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Eulsoo Park et al.
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prepared by Strecker synthesis.[14] Second, driving the
reaction to completion often requires an additional
enzymatic reaction. For example, cofactor depend-
ence of the DH reaction mandates coupling with a
second DH for cofactor recycling.[10] In contrast, TAs
do not pose such a cofactor problem, which adds up
to compelling features over the DH process. Howev-
er, equilibrium positions for the TA reactions are usu-
ally neutral between substrates and products, which
has made the industrial applications of TA reactions
lag behind in spite of their high turnover rate, high
enantioselectivity, broad substrate specificity and no
cofactor requirement.[11c,15]
TAs catalyze the transfer of an amino group from
an amino acid to an oxo acid and thereby play an im-
portant role in amino acid metabolism.[15,16] The
enzyme can be classified into a-TA and w-TA accord-
ing to the relative position of the amino group to be
transferred with respect to the carboxyl group of the
Scheme 1. Enzymatic coupled reactions to convert l-2 to l-
1. The oxo acid substrate 3a is supplied from l-2 by the
action of TD and then undergoes asymmetric amination by
(S)-specific w-TA with a concomitant deamination of 4a.
substrate.[15,16] Compared to an a-TA executing trans- pharmaceutically valuable unnatural amino acid with-
ACHTUNGTRENNUNGamination exclusively between amino acids and oxo out thermodynamic limitation to the final conversion.
acids, an w-TA exhibits distinctive substrate specificity
enabling the oxidative deamination of primary
amines.[15] Therefore, in contrast to the neutral equi- Results and Discussion
librium position for most a-TA reactions (i.e., Keq
ꢀ1), the thermodynamic equilibrium for w-TA reac- Cloning and Functional Expression of TD and w-TA
tions can be directed to completion using amines as
an amino donor.[17] This led us to develop several w- To carry out the coupled reactions shown in
TA processes for the production of enantiomerically Scheme 1, we set out to clone two genes encoding TD
pure chiral amines via kinetic resolution.[18]
and (S)-specific w-TA into expression vectors. We
In this report, we expand the product spectrum of chose a TD from Escherichia coli (ilvA) because the
w-TA by demonstrating a novel cost-effective process enzyme has been well characterized[21] and homolo-
for the production of l-homoalanine (l-1) which is gous expression in E. coli cells was expected to guar-
one of the pharmaceutically important unnatural antee highly functional expression. The ilvA gene was
amino acids. l-1 serves as a key intermediate for the amplified by colony PCR using E. coli DH5a cells as
production of the antituberculosis drug ethambutol[19] a template and then cloned into a pET21a(+) expres-
and the antiepileptic drug levetiracetam.[20] In this sion vector using NdeI and XhoI restriction sites,
study, the production process consists of two enzymat- leading to pET21-TD. Seven transformants were sub-
ic reactions as shown in Scheme 1: conversion of l- jected to DNA sequencing and sequence alignment
threonine (l-2) to 2-oxobutyrate (3a) by a threonine with the ilvA gene from E. coli K-12 showed that all
deaminase (TD) and asymmetric transfer of an amino transformants shared a single nucleotide mutation
group from benzylamine (4a) to 3a by an (S)-enantio- (G754A) which led to a D252N amino acid substitu-
selective w-TA. The net reaction employs l-2 and 4a tion. This seems to be a background mutation carried
as substrates, yielding ammonia, benzaldehyde (3b) by the E. coli DH5a strain in our laboratory. Cell-
and l-1. The coupled reactions overcome the afore- free extracts prepared with E. coli BL21ACTHUNRTGNE(GNU DE3) cells
mentioned obstacles to the asymmetric synthesis of transformed with pET21-TD displayed high TD activ-
amino acids. First, the TD reaction coupled with the ity, typically 300–750 U/mL, which was comparable to
w-TA reaction permits substitution of the expensive the expression result reported elsewhere.[21a]
oxo acid substrate (3a) by l-2 which is a cheap natural
In the case of w-TA, a BLASTP search (http://
amino acid produced on a million-ton scale annual- blast.ncbi.nlm.nih.gov/Blast.cgi) was carried out to
ly.[5a] Second, use of the amine compound (4a) as an identify a candidate gene using the protein sequence
amino donor unleashes the thermodynamic constraint of (S)-specific w-TA from Vibrio fluvialis JS17 (VF w-
of the TA reactions, allowing high conversion yield TA) which has been extensively studied.[22] Note that
without incorporation of another enzyme to remove E. coli lacks an w-TA activity. Among the homolo-
one of the products as reported elsewhere.[11] As a gous genes, we chose a putative class III aminotrans-
result, the coupled reaction system established a one- ferase from Paracoccus denitrificans PD1222 (94%
pot conversion of a cheap natural amino acid into a identity to VF w-TA) for gene cloning. Sequence
3392
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2010, 352, 3391 – 3398