Stereoselectivity of Four (R)‐Selective Transaminases for the Asymmetric Amination of Ketones

Abstract

Four (R)-w-transaminases originating from Hyphomonas neptunium (HN-wTA), Aspergillus terreus (AT-wTA) and Arthrobacter sp. (ArRwTA), as well as an evolved transaminase (ArRmut11-wTA) were successfully employed for the amination of prochiral ketones leading to optically pure (R)-amines. The first three transaminases displayed perfect stereoselectivity for the amination of all substrates tested (ee > 99%). Furthermore, the transaminase AT-wTA led in most cases to better conversion than ArR-wTA and HN-wTA using d-alanine as amine donor. a-Tetralone, which was the only substrate not accepted by HN-wTA, ArR-wTA, and AT-wTA, was successfully transformed with perfect enantioselectivity (ee > 99%) into the corresponding optically pure amine employing the variant ArRmut11-wTA.

Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

“…Therefore, use of a 1.5 molar equivalent of isopropylamine relative to the concentration of pyruvic acid affords a 97% theoretical conversion of pyruvic acid to L-or D-alanine, depending on the stereoselectivity of -TA. Moreover, acetone (i.e., the deamination product of isopropylamine) is highly volatile, and the resulting equilibrium shift by facile evaporation can drive even thermodynamically unfavorable reactions to completion, as demonstrated elsewhere with the reductive amination of ketones (23)(24)(25). Another advantage of using isopropylamine as an amino donor is that most -TAs show low levels of activity for acetone (26), which minimizes enzyme inhibition by the ketone product.…”

Metabolically driven equilibrium shift of asymmetric amination of ketones by ω-transaminase using alanine as an amino donor

“…acetone and pyruvate, respectively). Although acetone can be easily removed at a reduced pressure due to high volatility, 3,9) a drawback of using isopropylamine is its low reactivity towards most ω-TAs, unless modified by extensive protein engineering as demonstrated by Savile et al 3) In contrast, all known ω-TAs show high activities to alanine. Moreover, the removal of pyruvate can be easily achieved by coupling an additional enzyme, including alanine dehydrogenase, lactate dehydrogenase, acetolactate synthase, and pyruvate decarboxylase.…”

“…Moreover, the removal of pyruvate can be easily achieved by coupling an additional enzyme, including alanine dehydrogenase, lactate dehydrogenase, acetolactate synthase, and pyruvate decarboxylase. [9][10][11][12] The reason why several enzymatic options are available to convert pyruvate into other chemicals is that pyruvate is a pivotal metabolite connecting the glycolytic pathway and the TCA cycle to extract chemical energy from glucose as well as to provide carbon skeletons required for synthesizing diverse metabolites. We reasoned that the removal of pyruvate to cope with the unfavorable equilibrium could be implemented by exploiting the native cellular metabolism, instead of using the auxiliary enzymes.…”

Biocatálise aplicada à síntese de núcleos <i>&#946;</i>-hidroxi-1,2,3-triazólicos e síntese multienzimática do alcaloide diidropinidina

“…23 Na literatura, podem ser encontradas diferentes enzimas DHs, como as álcool desidrogenases (ADHs), ceto-redutases (KREDs), carbonil redutases (CRs) ou designações mais específicas (ex. chiquimato redutase).…”

“…O cofator NAD + é reciclado ao NADH pelo sistema formiato de amônio/formato desidrogenase (FDH), completanto a cascata enzimática (Esquema 30). 23,48,56 No sistema isopropilamina o tampão da amina foi empregada como amino doador em excesso para favorecer o equilíbrio no sentido da formação dos produtos e nenhum sistema de regeneração foi empregado, ao contrário do sistema AlaDH (Esquema 31).…”