Page 191 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

7.3 N-Carbamoyl-β-Alanine Amidohydrolase 167

A multiple sequence alignment of different NCβAAs together with an l-N-
α-carbamoylase and a d-N-α-carbamoylase from S. meliloti and A. tumefaciens,
respectively [51, 52] is shown in Figure 7.5. The highest sequence identity was
found with that from Saccharomyces kluyveri (SkNCβAA; 36.70%), but with all other
NCβAAs the similarity was less than 10%. A similar percentage was found between
Atβcar and d-N-α-carbamoylase of A. tumefaciens (AtDcar; 8.79%). However, the
amino acid sequence was quite similar with l-N-α-carbamoylase of S. meliloti
(SmLcar; 79.89%). This phenomenon was previously reported for SkNCβAA, which
shows a higher sequence identity with bacterial l-N-α-carbamoylases than with
mammalian NCβAAs [45]. The low similarity sequence among these enzymes led
to the hypothesis of a divergent evolution from the ancestral gene, allowing different
N-carbamoyl amidohydrolases to act on N-carbamoyl-substituted compounds and
divided the family of amidohydrolases into three subfamilies [45]. Additional
computational analysis has shown that several of these enzymes that have distantly
related primary structures share the same structural scaffold [6, 53, 54]. Thus, Atβcar
would be included in a first subfamily composed of bacterial l-N-carbamoylases
and the enzyme SkNCβAA from the eukaryotic S. kluyveri, a second subfamily with
the mammalian and most other eukaryotic NCβAAs, while d-N-α-carbamoylases
would constitute the third subfamily [45, 50].
N-carbamoyl amidohydrolases have been described as metalloenzymes [32, 50]
and for that reason a divalent cation is required in the reaction. It is worth noting
2+
that eukaryotic NCβAAs have been described as Zn dependent, but prokaryotic
ones and d-and l-carbamoylase activities are optimally active with cofactors other
2+
than Zn [32, 50]. The question remains as to whether eukaryotic NCβAAs would
2+ 2+ 2+ 2+
be more active with Mn ,Co ,orNi catalytic cofactors than with Zn .Atβcar
2+
showed the highest activity with Ni as cofactor in a 25 : 1 ion/protein ratio [50].
An enzyme’s substrate specificity indicates its potential application range as
a biocatalyst, and so the breakdown of different precursors to β-amino acids
by Atβcar has been evaluated. NCβAAs mainly hydrolyze β-ureidopropionic and
β-ureidoisobutyric acid to β-alanine and 3-AiBA, respectively. Atβcar is the first
NCβAA that has been shown to hydrolyze non-substituted substrate analogs in
which the carboxyl group is replaced by a sulfonic or phosphonic group to produce
taurine and ciliatine, respectively [50], but with catalytic efficiencies that are notably
lower than for β-ureidopropionic acid (N-carbamoyl-β-alanine) (Table 7.1). Both
3
2
monosubstitued β -and β -amino acids were both hydrolyzed by Atβcar, but
with better catalytic efficiency (k /K ) for the former (Table 7.1). Thus, for N-
cat
m
carbamoyl-α-methyl-β-alanine (β-ureido isobutyric acid) the k /K m ratio was 60
cat
times better than for N-carbamoyl-β-homoalanine. This value decreased drastically
2
with a larger substituent (phenyl group) for β -carbamoyl (N-carbamoyl-α-phenyl-
3
β-alanine) (Table 7.1) but no activity was detectable at all for the β -counterpart
2
(N-carbamoyl-β-phenylalanine) [50]. As mentioned above, β -amino acids have not
3
been as readily available as their β -counterparts, and must be prepared using multi-
step procedures [22]. However, the Atβcar enzyme would simplify their synthesis.
The natural activity of the many enzymes employed in industrial biotransforma-
tion is usually unknown; consequently the precursors used in those processes are