Necessary and sufficient conditions for the asymmetric synthesis of chiral amines using ω‐aminotransferases

Abstract

The half reactions of ω-aminotransferase (ω-AT) from Vibrio fluvialis JS17 (ω-ATVf) were carried out using purified pyridoxal 5'-phosphate-enzyme (PLP-Enz) and pyridoxamine 5'-phosphate-enzyme (PMP-Enz) complexes to investigate the relative activities of substrates. In the reaction generating PMP-Enz from PLP-Enz using L-alanine as an amine donor, L-alanine showed about 70% of the initial reaction rate of (S)-α-methylbenzylamine ((S)-α-MBA). However, in the subsequent half reaction recycling PLP-Enz from PMP-Enz using acetophenone as an amine acceptor, acetophenone showed nearly negligible reactivity compared to pyruvate. These results indicate that the main bottleneck in the asymmetric synthesis of (S)-α-MBA lies not in the amination of PLP by alanine, but in the amination of acetophenone by PMP-Enz, where conformational restraints of the enzyme structure is likely to be the main reason for limiting the amine group transfer from PMP-Enz to acetophenone. Based upon those half reaction experiments using the two amino acceptors of different activity, it appears that the relative activities of the two amine donors and the two acceptors involved in the ω-AT reactions can roughly determine the asymmetric synthesis yield of the target chiral amine compound. Predicted conversion yields of several target chiral amines were calculated and compared with the experimental conversion yields. Approximately, a positive linear correlation (Pearson's correlation coefficient = 0.92) was observed between the calculated values and the experimental conversion yields. To overcome the low (S)-α-MBA productivity of ω-ATVf caused by the possible disadvantageous structural constraints for acetophenone, new ω-ATs showing higher affinity to benzene ring of acetophenone than ω-ATVf were computationally screened using comparative modeling and protein-ligand docking. ω-ATs from Streptomyces avermitilis MA-4680 (SAV2612) and Agrobacterium tumefaciens str. C58 (Atu4761) were selected, and the two screened ω-ATs showed higher asymmetric synthesis reaction rate of (S)-α-MBA and lower (S)-α-MBA degradation reaction rate than ω-ATVf. To verify the higher conversion yield of the variants of ω-ATs, the reaction with 50 mM acetophenone and 50 mM alanine was performed with coupling of lactate dehydrogenase and two-phase reaction system. SAV2612 and Atu4761 showed 70% and 59% enhanced yield in the synthesis of (S)-α-MBA compared to that of ω-ATVf, respectively.

Amination of Ketones by Employing Two New (S)‐Selective ω‐Transaminases and the His‐Tagged ω‐TA from Vibrio fluvialis

“…The pyruvate coproduct had to be removed to shift the equilibrium to the product side. [21] For this purpose, several types of enzymes have already been applied such as lactate dehydrogenase, [13,22] alanine dehydrogenase, [12,23] pyruvate decarboxylase [24] and acetolactate synthase. [25] In this study, commercial lactate dehydrogenase from rabbit muscle (LDH system) and l-alanine dehydroge-nase from Bacillus subtilis [26] (AlaDH system) were employed (Scheme 1).…”

“…The ω-TA from Vibrio fluvialis (VF-ωTA) has been investigated previously, and comprehensive data for initial reaction rates are available for various substrates, [21,27] which employ the enzyme as a whole-cell catalyst in aqueous and biphasic systems [28] and immobilized enzymes. [29] Additionally, the applicability of VF-ωTA has been evaluated on a pilot-plant scale with a membrane reactor [30] and a packedbed reactor.…”

Process considerations for the asymmetric synthesis of chiral amines using transaminases

“…Jankowski et al (2008) have developed a group contribution method for estimating Gibbs free energies for biochemical reactions in aqueous solutions at pH 7 and 258C, having a standard error of AE2 kcal/mol. This methodology was recently applied by Seo et al (2011) in the comparison of the transamination potential of different amine donors, where 1-aminoindan was estimated to be thermodynamically favorable for the transamination of acetophenone. Considering the uncertainty in the group contribution method it was within the standard error, that an experimental yield of only 37% was obtained using four equivalents of amine donor, indicating a thermodynamically unfavorable reaction (Seo et al, 2011).…”

A subfamily of PLP-dependent enzymes specialized in handling terminal amines

“…As noted above, the amino acceptor in this case is not an aldehyde but a ketone and it is commonly accepted that transaminations of ketones are thermodynamically unfavorable [51][52][53][54][55]. Ketones have in fact a relatively low electrophilicity of the carbonyl carbon, as compared with α-keto acids and aldehydes, and are more stable than the corresponding amines.…”