Characterization of a Novel Methylaspartate Ammonia Lyase from E. coli O157:H7 for Efficient Asymmetric Synthesis of Unnatural Amino Acids

Abstract

The asymmetric addition of ammonia to unsaturated acids using engineered methylaspartate ammonia lyase (MAL) is a particularly attractive and atom-economic method for the synthesis of unnatural amino acids. However, owing to insufficient enzyme gene mining of MALs, the catalytic performances of MALs have only been characterized in a few organisms. Herein, we describe a novel MAL from Escherichia coli (E. coli) O157:H7, whose gene was derived from a genome mining strategy. The enzyme (designated as El-MAL) has been successfully expressed in E. coli BL21­(DE3) and isolated and purified to homogeneity by using 6 × polyhistidine tag. El-MAL existed as a dimer in solution, consisting of two identical subunits (ca. 45 kDa). Enzymatic properties indicated that the enzyme performed maximum activity in the presence of Mg2+ at pH 8.5 and 25 °C. El-MAL accepted fumarate, mesaconate, maleate, citraconic acid, and itaconic acid as substrates in the amination reaction. To the best of our knowledge, such catalytic activity toward citraconic acid and itaconic acid has not been reported previously. Therefore, this novel MAL displayed with high stereoselectivity in an asymmetric amination reaction for the synthesis of unnatural amino acids may become a promising biocatalyst in further exploitation.

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

“…Based on our previous studies, we found that Ec MAL 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 ). However, Ec MAL showed low activity in the catalysis of bulky unsaturated carboxylic acids.…”

“…Notably, there was trace or even no specific activity observed for the WT. Moreover, compared with the bulky caffeic acid, different mutants exhibited obviously different catalytic activities (Ni et al 2020 ). Interestingly, the triple site mutant M6 showed significant improvement in activity toward 1b and was even better than M8 (Fig.…”

Creation of a (R)-β-Transaminase by Directed Evolution of d-Amino Acid Aminotransferase

“…Optically pure unnatural amino acids have emerged as a class of key building blocks for the synthesis of several fine chemicals, pharmaceuticals, agrochemicals, and pharmaceutically important peptides . Numerous biocatalytic routes employing wild-type or engineered enzymes, including acylase, aldolase, amidase, l - erythro -3,5-diaminohexanoate dehydrogenase, methylaspartate ammonia-lyase, aspartate ammonia-lyase, hydantoinase, lipase, nitrilase, amine dehydrogenase, imine reductase, monoamine oxidase, Baeyer–Villiger monooxygenase, and reductive aminase, have been developed to access chiral unnatural amino acids. Still, developing an efficient biocatalytic process for the synthesis of chiral β- and γ-amino acids remains a challenge …”