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304 14 Enzymatic Stereoselective Synthesis of -Amino Acids

Fe-(III) Nitrile Hydratases Nitrile hydratases function as Lewis acids, in contrast to
the mode of action of most nonheme iron center enzymes, which catalyze redox
type reactions. The nitrile hydratase activity found in Rhodococcus N-771 [54–56]
was found to be photoregulated, with light reversing the in vivo inhibitory nitric
oxide (NO) binding and thereby activating the nitrile hydratase [56].

Co-(III) Nitrile Hydratases Results obtained from X-ray absorption studies identi-
fied sequence homology at the metal binding region of both the Fe and the Co
nitrile hydratases. This strongly suggests that the active site structure is similar
in both types of enzymes. The precise nitrile hydratase mode of action remains
unknown, but Huang and coworkers [57] suggested three possible mechanisms for
hydration of nitrile substrates to their corresponding amides.

14.3.3
Amidases

Amidase enzymes (EC 3.5.1.4) belong to the hydrolase family of enzymes and
catalyze the hydrolysis of monocarboxylic amides to their corresponding acid
products. Conventional amidase classification is done according to substrate profile
or is based on their amino acid sequence homology [58].
Amidases play a significant role in both prokaryotes and eukaryotes, functioning
in the production of growth regulators such as auxin and biotin in plants [59],
in nutrient metabolism, in the degradation of toxic cyanogenic compounds, and
finally in posttranslational modification of amino acids and proteins.

14.3.3.1 Amidase Structure and Mechanism
Amidase enzymes catalyze the hydrolysis of amide bonds with considerable diver-
gence noted within the family with respect to substrate specificity. All amidase
enzymes, however, maintain the core α,β,α structure, where the topologies of the
C and N terminal halves are similar.
Aliphatic amidase enzymes demonstrate sequence similarity to the nitrilase
superfamily thus indicating some form of evolutionary relationship. These ami-
dases contain a Glu-Lys-Cys catalytic triad and exist as homotetrameric or
homohexameric structures that function via a ping-pong (bi-bi) reaction mech-
anism [60, 61].

14.3.4
Nitrile Hydratase and Amidase Cascade Substrate Selectivity
Winkler and coworkers tested the substrate specificity of three different Rhodococcus
sp. strains on five- and six-membered cis-or trans-alicyclic β-aminonitriles. They
found that the five-membered substrates were hydrolyzed significantly faster than
their corresponding six-membered counterparts, and the reaction products from
the trans-aminonitriles were formed considerably faster than for the cis-substrates
[62]. In addition, the Rhodococci exhibited far greater enantioselectivity for the