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164 7 Synergies of Chemistry and Biochemistry for the Production of -Amino Acids

together with a suitable enantiospecific carbamoylase and a hydantoin racemase
(Figure 7.2a) [34, 35]. Although this process has been used industrially since the
1970s [25], the ever increasing demand for d-amino acids with various side chains
has maintained the interest in this industrial process, even more so because the
use of hydantoin racemase allows the total conversion of racemic hydantoins when
chemical racemization in situ is not favored [36–38].
Besides the hydantoinases (dihydropyrimidinases) application in the context of
the ‘‘hydantoinase process,’’ the first attempt to simulate this industrial method
using structurally analogous DHU derivatives was carried out by Kanegafuchi in the
1990s using a Pseudomonas strain [39]. However, it was not until the following decade
that two dihydropyrimidinases belonging to Arthrobacter and Sinorhizobium genera
were shown to be able to hydrolyze different 5- and 6-monosubstituted DHUs
[26, 27]. Concerning the second enzyme (SmelDhp), an extensive biochemical and
biophysical characterization, together with the elucidation of its X-ray structure
(Figure 7.3), has been carried out by our group, allowing us to gain new insight into
the amidohydrolase superfamily of enzymes [27, 40–42]. SmelDhp hydrolyzed the
two natural DHU derivatives produced in the reductive catabolism of pyrimidines
(DHU and 5-methyl-dihydrouracil (5-METDHU)) and 6-monosubstituted DHUs
such as those with methyl, iso-propyl, propyl, and iso-butyl substituents. From the
kinetic results obtained for 5- and 6-methyl-DHU, substitution at the 6-position of
the substrate produced a decrease in the affinity of the enzyme by two orders of
magnitude when compared to the 5-substituted DHU [42]. Manual docking of the
substrate suggested unfavorable interactions of the substituent with His58, Met61,
Phe63, and/or Cys315, which could explain the lower efficiency of the enzyme
with substrates bearing bulky substituents such as phenyl or isobutyl as compared
to other substrates, such as 5- and 6-methyl-DHU [42]. Interestingly, some of
these residues are located in the well-known Stereo-Gate Loops, which govern the
enantioselectivity of hydantoinases toward 5-monosubstituted hydantoins [43].
Prior to this decade only three genera were known to hydrolyze different 6-
monosubstituted DHUs (Pseudomonas, Sinorhizobium,and Arthrobacter, see earlier
text). However, recent works have proved that this ability is also available in several
other organisms, such as Vigna, Ochrobactrum, Delftia, Aminobacter,or Rhizobium
[28, 29]. Because the chemoenzymatic method of producing β-amino acids from
monosubstituted DHUs (Figure 7.4) depends on the enantioselectivity of dihy-
dropyrimidinases, recent studies have tried to shed some light on this aspect [28,
29, 44]. The results to date demonstrate that, in general, the enantioselectivity of
the known dihydropyrimidinases is low, with the exception of the particular case
of DHUs with differently substituted phenyl moieties in position 6, for which the
enzymes belonging to Vigna angularis, Arthrobacter polychromogenes DSM20136, and
A. polychromogenes DSM342 presented high enantioselectivity [28, 29]. Interestingly
enough, the enzymes from different organisms present different enantioselectivies:
whereas the enzymes from V. angularis, S. meliloti,and Arthrobacter crystallopoi-
etes are reported as S-enantioselective for 6-monosubstituted DHUs [28, 29, 44],
biotransformation experiments of whole cells of A. polychromogenes DSM20136,
A. polychromogenes DSM342, Arthrobacter sp. E7, Bacillus sp. A16, Aminobacter sp.