Biocatalytic Enantioselective Synthesis of N‐Substituted Aspartic Acids by Aspartate Ammonia Lyase

Abstract

The gene encoding aspartate ammonia lyase (aspB) from Bacillus sp. YM55‐1 has been cloned and overexpressed, and the recombinant enzyme containing a C‐terminal His6 tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His6 tag does not affect AspB activity. The enzyme processes L‐aspartic acid, but not D‐aspartic acid, with a Km of ≈15 mM and a kcat of ≈40 s−1. By using this recombinant enzyme in the reverse reaction, a set of four N‐substituted aspartic acids were prepared by the Michael addition of hydroxylamine, hydrazine, methoxylamine, and methylamine to fumarate. Both hydroxylamine and hydrazine were found to be excellent substrates for AspB. The kcat values are comparable to those observed for the AspB‐catalyzed addition of ammonia to fumarate (≈90 s−1), whereas the Km values are only slightly higher. The products of the enzyme‐catalyzed addition of hydrazine, methoxylamine, and methylamine to fumarate were isolated and characterized by NMR spectroscopy and HPLC analysis, which revealed that AspB catalyzes all the additions with excellent enantioselectivity (>97 % ee). Its broad nucleophile specificity and high catalytic activity make AspB an attractive enzyme for the enantioselective synthesis of N‐substituted aspartic acids, which are interesting building blocks for peptide and pharmaceutical synthesis as well as for peptidomimetics.

New insights into the biosynthesis of fosfazinomycin

“…We anticipated that N -hydroxyaspartic acid ( N- hydroxy-Asp) would be the product of the FzmM reaction with L-Asp. Because of its reported instability, 20 we derivatised the enzymatic product with Fmoc-Cl followed by LC-MS analysis (Fig. S11–S14 † ), which showed that Fmoc- N- hydroxy-Asp was indeed produced (Scheme 1).…”

Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans

“…In some cases, the use of biocatalysts is an attractive alternative option (Wohlgemuth 2010). Recently, the ammonia lyases and aminomutases (which exhibit ammonia lyase activity) have gained a lot of interest for the asymmetric synthesis of chiral α- and β-amino acids, with the advantage that they use readily available unsaturated acids as substrates (Turner 2010; Verkuijl et al 2010; Szymanski et al 2009; Wu et al 2009, 2010; Weiner et al 2008). However, most known ammonia lyases show low stability and therefore they are not compatible with the harsh reaction conditions that are usually required for industrial processes, such as high temperature, high pH, and high ammonia concentrations needed to catalyze the reverse reactions (Turner 2010).…”

Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

“…The Class II fumarases, encoded by the fumC gene, are thermostable homotetramers with no requirement for cofactors and catalyze the interconversion of fumarate to L -malate [2]. FumC proteins are widely distributed in nature, from prokaryotes like Bacillus subtilis , Pseudomonas aeruginosa , Sulfolobus solfataricus , and Saccharomyces cerevisiae , to mammals [3,8]. …”