyclic primary amines

Wu, Tao; Xu, Yan; Nie, Yao; Mu, Xiaoqing

doi:10.1016/j.cej.2024.151735

Chemical Engineering Journal

2024

DOI: 10.1016/j.cej.2024.151735

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Engineering the substrate acceptance of l-amine dehydrogenase enables the collective biocatalytic synthesis of N-heterocyclic primary amines

Tao Wu,

Yan Xu,

Yao Nie

et al.

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Computer-Aided Flexible Loops Engineering of Glutamate Dehydrogenase for Asymmetric Synthesis of Chiral Pesticides l-phosphinothricin

Yang,

Huang,

Amanze

et al. 2024

J. Agric. Food Chem.

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The access to the enantiopure noncanonical amino acid L-phosphinothricin (L-PPT) by applying biocatalysts is highly appealing in organic chemistry. In this study, a NADH-dependent glutamate dehydrogenase from Lachnospiraceae bacterium (LbGluDH) was chosen for the asymmetric synthesis of L-PPT. Three flexible loops undergoing big conformational shifts during the catalysis were identified and rationally engineered following the initial mutagenesis. The enzyme's specific activity toward the key precursor of L-PPT, 2-oxo-4-[(hydroxy) (methyl) phosphinyl] butyric acid (PPO), was improved from negligible to 9 U/mg, and the K m value was reduced to 17 mM. The computational analysis showed that the modified loops broadened the enzyme's narrow tunnels, allowing the substrate to access the binding pocket and get closer to the crucial residue D165, thereby enhancing the catalytic process. Utilizing the variant as the catalyst, the preparation of L-PPT achieved a 100% conversion rate within 60 min, coupled with a stereoselectivity exceeding 99.9%, demonstrating its practical capacity for industrial application. Similar enhancement in catalytic activity was obtained applying the same strategy to a typical NADH-dependent GluDH from Pyrobaculum islandicum (PisGluDH), indicating the effectiveness of our strategy for the protein engineering of GluDHs targeted to the biosynthesis of unnatural compounds.

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Computer-Aided Flexible Loops Engineering of Glutamate Dehydrogenase for Asymmetric Synthesis of Chiral Pesticides l-phosphinothricin

Yang,

Huang,

Amanze

et al. 2024

J. Agric. Food Chem.

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show abstract

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Engineering the substrate acceptance of l-amine dehydrogenase enables the collective biocatalytic synthesis of N-heterocyclic primary amines

Cited by 1 publication

References 82 publications

Computer-Aided Flexible Loops Engineering of Glutamate Dehydrogenase for Asymmetric Synthesis of Chiral Pesticides l-phosphinothricin

Computer-Aided Flexible Loops Engineering of Glutamate Dehydrogenase for Asymmetric Synthesis of Chiral Pesticides l-phosphinothricin

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