Structure-guided protein engineering of ammonia lyase for efficient synthesis of sterically bulky unnatural amino acids

Zi-Fu Ni,Min-Hua Zong,Wen-Yong Lou,Pei Xu

doi:10.1186/s40643-021-00456-5

Zi-Fu Ni, Min-Hua Zong + Show 2 more

Open Access

https://doi.org/10.1186/s40643-021-00456-5

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Abstract

Enzymatic asymmetric amination addition is seen as a promising approach for synthesizing amine derivatives, especially unnatural amino acids, which are valuable precursors to fine chemicals and drugs. Despite the broad substrate spectrum of methylaspartate lyase (MAL), some bulky substrates, such as caffeic acid, cannot be effectively accepted. Herein, we report a group of variants structurally derived from Escherichia coli O157:H7 MAL (EcMAL). A combined mutagenesis strategy was used to simultaneously redesign the key residues of the entrance tunnel and binding pocket to explore the possibility of accepting bulky substrates with potential application to chiral drug synthesis. Libraries of residues capable of lining the active center of EcMAL were then constructed and screened by an effective activity solid-phase color screening method using tyrosinase as a cascade catalyst system. Activity assays and molecular dynamics studies of the resultant variants showed that the substrate specificity of EcMAL was modified by adjusting the polarity of the binding pocket and the degree of flexibility of the entrance tunnel. Compared to M3, the optimal variant M8 was obtained with a 15-fold increase in catalytic activity. This structure-based protein engineering of EcMAL can be used to open new application directions or to develop practical multi-enzymatic processes for the production of various useful compounds.

Highlights

The hydroamination of olefins and carbon–nitrogen bond-forming reactions of unsaturated carboxylic acids offer a vast range of applications in the synthesis of fine chemicals (Raj et al 2012)
Selection and grouping of key amino acids of Ecmal Based on our previous studies, we found that Escherichia coli O157:H7 MAL (EcMAL) could accept a range of short-chain unsaturated carboxylic acids, including fumaric acid, mesaconic acid, and itaconic acid, for the asymmetric synthesis of unnatural amino acids (Ni et al 2020)
We chose caffeic acid (1a) as the model compound for assessing the activity of EcMAL mutants, the basic reaction of which is shown in Additional file 1: Scheme S1

Summary

Introduction

The hydroamination of olefins and carbon–nitrogen bond-forming reactions of unsaturated carboxylic acids offer a vast range of applications in the synthesis of fine chemicals (Raj et al 2012). ALs, belonging to the enolase family, use unsaturated carboxylates as substrates for the hydroamination reaction to generate unnatural amino. MAL and its variants have been reported to catalyze C–N bond formation in the synthesis of artificial dipeptide sweeteners, showing that they possess broad amine scope in accepting unnatural substrates (Zhang et al 2020b). Despite these notable advances, unnatural amino acids based on large-frame organic acids remain largely unstudied. They can be envisaged as playing an important role in the synthesis of many chemicals, such as 3,4-dihydroxycinnamic acid, a precursor in the production of levodopa (l-dopa) (Fordjour et al 2019)

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