Enzymatic nitrile hydrolysis catalyzed by nitrilase ZmNIT2 from maize. An unprecedented β-hydroxy functionality enhanced amide formation

Article abstract of DOI:10.1016/j.tet.2006.04.069

To explore the synthetic potential of nitrilase ZmNIT2 from maize, the substrate specificity of this nitrilase was studied with a diverse collection of nitriles. The nitrilase ZmNIT2 showed high activity for all the tested nitriles except benzonitrile, producing both acids and amides. For the hydrolysis of aliphatic, aromatic nitriles, phenylacetonitrile derivatives and dinitriles, carboxylic acids were the major products. Unexpectedly, amides were found to be the major products in nitrilase ZmNIT2-catalyzed hydrolysis of β-hydroxy nitriles. The hydrogen bonding between the hydroxyl group and nitrogen in the enzyme-substrate complex intermediates that disfavors the loss of ammonia and formation of acyl-enzyme intermediate, which was further hydrolyzed to acid, was proposed to be responsible for the unprecedented β-hydroxy functionality assisted high yield of amide formation.

Full text of DOI:10.1016/j.tet.2006.04.069

Tetrahedron 62 (2006) 6150–6154
Enzymatic nitrile hydrolysis catalyzed by nitrilase ZmNIT2
from maize. An unprecedented b-hydroxy functionality
enhanced amide formation
*
Chandrani Mukherjee, Dunming Zhu, Edward R. Biehl, Rajiv R. Parmar and Ling Hua
Department of Chemistry, Southern Methodist University, Dallas, TX 75275, USA
Received 15 February 2006; revised 13 April 2006; accepted 21 April 2006
Available online 12 May 2006
Abstract—To explore the synthetic potential of nitrilase ZmNIT2 from maize, the substrate specificity of this nitrilase was studied with a
diverse collection of nitriles. The nitrilase ZmNIT2 showed high activity for all the tested nitriles except benzonitrile, producing both acids
and amides. For the hydrolysis of aliphatic, aromatic nitriles, phenylacetonitrile derivatives and dinitriles, carboxylic acids were the major
products. Unexpectedly, amides were found to be the major products in nitrilase ZmNIT2-catalyzed hydrolysis of b-hydroxy nitriles. The
hydrogen bonding between the hydroxyl group and nitrogen in the enzyme–substrate complex intermediates that disfavors the loss of ammo-
nia and formation of acyl–enzyme intermediate, which was further hydrolyzed to acid, was proposed to be responsible for the unprecedented
b-hydroxy functionality assisted high yield of amide formation.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Several nitrilases have been characterized from plants and
microorganisms.16–22 Recently, Glawischnig et al. cloned
and characterized a nitrilase ZmNIT2 from maize (Zea
mays), and demonstrated that it was involved in the biosyn-
thesis of a plant hormone auxin.23 This nitrilase was reported
to convert indole-3-acetonitrile to indole-3-acetic acid at
least 7–20 times more efficiently than AtNIT1/2/3 from
Arabidopsis thaliana. The nitrilase ZmNIT2 also showed
no substrate inhibition for indole-3-acetonitrile that was
observed for nitrilases from A. thaliana.19 This characteris-
tic property is particularly important for enzymes to be used
as an efficient catalyst in organic synthesis, because useful
processes usually require high concentration of reactants
to achieve optimal space productivity. These unique features
stimulated us to explore the synthetic application potential
of the nitrilase ZmNIT2 from maize. Therefore, we studied
the substrate specificity of this nitrilase with a collection of
nitriles with structural diversity. The nitrilase ZmNIT2 was
found to be active toward aliphatic nitriles, phenylaceto-
nitrile derivatives, and aromatic nitriles. In these cases, acids
were obtained as the major products with concomitant for-
mation of amides. On the contrary, nitrilase ZmNIT2 cata-
lyzed the hydrolysis of b-hydroxy nitriles, producing
amides as the major products instead of acids.
Biocatalysts have attracted much attention in environmental
science, synthetic organic chemistry, microbiology, and bio-
technology. Hydrolytic biocatalysts are becoming optimal
tools for the synthesis of pharmaceutically and biologically
important molecules.1 In particular, among hydrolytic bio-
catalysts, the nitrilase superfamily enzymes have evoked
substantial commercial interest because of their very high
synthetic potential.2–4 Two important enzymes of this super-
family are nitrilase (EC 3.5.5.1) and nitrile hydratase (EC
4.2.1.84). Nitrilases catalyze hydrolysis of nitriles to their
corresponding carboxylic acids and ammonia, while nitrile
hydratases hydrolyze nitriles to amides.5 These enzymes
are highly demanding biocatalysts in drug synthesis, biodeg-
radation of nitrile wastes, and agricultural development.6,7
The chemical industry has considerable interest in the utili-
zation of nitrilases for the chemo-, regio-, or enantioselective
production of carboxylic acids from nitriles under mild con-
ditions,8–13 because chemical hydrolysis of nitriles usually
requires strong basic or acidic conditions and elevated reac-
tion temperature, that often results in the undesirable side
reactions. For example, elimination of the OH group occurs
in the chemical hydrolysis of b-hydroxy nitriles to yield un-
saturated by-products.14 However, the total synthetic appli-
cability of these biocatalysts has not been fully achieved
because of the paucity of available enzymes.7,15
2. Results and discussion
Nitrilase ZmNIT2 gene from maize was over-expressed in
Escherichia coli and the encoded protein was purified
* Corresponding author. Tel.: +1 214 768 1609; fax: +1 214 768 4089;
e-mail: lhua@smu.edu
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.04.069

C. Mukherjee et al. / Tetrahedron 62 (2006) 6150–6154
6151
from cell-free extract by following the literature method.23
This His-tagged enzyme was stable during storage under dif-
ferent conditions. For example, the lyophilized enzyme was
stable for months at 4 ꢀC. Ninety percent of the initial activ-
ity was recovered after being stored in potassium phosphate
buffer (pH 7.2, 10 mM) with 50% glycerol at À20 ꢀC for
a month.
on the substituent on the benzene ring. Racemic mandelo-
nitrile was hydrolyzed by nitrilase ZmNIT2 to enantiopure
(R)-mandelic acid.
As shown in Table 1, nitrilase ZmNIT2 also efficiently cat-
alyzed the hydrolysis of aliphatic nitriles to the correspond-
ing carboxylic acids as major products. The acid and amide
product distribution was affected by the structure of aliphatic
nitriles. It appeared that the amount of amide increased as
the chain length became longer. Dinitriles were converted
to di-acids and di-amides, and no mixed amide–acid was
detected. Selective hydrolysis to mono-acid or amide was
not achieved. Methylthioacetonitrile was hydrolyzed to
methylthioacetic acid and methylthioacetamide.
The substrate specificity of this nitrilase was studied by treat-
ing nitrile substrates with the purified enzyme in potassium
phosphate buffer as described in Section 4. The products
were analyzed and characterized by GC, HPLC, and NMR
spectroscopy. The results are summarized in Tables 1 and
2. It can be seen that nitrilase ZmNIT2-catalyzed hydrolysis
of various nitriles with diverse structures to afford the corre-
sponding acids with concomitant amide formation. It was
surprising that nitrilase ZmNIT2 efficiently catalyzed the
hydrolysis of substituted benzonitrile derivatives and croto-
nitrile to give acids as the major products, although it showed
low activity for benzonitrile (entries 1–4 in Table 1). Benzyl
cyanide and its substituted counterparts were also efficiently
hydrolyzed to acids with concomitant formation of amides.
The amount of amide formed in the reaction was dependent
Nitrilase-catalyzed hydrolysis of b-hydroxy nitriles results
in b-hydroxy carboxylic acids, which are important pre-
cursors of b-blockers and b-amino alcohols.14 Although
biocatalytic transformation of b-hydroxy nitriles to the cor-
responding b-hydroxy carboxylic acids and/or amides has
been achieved with nitrile hydratase/amidase-based micro-
bial biocatalysts,24–27 to our best knowledge, the hydrolysis
of b-hydroxy nitriles catalyzed by isolated nitrilases has not
been reported. Therefore, the activity of nitrilase ZmNIT2
toward a series of b-hydroxy nitriles was examined. In con-
trast to the nitriles in Table 1, when b-hydroxy nitriles were
treated with nitrilase ZmNIT2 under the same condition
the major products were amides with the conversion of 63
to 88%, along with acids as minor products (Table 2).
Although the hydrolysis of mandelonitrile was highly enan-
tioselective, nitrilase ZmNIT2 showed little enantioselectiv-
ity for the hydrolysis of b-hydroxy nitriles (less than 40% ee,
data were not shown and the absolute configurations were
not determined). This is consistent with the observation in
most cases that a chiral carbon atom at the b-position to
the reaction center would be recognized with much more dif-
ficulty than the one at a-position.25
Table 1. Hydrolysis of various nitriles catalyzed by nitrilase ZmNIT2
Entry Nitrile
Conversion (%)a
Acid
Amide
1
Benzonitrile
Overall yield <5
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
4-Hydroxy-3-methoxybenzonitrile
4-Acetylbenzonitrile
Crotonitrile
Benzyl cyanide
3,4-(Methylenedioxy)-phenylacetonitrile 74
2,4-Dimethoxyphenylacetonitrile
Mandelonitrile
Allyl cyanide
n-Butyronitrile
Valeronitrile
83
68
79
90
17
31
20
10
26
33
<2
20
19
26
36
40
16
17
24
10
17
67
45b
80
80
73
64
60
84
83
76
89
83
There are several reports on the formation of amides as
minor products in nitrilase-catalyzed hydrolysis of nitrile
substrates.15,16,28–31 The unexpected large amount of amides
from b-hydroxy nitriles catalyzed by nitrilase ZmNIT2 was
worthy to be further investigated. 3-(4-Fluorophenyl)-3-
hydroxypropionitrile and 3-(4-methylphenyl)-3-hydroxy-
propionitrile were used for detailed study. To gain insight
on the mechanism of amide formation, reaction mixtures
were analyzed at different time intervals and the conversions
of acid and amide were determined. The ratios of amide/acid
in the hydrolysis of 3-(4-fluorophenyl)-3-hydroxypropio-
nitrile after 2 and 6 h were 4.7 and 4.5, respectively, while
those for hydrolysis of 3-(4-methylphenyl)-3-hydroxy-
propionitrile were 7.1 and 6.5. The results indicated that
the ratio of product amide to acid kept fairly constant as
the reaction proceeded. The same time independence of
amide/acid ratio was observed for the hydrolysis of 3-phe-
nylpropionitrile. The product mixture, which was obtained
from the hydrolysis of 3-(4-fluorophenyl)-3-hydroxypropio-
nitrile after 6 h, was incubated with a fresh enzyme over-
night. The amide/acid ratio did not show meaningful
change. This ruled out the possibility that enzyme deactiva-
tion was responsible for the nearly constant amide/acid ratio.
The concurrent formation of amide and acid clearly sug-
gested that the acid was not produced from amide as in the
Hexanenitrile
Heptanenitrile
3-Phenylpropionitrile
4-Phenylbutyronitrile
2-Methylthioacetonitrile
1,4-Dicyanobutane
2-Methylglutaronitrile
a
The conversion was determined by GC, HPLC, and NMR analysis.
The product was (R)-mandelic acid with 100% ee.
b
Table 2. Hydrolysis of b-hydroxy nitriles catalyzed by nitrilase ZmNIT2
OH
OH
OH
CONH2
COOH
CN
ZmNIT2
+
X
X
X
Entry
Substrate
Conversion (%)a
Amide
Acid
1
2
3
4
5
6
4-CH3
4-F
4-Cl
4-Br
4-OCH3
4-CH3CO
14
17
13
12
37
27
85
73
85
88
63
68
a
The conversion was determined by HPLC and NMR analysis.

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C. Mukherjee et al. / Tetrahedron 62 (2006) 6150–6154
sequential hydrolysis of nitrile catalyzed by nitrile hydratase
and amidase. This was further supported by the observation
that hexanoamide was not a substrate of nitrilase ZmNIT2.
Nitrilase possesses a cysteine residue in its catalytic site and
the proposed reaction mechanism for the hydrolysis of
nitriles involves the formation of an enzyme–substrate
complex where the thiol residue of the enzyme forms a
covalent bond with nitrile carbon.29,32,33 Addition of a
H2O molecule to the complex generates a tetrahedral inter-
mediate. Loss of ammonia from this tetrahedral intermediate
gives an acyl–enzyme intermediate, which is further hydro-
lyzed to produce carboxylic acid and release the enzyme for
next catalytic cycle. In a few cases, small amount of amide
was also obtained, and it was due to cleavage of the C–S
bond in the tetrahedral intermediate, that furnished amide
and released enzyme.1
We also investigated effect of temperature, pH, and solvent
on the product distribution of amide and acid. The results
are presented in Figures 1–3. From the results it can be
seen that temperature, pH, and solvent did not exert signifi-
cant impact on the product distribution of amide and acid.
This is consistent with the reported independence of the
amide to acid ratio on reaction conditions.28
3-(4-fluorophenyl)-3-hydroxypropionitrile
3-(4-methylphenyl)-3-hydroxypropionitrile
Recently, Effenberger et al. have found that amides were
major products in the hydrolysis of a-fluoroarylacetonitriles
and p-conjugated nitriles with acceptor groups (CN, NO2)
catalyzed by AtNIT1 from A. thaliana. The stabilization of
the tetrahedral intermediate in the enzyme–substrate com-
plex by these electron-withdrawing groups was proposed
to be responsible for the preference of amide formation.15
Hydrolysis of nitriles catalyzed by nitrilase ZmNIT2 should
also involve participation of cysteine thiolate as a nucleo-
philic entity to form an enzyme–substrate complex.29,32,33
Hydrogen bond formation is a very common feature in
enzyme catalysis, when polar amine group either from
substrate or from enzyme residue is in close proximity to
hydroxy group from similar environment. It has also been
observed that hydrogen bonding could greatly enhance the
reaction rate in enzyme catalysis.34,35 Therefore, when b-
hydroxy nitriles were substrates, it was possible that the
hydrogen bond between the lone pair of electrons on the ni-
trogen atom and the b-OH group was formed in the enzyme–
substrate complex (1) because they were in close proximity,
as shown in Scheme 1. Addition of H2O generated the tetra-
hedral intermediate (2), which had two fates. One was loss of
ammonia to form intermediate (3), which was further hydro-
lyzed to carboxylic acid (pathway B). The other one was
cleavage of the C–S bond to yield amide (pathway A).
These two pathways were competitive and the existence of
hydrogen bond in intermediate (2) made pathway A favor-
able over pathway B, because loss of ammonia required
extra energy to break down the six-membered cyclic hydro-
gen bond system. Therefore, amides were the major products
in the hydrolysis of b-hydroxy nitriles catalyzed by nitrilase
ZmNIT2. To assess the importance of intra-molecular
bonding in promoting amide formation, the hydrolysis of
8
6
4
2
0
20
30
40
50
Temperature (°C)
Figure 1. Temperature effect on the product distribution in hydrolysis of
b-hydroxy nitriles.
3-(4-fluorophenyl)-3-hydroxypropionitrile
3-(4-methylphenyl)-3-hydroxypropionitrile
8
6
4
2
0
6
7
8
9
pH
Figure 2. pH effect on the product distribution in hydrolysis of b-hydroxy
nitriles.
3-(4-fluorophenyl)-3-hydroxypropionitrile
3-(4-methylphenyl)-3-hydroxypropionitrile
6
4
2
0
1
2
3
4
Solvent
Figure 3. Solvent effect on the product distribution in hydrolysis of
b-hydroxy nitriles (1. t-BuOH; 2. MeOH; 3. acetonitrile; 4. acetone).
H
H
OH
O
O
N
NH2
OH
H
O
N
NH2
O
SEnz
H2O
EnzSH
-EnzSH
A
EnzS
1
2
B
-NH3
OH
OH
O
O
H2O
SEnz
OH
EnzSH
3
Scheme 1. Proposed hydrogen bonding facilitated amide formation in ZmNIT2-catalyzed hydrolysis of b-hydroxy nitriles.

C. Mukherjee et al. / Tetrahedron 62 (2006) 6150–6154
6153
4-phenylbutyronitrile and 4-hydroxy-4-phenylbutyronitrile
was tested. As predicted, the carboxylic acids were the major
products with amide/acid ratios being 0.2 for both substrates.
Because formation of seven-membered ring was disfavored
in this case, g-hydroxyl groups did not promote the amide
formation. This indicated that b-hydroxyl group played
a critical role in the formation of large amount of b-hydroxy
amides. Since the tetrahedral intermediate (2) was stabilized
by intra-molecular hydrogen bonding, which was minimally
affected by media, the reaction conditions did not have great
effect on the product distribution as shown in Figures 1–3.
the cells were induced with 0.1 mM of IPTG when optical
density at 595 nm was 0.6. The bacterial cultures were incu-
bated at 30 ꢀC on an orbital shaker at 180 rpm for another
4 h. The cells were harvested.
4.2. Preparation of cell-free extract and purification of
nitrilase ZmNIT2 enzyme
The cultures of E. coli BL21(DE3)pLysS were harvested by
centrifugation. The cell pellet was resuspended in potassium
phosphate lysis buffer (10 mM, pH 7.2, 1 mM DTT), and the
cell was lysed by homogenizer. The cell-free extract was
mixed with equal volume of PEI solution (0.25% polyethyl-
eneimine MW 40–60K, 6% NaCl, 100 mM Borax, pH 7.4)
to remove lipids.36 After centrifugation, the supernatant was
precipitated with 30% ammonium sulfate. The resulting pre-
cipitate was collected after centrifugation and dissolved in
potassium phosphate buffer (10 mM, pH 7.2, 1 mM DTT).
The lysate was desalted by gel filtration into potassium phos-
phate buffer (10 mM, pH 7.2, 1 mM DTT), and lyophilized.
One more concern was that the tetrahedral intermediate (2)
in Scheme 1 might be bound in a metal coordinative struc-
ture replacing the proton by a metal ion. This structure could
be destructed by a strong chelating ligand such as EDTA.
Thus addition of EDTA to the reaction mixture should
reduce the amide formation. However, the amount of amide
was not reduced when the reaction was carried out in the
presence of EDTA, indicating metal ion was not involved
in ZmNIT2-catalyzed nitrile hydrolysis.
4.3. Hydrolysis of nitriles catalyzed by nitrilase ZmNIT2
3. Conclusions
A typical experimental procedure was as follows: the nitrile
(45 mM) in 1 ml of potassium phosphate buffer (100 mM,
pH 7.15) was treated with 1 mg of lyophilized enzyme
ZmNIT2. The reaction mixture was shaken for 14 h at
30 ꢀC, and then acidified with a few drops of 1 N HCl solu-
tion to adjust pH to 3–4. The products and/or unreacted
nitriles were extracted into 1 ml of ethyl acetate. The extract
was dried over sodium sulfate and subjected to HPLC or GC
analysis to determine the conversion. For GC analysis, the
acids were converted to methyl ester derivatives using
freshly prepared ethereal solution of diazomethane at 0 ꢀC.
For NMR analysis, the solvent was removed from the extract
and the residue was dissolved in chloroform-d or acetone-d6.
The products were identified by comparing the retention
Nitrilase ZmNIT2 from maize efficiently catalyzed the
hydrolysis of a broad range of nitriles with diverse structures
to afford the corresponding carboxylic acids and amides. For
most nitriles such as aliphatic, aromatic nitriles, and phenyl-
acetonitrile derivatives, carboxylic acids were produced as
the major products. In contrast, nitrilase ZmNIT2 catalyzed
the hydrolysis of b-hydroxy nitriles to afford amides as the
major products. The unprecedented b-hydroxy functionality
enhanced amide formation might be due to the hydrogen
bonding between the hydroxyl group and nitrogen in the
reaction intermediates, that prevented the loss of ammonia
and formation of acyl–enzyme intermediate, thus led to
the formation of amide as major product. Because the crystal
structure of nitrilase ZmNIT2 is not known, how the intra-
molecular hydrogen bonding in the tetrahedral intermediate
(2) facilitates the elimination of cysteine to give amide needs
to wait for further studies.
1
time with authentic sample, or H and 13C NMR data with
those in the literature.14,25,37
Acknowledgements
We thank Professor Erich Glawischnig at Technische
4. Experimental
Universitat Munchen for providing us the plasmid of nitril-
¨
¨
ase ZmNIT2 gene from maize (Zea mays), and Southern
The GC analysis was performed on a Hewlett-Packard 5890
series II plus gas chromatograph. The HPLC analysis was
performed on an Agilent 1100 series high-performance liq-
uid chromatography system. 1H and 13C NMR spectra were
recorded on a 400 MHz Bruker AVANCE DRX-400
Multinuclear NMR spectrometer. All the nitriles, amide,
and acid standards were purchased from Aldrich or prepared
by following the literature procedures.14,25
Methodist University for start-up support.
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