Page 325 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

14.3 Nitrile Hydrolysis Enzymes 301

NH NH O NH 2 O
2
N 2
R β α R NH 2 R OH
Nitrile hydratase Amidase
β-Aminonitrile β-Amino amide β-Amino acid

Nitrilase

Scheme 14.4 Biocatalytic conversion of β-aminonitriles to β-amino acids by nitrile hydrolyz-
ing enzymes.
14.3
Nitrile Hydrolysis Enzymes

14.3.1
Nitrilase

Nitrilases (EC 3.5.5.1, α, β hydrolase enzymes) are thiol enzymes capable of
hydrolyzing nitriles to their corresponding acids with the concomitant release of
ammonia. They belong to the first branch of the nitrilase superfamily and are found
in a variety of plants, animals, as well as prokaryotes [37]. The nitrilase superfamily
consists of 13 different enzyme classes with considerable structural homology,
despite demonstrating significant sequence variation. Members included in this
listing are (i) nitrilase, (ii) aliphatic amidase, (iii) amino-terminal amidase, (iv)
biotinidase, (v) β-ureidopropionate, (vi) carbamylase, (vii) prokaryote nicotinamide
adenine dinucleotide (NAD) synthetase, (viii) eukaryote NAD synthetase, (ix)
apolipoprotein N-acyltransferase, (x) Nit and NitFhit, (xi) NB11, and finally (xii) NB
12 [37].
Nitrilases can be used on a commercial scale as demonstrated by the production
of acrylic acid, (R)-(−)-mandelic acid (Mitsubishi Rayon-Japan, BASF-Germany) as
well as nicotinic acid (Lonza-China), thus proving the economic potential of this
class of enzyme [38–40].

14.3.1.1 Nitrilase Structure and Mechanism
Brenner [41] showed that a nitrilase folded protein consisted of a polypeptide of
α-helices and β-sheets that formed a novel α-β-β-α sandwich fold, with a triad
of residues in the active site (Glu-Lys-Cys) essential for the covalent catalysis.
Structural elucidation of a nitrilase produced by Rhodococcus rhodochrous J1 showed
that it formed a multimeric extended helix [42].
Mahadevan and Thimann [43] postulated the first nitrilase reaction mechanism,
suggesting that the nitrile carbon present in the substrate displays a partial positive
charge that is subject to nucleophilic attack by one of the two SH groups in the
nitrilase active site. The resulting thioimidate is then hydrolyzed to a thioester,
with the release of ammonia as a by-product. Hydrolysis of the acyl-enzyme then
results in the release of the final acid product.