Characterization and synthetic applications of recombinant AtNIT1Steffen Osswald,1Harald Wajant2and Franz Effenberger1 1 Institut fuÈr Organische Chemie, and 2 Institut fuÈr Zellbiologie
Trang 1Characterization and synthetic applications of recombinant AtNIT1
Steffen Osswald,1Harald Wajant2and Franz Effenberger1
1 Institut fuÈr Organische Chemie, and 2 Institut fuÈr Zellbiologie und Immunologie, UniversitaÈt Stuttgart, Germany
The nitrilase AtNIT1 from Arabidopsis thaliana was
over-expressed in Escherichia coli with an N-terminal His6tag
and puri®ed by zinc chelate anity chromatography in a
single step almost to homogeneity in a 68% yield with a
speci®c activity of 34.1 Uámg)1 The native enzyme
( 450 kDa) consists of 11±13 subunits (38 kDa) The
temperature optimum was determined to be 35 °C, and a
pH optimum of 9 was found Thus, recombinant AtNIT1
resembles in its properties the native enzyme and the
nitrilase from Brassica napus The stability of AtNIT1
could be signi®cantly improved by the addition of
dithiothreitol and EDTA The substrate range of AtNIT1
diers considerably from those of bacterial nitrilases
Aliphatic nitriles are the most eective substrates, showing increasing rates of hydrolysis with increasing size of the residues, as demonstrated in the series butyronitrile, octanenitrile, phenylpropionitrile In comparison with 3-indolylacetonitrile, the rate of hydrolysis of 3-phenyl-propionitrile is increased by a factor of 330, and the Km value is reduced by a factor of 23 With the exception of
¯uoro, substituents in the a position to the nitrile function completely inhibit the hydrolysis
Keywords: Arabidopsis thaliana; enzymatic properties; nitrile; substrate speci®city
Nitriles are found in a variety of naturally occurring
compounds such as cyanolipids, cyanoglucosides, and
simple aliphatic or aromatic nitriles as metabolites of
micro-organisms [1] In nature, the hydrolysis of nitriles to
the corresponding carboxylic acid and NH3is catalyzed by
nitrilases (EC 3.5.5.1) or based on the sequential action of
a nitrile hydratase (EC 4.2.1.84)±amidase (EC 3.5.1.4)
system [2,3] Most nitrilases described so far have been
isolated from fungi or bacteria In recent years, however,
four nitrilases (AtNIT1±AtNIT4) have been cloned from
Arabidopsis thaliana, a member of the brassicaceae family
[4,5] The genes of AtNIT1±3 are clustered on chromosome 3
and have sequence identities of more than 80% at the
amino acid level, whereas AtNIT4 has a distinct
chromo-somal localization and is only 65% identical with AtNIT1±3
[5] The subdivision of the Arabidopsis nitrilases into
AtNIT1±3 and AtNIT4 is also re¯ected by functional
differences between these enzymes Whereas AtNIT1±3
convert 3-indolylacetonitrile (IAN) into the plant hormone
3-indolylacetic acid, IAN is not a substrate for AtNIT4
[6,7] Moreover, homologs of AtNIT1±3 have exclusively
been found in Arabidopsis and other members of the
brassicaceae, whereas AtNIT4 isoforms have also been reported in species from other taxonomic groups such as tobacco [8] and rice [7] In accordance with the brassi-caceae-restricted occurrence of nitrilases of the AtNIT1±3 type, these enzymes seem to be involved in the degradation
of nitriles released from glucosinolates, which can be found
in high concentrations in various species of the brassicaceae [9] Recent studies have shown that AtNIT4 and two related nitrilases from tobacco are b-cyano-(L)-alanine nitrilases [7] As nitrilases of the AtNIT4 type have been found in taxonomically quite distinct groups, it seems likely that AtNIT4 homologs may exist in all higher plants
In accordance with this is the fact that the substrate of the AtNIT4-type nitrilases, b-cyano-(L)-alanine, seems to occur
in all plants as the result of detoxi®cation of cyanide, which
is inevitably produced during biosynthesis of the plant hormone ethylene [10]
In general, nitriles are synthetically more accessible than the corresponding carboxylic acids Chemical hydrolysis of nitriles to carboxylic acids, however, requires drastic conditions (strong mineral acids and bases and relatively high reaction temperature) Biocatalysts for the transfor-mation of nitriles to carboxylic acids are therefore of particular interest
Up until now, hydratase±amidase systems, not nitri-lases, have mainly been used in practice as nitrile-hydrolyzing enzymes [3,11±14] In this paper, we report
on basic investigations of the nitrilase AtNIT1 from
A thaliana, in particular, the substrate range required for the hydrolysis of nitriles to carboxylic acids Cloning and overexpression of AtNIT1 [4,5] (EC 3.5.5.1) will provide
an interesting plant nitrilase in suf®cient quantities for synthetic applications The application of AtNIT1 to the hydrolysis of several speci®c substrates such as aliphatic dinitriles and 2-¯uoroarylacetonitriles has been published
in detail [15,16]
Correspondence to F Eenberger, Institut fuÈr Organische Chemie,
UniversitaÈt Stuttgart, Pfaenwaldring 55, D-70569 Stuttgart,
Germany Fax: + 49 711685 4269, Tel.: + 49 711685 4265,
E-mail: franz.eenberger@po.uni-stuttgart.de
Abbreviations: AtNIT1, nitrilase from Arabidopsis thaliana; IAN,
3-indolylacetonitrile.
Note: This is part 42 of the series of publications Enzyme catalyzed
reactions Part 41 is Eenberger, F & Osswald, S (2001) Selective
hydrolysis of aliphatic dinitriles to monocarboxylic acids by a nitrilase
from Arabidopsis thaliana Synthesis 1866±1872.
(Received 3 September 2001, revised 23 November 2001, accepted 26
November 2001)
Trang 2M A T E R I A L S A N D M E T H O D S
Expression cloning of AtNIT1
AtNIT1 cDNA was cloned in the expression vector pQE10
(Qiagen), which allows isopropyl b-D
-thiogalactoside-induced expression of N-terminally His-tagged recombinant
protein In brief, the coding region and part of the
3¢-noncoding region of AtNIT1 cDNA were ampli®ed
from an A thaliana cDNA library (Stratagene) with an
advanced polymerase system (Clontech) using the primers
AtNIT1-for (5¢-GCTGCTAGATCTTATGTCAACTGT
CCAAAA CGCAACTCCTTTTAACGGCGTTGCCCC
ATCCACC -3¢; start codon according to [4] in bold) and
AtNIT1-rev (5¢-ACAATTGATGATTCAACGCCCAAC
3¢) Using the BglII sites in the 5¢ overhang of AtNIT1-for
and the 3¢-noncoding region of the cDNA, the AtNIT1
cDNA was inserted in-frame in the BamHI site of pQE10
The resulting expression plasmid pQE10-AtNIT1 was
sequenced to con®rm the identity of the AtNIT1 sequence
after PCR ampli®cation pQE10-AtNIT1 was transformed
in Escherichia coli M15[pREP4] cells (Qiagen) for
over-expression of AtNIT1 For induction of recombinant
AtNIT1, an overnight culture was performed at 37 °C in
Luria±Bertani medium supplemented with ampillicin
(50 lgámL)1) and kanamycin (20 lgámL)1), diluted 1 : 20
with Luria±Bertani medium supplemented again with
ampillicin and kanamycin, and grown at 30 °C After 4 h,
isopropyl b-D-thiogalactoside was added to a ®nal
concen-tration of 0.5 mM for induction of AtNIT1 expression
After an additional 6 h, cells were harvested
Preparation of the crude extract and puri®cation
of recombinant AtNIT1
Cells were separated from the nutrient medium by
centri-fugation (30 min, 4 °C, 5700 g), and washed with sodium
phosphate buffer A (50 mM, pH 7.8) The pellet was
resuspended in buffer A (100 mL per 10 g wet weight)
and sonicated (3 ´ 5 min, 0 °C) The homogenate was
centrifuged (40 min, 4 °C, 186 000 g) The supernatant
(100 mL) was degassed with argon, ®ltered through a
membrane (70 lm) and applied to a Zn2+-charged HiTrap
metal chelate af®nity chromatography column (Pharmacia)
The column was rinsed successively with 20 mL each of
sodium phosphate buffer B (50 mM, 100 mMNaCl, pH 7.8)
and buffer A until the absorbance reached the base line of
column equilibration Nonspeci®cally bound proteins were
eluted at a ¯ow rate of 2 mLámin)1 in a 22.5-mL linear
gradient of 0±100 mM imidazole in buffer A, and
succes-sively in 5 mL of sodium phosphate buffer C (50 mM,
100 mM imidazole, pH 7.8) After additional rinsing with
11.25 mL buffer A, AtNIT1 was eluted with 11.25 mL
sodium phosphate buffer (50 mM, 100 mMEDTA, pH 7.8)
To the collected fractions (2.5 mL), 25 lL sodium
phos-phate buffer (50 mM, 100 mM dithiothreitol, pH 7.8) was
added, and after measurement of enzyme activity, fractions
were pooled
Gel-®ltration analysis
Recombinant puri®ed AtNIT1 (200 lL) was separated by
size-exclusion chromatography on a Superdex 200 HR10/30
column (Pharmacia) in 50 mM sodium phosphate buffer, containing 100 mM EDTA and 1 mM dithiothreitol,
pH 7.8, at a ¯ow rate of 0.5 mLámin)1 For calibration
of the column, thyroglobulin (663 kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa) (all from Sigma) were used
Enzyme assay Enzyme activity towards 3-phenylpropionitrile was assayed using bacterial protein (0.34±135 mg) in 5 mL Tris/HCl buffer (70 mM, pH 8.5) and 50 lL 3-phenylpropionitrile in methanol (0.25M) The reaction was carried out for 1 h at
35 °C An aliquot of 1 mL was acidi®ed with 50 lL HCl (5M) and extracted with diethyl ether (5 mL) After centrifugation (5 min, 2000 g) and cooling at )30 °C for
30 min to freeze the aqueous layer, the organic layer was decanted and derivatized with ethereal diazomethane (0.2M) After concentration, the residue was taken up in
1 mL diethyl ether and subjected to gas chromatography on
a Carlo Erba Fractovap 4160 with FID and Spectra Physics minigrator using a capillary glass column (50 m) with PS086 and carrier gas 50 kPa hydrogen Peak areas were calibrated
as follows A volume of 5 mL each of a solution of 3-phenylpropionitrile (181.5 mg) and 3-phenylpropionic acid (205.2 mg) in methanol (10 mL), and Tris/HCl buffer (990 mL, 70 mM, pH 8.5) were mixed, and 5 mL from this mixture was added to 5 mL of the 3-phenylpropionitrile solution This procedure was repeated three times A sample
of 1 mL from each solution was treated as described above and analyzed by gas chromatography The conversion factor was determined from the plot of ratio areas vs ratio concentrations One unit is de®ned as 1 lmol convert-edámin)1
Determination of temperature and pH optimum
of AtNIT1 Temperature dependence Nitrilase activity towards 3-phenylpropionitrile was assayed as described above using puri®ed enzyme (55.2 UámL)1, 1.98 mg proteinámL)1) in a
1 : 5000 dilution with Tris/HCl buffer (70 mM, pH 8.5) and
50 lL 3-phenylpropionitrile in methanol (0.25M) The reaction was initiated by the addition of substrate either directly after preliminary heating at the respective tempera-ture for 10 min or cooling at 7 °C for 30 min and after 24 h, respectively
PH dependence Enzyme activity was assayed as described above using puri®ed enzyme (56.8 UámL)1, 1.78 mg pro-teinámL)1) in phosphate buffer (50 mM, pH 7.8), which was diluted (1 : 5000) at 4 °C with the respective buffer After preliminary warming at room temperature, the reaction was initiated by the addition of 50 lL 3-phenylpropionitrile in methanol
R E S U L T S
Puri®cation and determination ofKmvalues Recombinant AtNIT1 was puri®ed from E coli lysates by metal chelate af®nity chromatography using a Zn2+-charged
Trang 3HiTrap column After a wash step with 100 mMimidazole, the tightly bound AtNIT1 was eluted with high recovery by
100 mM EDTA (Fig 1A) This single-step puri®cation yielded almost pure AtNIT1 (Fig 1B) with a speci®c activity
of 34.1 Uámg)1(Table 1) and a subunit mass of 38 kDa (Fig 1B) Recombinant AtNIT1 was eluted during gel-®ltration chromatography (Fig 1C) in fractions corre-sponding to a molecular mass of 450 kDa, suggesting that native AtNIT1 occurs as a homomeric protein complex of 11±13 subunits (data not shown)
Recombinant AtNIT1 showed a Kmvalue of 3.67 mMfor 3-indolylacetonitrile and 0.159 mMfor 3-phenylpropionit-rile (Fig 2A,B) The Kmvalue for 3-indolylacetonitrile is in good agreement with a reported value of 5 mM[4] Enzyme stability
As a crude extract of recombinant AtNIT1 had a half-time
of 2 days at pH 8 and 4 °C, the in¯uence of antioxidants and protease inhibitors on enzyme stability was investi-gated
Of the applied thiols that proved to be good antioxidants (mercaptoethanol and dithiothreitol) [4,17±21], dithiothre-itol had the better stabilizing effect (Table 2) The loss of enzyme activity on the addition of 2 mMdithiothreitol was 20% compared with 63% for the reference without thiol However, increasing the dithiothreitol concentration to
5 mM did not further improve enzyme stability The best result with protease inhibitors was achieved using EDTA at
a concentration of 2 mM (Table 2) Thus, all buffers used for cell disintegration and conversions were supplemented with dithiothreitol and EDTA (2 mMeach) In this way, we succeeded in signi®cantly increasing the enzyme stability of both crude extract and puri®ed enzyme: after 2 days at room temperature and 3 months at 4 °C, 95% and 90% enzyme activity, respectively, remained
Temperature optimum The nitrilases investigated so far generally show highest activity in the temperature range 35±40 °C, no matter what the enzyme source [18,21±23] However, as little is known about their stability at higher temperatures, which is a decisive factor in their application as biocatalysts in chemical reactions, the effect of temperature on AtNIT1 stability was investigated Recombinant AtNIT1 shows a sharp temperature optimum at 35 °C, determined after 1 h
of incubation, with a gentle slope at < 35 °C and a steeper slope at > 35 °C (Fig 3) Enzyme stability at different temperatures was determined after 24 h of incubation At
25 °C and 35 °C, only a slight decrease in activity was found At 35 °C, the relative enzyme activity amounts to
80%, whereas the enzyme was almost completely deac-tivated at 40 °C The highest absolute enzyme activity,
Fig 1 Puri®cation and characterization of recombinant AtNIT1 in
E coli (A) Lysate of isopropyl b- D -thiogalactopyranoside-induced
E coli-pQE10-AtNIT1 was applied to a Zn 2+ -charged HiTrap
column The column was washed with 100 m M imidazole (- - - -), and
bound AtNIT1 was eluted with 100 m M EDTA (± ± ± ±) Fractions were
analyzed for nitrilase activity as described in Materials and methods.
The elution pro®le was detected at 280 nm (B) Fractions obtained in
(A) were separated by SDS/PAGE and stained with Coomassie Last
lane, molecular masses of standards (kDa); lanes 17±19, active fractions
after HiTrap chromatography (C) Estimation of native molecular mass
of recombinant AtNIT1 by gel-®ltration chromatography on a
Superdex 200 HR10/30 column A 200 lL volume of puri®ed
AtNIT1 (d) and various mass standards [s; thyroglobulin (663 kDa),
apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), BSA
(66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa)]
were separated at a ¯ow rate of 0.5 mLámin )1 AtNIT1 was eluted The
AtNIT1 elution volume corresponds to molecular mass of 450 kDa.
The dotted line shows the 95% con®dence interval of the linear
regression of the semilogarithmic molecular mass±elution volume plot.
Table 1 Summary of puri®cation of the nitrilase from A thaliana.
Fraction Totalactivity (U) Totalprotein (mg) Speci®cactivity (Uámg )1 ) Puri®cation(fold) Yield (%)
Trang 4however, was found at 35 °C, and, moreover, the stability at
this temperature is suf®cient for applications in longer
lasting biotransformations
PH dependence of AtNIT1
The pH dependence of AtNIT1 was investigated with
different buffer systems in order to guarantee suf®cient
buffering capacity in the range pH 6±10 (Fig 4)
As can be seen from Fig 4, the choice of the buffer
system affects the enzyme activity slightly, changing from
Tris/HCl to glycine/NaOH With both buffer systems,
however, an activity optimum of pH 9.0 was found, with 97% of the maximum activity being measured at pH 8.5 The decrease in enzyme activity at pH values > 9 is not an irreversible process: acidifying an enzyme solution with
pH 10 back to pH 9 resulted in > 80% recovery of activity The pH optimum measured in this way is in slight contrast with the value of pH 7.5 reported for the nitrilase from
A thaliana [4], possibly arising from the deviant structure at the N-terminus However, several bacterial nitrilases also clearly have a basic pH optimum [23±27]
Substrate range of recombinant AtNIT1 The substrate range of recombinant AtNIT1 was investi-gated using structurally varied aromatic and aliphatic nitriles (Table 3) The activities given in Table 3 are referred
to the speci®c nitrilase activity towards butyronitrile As can
be seen, aliphatic nitriles are the most effective substrates, showing increased rates of hydrolysis with increasing size
of the hydrophobic residue, in the order butyronitrile, octanenitrile, phenylpropionitrile In contrast with 3-phe-nylpropionitrile, arylacetonitriles such as benzyl cyanide were converted 20 times more slowly Aromatic nitriles, such as benzonitrile, were converted even more slowly (270 times) than phenylpropionitrile The assumed natural substrate of AtNIT1, 3-indolylacetonitrile [4], was found
Fig 3 Determination of the temperature optimum of AtNIT1.
Table 2 Eect of antioxidants and protease inhibitors on enzyme
activity Enzyme activity was determined after incubation of 5 mL
crude enzyme extract in Tris/HCl buer (70 m M , pH 8.0) with the
respective antioxidant (neat) or a stock solution of protease inhibitors
(50-fold concentration; Protease-Inhibitor-Set,
Boehringer-Mann-heim) The reaction was carried out for 48 h at room temperature with
vigorous stirring The initial activity of 112 UáL )1 is 100% in the case
of antioxidants and 97 UáL )1 in the case of inhibitors.
Reagent Concn(m M )
Relative activity (%) Mercaptoethanol a 1 62
2 64
5 54 Dithiothreitol a 1 61
2 80
5 78 Aminophenylmethanesulfonyl ¯uoride b 1 76
5 23
2 96
10 94
100 97
a Activity of the reference, 37%; b activity of the reference, 82%.
Fig 2 Experimentally determined rates for the hydrolysis of
3-ind-olylacetonitrile (100 mg proteináL )1 ) (A) and 3-phenylpropionitrile
(0.3 mg proteináL )1 ) (B) plotted against initial substrate concentrations.
A Lineweaver±Burk plot of the data was used to calculate K m
values: 3.67 m M for 3-indolylacetonitrile and 0.159 m M for
3-phenyl-propionitrile.
Fig 4 Determination of the pH optimum of AtNIT1 using dierent buer systems (j) Tris/HCl; (m) glycine/NaOH; (.) KH 2 PO 4 /
K 2 HPO 4
Trang 5to be a poor substrate (Table 3) Also the hydrolytic rate of
cinnamonitrile, an a,b-unsaturated system, is signi®cantly
diminished compared with the corresponding saturated
phenylpropionitrile A double bond in the b,c-position,
however, has almost no effect on enzyme activity, as can be
seen if 3-butenenitrile is compared with
4-phenyl-butyronitrile Suitability as a substrate is strongly in¯uenced
by the substituents in the 2-position All substituents other
than ¯uoro inhibit enzymatic hydrolysis almost completely
(Table 3) Nitriles with substituents in the 3-position, for
example 3-methylbutyronitrile, are also poor substrates, but
the decrease in the hydrolytic rate is less pronounced
Interestingly, benzoylglycine nitrile is a much better
substrate for AtNIT1 than glycine nitrile itself
Acid amides as byproducts of AtNIT1-catalyzed
nitrile hydrolysis
Acid amide was ®rst detected as a major product of
AtNIT1-catalyzed nitrile hydrolysis with fumaronitrile as
substrate In this reaction, which was followed by gas
chromatography, less than 10% of the expected amount of
3-cyanoacrylic acid, estimated from the calibration, was
formed A product mixture of an unidenti®ed product and
3-cyanoacrylic acid in the ratio 93 : 7 (Table 4) was found
by HPLC As demonstrated by co-injection, fumaric acid
was not formed in the reaction After extractive separation
from 3-cyanoacrylic acid and subsequent recrystallization
from chloroform, the unknown product was isolated in
68% yield and unambiguously characterized as
3-cyano-acrylamide by elemental analysis and NMR spectroscopy
The amide to acid ratio was independent of conversion The
isolated amide was not hydrolyzed to 3-cyanoacrylic acid
under these reaction conditions Moreover, the hydrolytic
rate and ratio of acid to amide did not depend on enzyme
purity, giving similar results with both crude extract and
highly puri®ed enzyme From blank experiments, it could
also be excluded that it was an impurity of nitrile hydratase
As 3-cyanoacrylamide had been identi®ed as a byproduct
of fumaronitrile hydrolysis, the AtNIT1-catalyzed
hydro-lysis of other substrates with donor and acceptor
substi-tuents was investigated with respect to amide formation
Table 4 Product distribution of amide and acid in the AtNIT1-cata-lyzed nitrile hydrolysis The reactions were performed as described (Table 3) in Tris/HCl buer (70 m M , containing dithiothreitol and EDTA, 2 m M each, pH 8) at room temperature with 10 m M substrate concentration The samples were analyzed by gas chromatography and/or HPLC (RP C 18 Vertex column; 4 ´ 250 mm; Nucleosil 100;
5 lm; Knaur ( ); ¯ow rate 1 mLámin )1 ; 220 nm detection wavelength) Relative activities are referred to the speci®c nitrilase activity towards butyronitrile [8.931 lmolámin )1 á(mg protein) )1 100% at pH 8.0; 1.736 lmolámin )1 á(mg protein) )1 100% at pH 6.0].
Substrate
Relative activity (%)
Product distribution (amide : acid)
a Relative activity referred to the E-isomer b Conversion at
pH 6.0, relative activity referred to butyronitrile under these conditions.
Table 3 Relative activities of recombinant AtNIT1-catalyzed hydrolysis of nitriles The reactions were performed in Tris/HCl buer (70 m M , with dithiothreitol and EDTA, 2 m M each, pH 8) at room temperature At a concentration of 1.25 m M , all substrates were completely soluble; the enzyme concentration was varied so that the reaction time for all substrates was in the range 2±4 h for conversion of 15±40% Relative activities are referred to the speci®c nitrilase activity towards butyronitrile [1.393 lmolámin )1 á(mg protein) )1 100%].
Substrate
Relative activity (%) Substrate
Relative activity (%) Butyronitrile 100 2-Methylbutyronitrile < 0.01 b
Octanenitrile 291 2-Fluoropentanenitrile 131
3-Indolylacetonitrile a 2.2 2-Phenylpropionitrile < 0.01 b
Benzonitrile 2.7 3-Methylbutyronitrile 4.0
Benzyl cyanide a 31 Cyclopropylacetonitrile 15
3-Phenylpropionitrile a 729 2-Hydroxypentanenitrile 0.2 c
4-Phenylbutyronitrile a 154 Glycine nitrile 0.4
Cinnamonitrile 48 2-Amino-4-methylpentanenitrile < 0.03 b
4-Phenylbut-3-enenitrile 188 Benzoylglycine nitrile 65
a See also literature data [9] b 24 h reaction time c Hydrolysis at pH 7.0.
Trang 6(Table 4) Amides have also been found as major products
in the AtNIT1-catalyzed hydrolysis of
a-¯uoroarylaceto-nitriles [15] An a-¯uoro substituent, however, does not
conclusively result in amide formation as can be seen in the
hydrolysis of a-¯uorobutyronitrile, yielding 95% of the
corresponding acid (Table 4) Nevertheless, both a ¯uoro
substituent in the a position and a second nitrile group
conjugated to the nitrile (fumaronitrile) seem to play a
decisive role in amide formation
The assumption that electron-withdrawing substituents
favor the formation of amides was supported by the
hydrolysis of differently 3-substituted acrylonitriles
(Table 4) Whereas 3-nitroacrylonitrile was hydrolyzed to
3-nitroacrylamide as sole product, in the case of the
donor-substituted 3-methoxyacrylonitrile and crotononitrile, the
corresponding acids were formed almost quantitatively
(Table 4) As 3-nitroacrylonitrile tends to decompose under
basic conditions, the reaction was performed at pH 6
(Table 4), where the amide was formed only by
enzyme-catalyzed hydrolysis and not by chemical reaction, as
con®rmed by a blank experiment Table 4 reveals that the
relative activity is almost completely independent of the
kind of substituent
D I S C U S S I O N
Substrate range
Analysis of the substrate range with a variety of structurally
different aromatic and aliphatic nitriles revealed that
aliphatic nitriles are hydrolyzed more ef®ciently than the
natural substrate IAN or structurally related aromatic
nitriles With a relative activity of only 2.2%, compared with
butyronitrile, 3-indolylacetonitrile is a poorer substrate for
AtNIT1 than benzyl cyanide (31% relative activity) This
®nding is in agreement with literature data, showing that
IAN is one of the weakest substrates [9] The order of the
relative AtNIT1 activity towards the substrates
3-phenyl-propionitrile, 4-phenylbutyronitrile and benzyl cyanide
(Table 3) also corresponds to that just recently reported
[9] 2-Substituted substrates such as 2-methylbutyronitrile
and 2-phenylpropionitrile, however, were almost completely
unacceptable for AtNIT1, indicating that substituents in the
2-position, other than ¯uoro, inhibit the hydrolysis The
broad substrate range observed for AtNIT1 in this study is
in good agreement with reports showing that AtNIT1 acts
on a variety of aliphatic and aromatic substrates [4,9] and is
in contrast with the high speci®city of AtNIT4 for
b-cyano-(L)-alanine [7] Its broad substrate range, recombinant
accessibility and reasonable stability make AtNIT1 a
promising candidate for applications in organic chemistry,
in particular the synthesis of optically active
2-¯uorocarb-oxylic acids, which are very useful as analogs of pheromones
and antirheumatics, for example [15] Also the
mono-hydrolysis of aliphatic dinitriles to monocarboxylic acids is
of great industrial interest because selective chemical
hydrolysis is virtually impossible [16]
Amide formation
The formation of amides as byproducts of
nitrilase-catalyzed reactions was ®rst reported as early as 1964
[28,29] Furthermore, in subsequent publications [19,30±
32], small amounts of amides (< 15%) could be detected besides the carboxylic acids during nitrilase catalysis In all cases, the amide to acid ratio was independent of reaction conditions (temperature and pH) and the applied enzyme concentrations In their basic work on the four A thaliana nitrilases NIT1±4, Bartel & Fink [5] described the conversion of IAN into 3-indolylacetic acid and indole-3-acetamide and found that the latter is not a substrate for these enzymes For the hydrolysis of b-cyano-(L)-alanine, catalyzed by NIT4, Piotrowski et al [7] reported the simultaneous formation of asparagine and aspartic acid in a ratio of 1.5 : 1, independent of reaction conditions A dependence of the amide to acid ratio on the substituents, however, has not been reported in the literature so far
Until now the reaction mechanism of nitrilase-catalyzed hydrolysis has not been con®rmed experimentally The mechanism postulated [19,28,29,33] involves the donation
of a cysteine from the enzyme to the nitrile group to yield a thioimidate, which subsequently forms a tetrahedral inter-mediate A by addition of water Generally, NH3 is eliminated from this intermediate A to give a thioester, which reacts with a further water molecule to give the carboxylic acid (Fig 5) Therefore, the formation of the acid amide from A logically arises from the elimination of cysteine
It has been shown for the chemical hydrolysis of thioimidate esters [34,35] that the formation of thiol ester
is favored in acidic medium (pH < 2.7), whereas at higher
pH values (pH > 2.7) the formation of amide dominates This result was explained by a facilitated elimination of NH3 caused by protonation of the amino group in the tetrahedral intermediate
Although, as mentioned, some papers have dealt with the mechanism of the nitrilase-catalyzed hydrolysis of nitriles, a relationship between the chemical structure of the substrate and the amount of acid amide formation has not so far been described For AtNIT1-catalyzed nitrile hydrolysis, we could demonstrate for the ®rst time such a structural
Fig 5 Postulated mechanism for acid amide formation in the nitrilase-catalyzed hydrolysis of nitriles.
Trang 7relationship, because the amide formation clearly depends
on the kind of substituent The preference for acid amide
formation by a-¯uoro substituents or by acceptor groups
(CN, NO2) in p-conjugated nitriles is clear evidence of an
electronically preferred formation and stabilization of the
tetrahedral intermediate A in the enzyme±substrate
com-plex Because the crystal structure of the active site of
AtNIT1 is not known, how the stabilization of the
tetrahedral intermediate assists the elimination of cysteine
to yield the acid amide cannot be explained
C O N C L U S I O N S
Chemical hydrolysis of many nitriles with labile substituents
catalyzed by acid or base is virtually impossible because of
the drastic reaction conditions required Therefore, over the
last few years, biocatalysts capable of hydrolyzing nitriles to
carboxylic acids have been intensively investigated [36]
In most cases, however, nitrile hydratase±amidase systems
have been described, although not exclusively [36] The
nitrilase AtNIT1 from A thaliana is the ®rst plant nitrilase to
be investigated with respect to its synthetic potential Because
of optimized expression, the enzyme is now accessible in
suf®cient quantities Clear optimization of enzyme stability
under the reaction conditions, which is important for
practical application, could be achieved by addition of the
protease inhibitor EDTA (Table 2) Therefore, slowly
reacting nitriles can also be hydrolyzed without problem
The most important criteria for practical applications,
however, are the substrate range and selectivity of an
enzyme In contrast with other nitrile-hydrolyzing enzymes,
the nitrilase AtNIT1 stands out as having a very broad
substrate range (Table 3) Although longer-chain aliphatic
nitriles are the most effective substrates, hydrolysis of
aromatic nitriles is also catalyzed Because of the clearly
improved enzyme stability, AtNIT1-catalyzed hydrolysis is
also applicable to aromatic nitriles Moreover, AtNIT1
shows a very interesting stereoselectivity and
chemoselec-tivity The in¯uence of substituents in the a position to the
nitrile function has already been mentioned Because the
enzyme does not accept any substituents at the a position
except ¯uoro, compounds bearing several different cyano
groups can be selectively hydrolyzed Hydrolysis of racemic
2-¯uoroarylacetonitriles proceeds enantioselectively [15] In
dinitriles with chemically comparable cyano groups (e.g
adiponitrile), only one cyano group is hydrolyzed
exclu-sively to give the corresponding cyanocarboxylic acids
[16,31,37], opening up interesting possibilities for organic
synthesis, for example the preparation of certain lactams
[31] Furthermore, AtNIT1 exhibits cis/trans selectivity with
a,b-unsaturated nitriles [38], as also reported for other
enzymes [19,39,40]
Because of its broad substrate range on the one hand and
unusual regioselectivities and stereoselectivities, the nitrilase
AtNIT1 from A thaliana is a very interesting biocatalyst in
organic synthesis
A C K N O W L E D G E M E N T S
This work was generously supported by the Fonds der Chemischen
Industrie We acknowledge Dr K Trummler for assistance in enzyme
puri®cation, Dr S FoÈrster for fermentation, and Dr A Baro for
preparing the manuscript.
R E F E R E N C E S
1 Legras, J.I., Chuzel, G., Arnaud, A & Galzy, P (1990) Natural nitriles and their metabolism World J Microbiol Biotechnol 6, 83±108.
2 Faber, K (1995) Biotransformations in Organic Chemistry, 2nd edn Springer, Berlin.
3 Drauz, K & Waldmann, H (1995) Enzyme Catalysis in Organic Synthesis Verlag Chemie, Weinheim.
4 Bartling, D., Seedorf, M., MithoÈfer, A & Weiler, E.W (1992) Cloning and expression of an Arabidopsis nitrilase which can convert indole-3-acetonitrile to the plant hormone, indole-3-acetic acid Eur J Biochem 205, 417±424.
5 Bartel, B & Fink, G.R (1994) Dierential regulation of an auxin-producing nitrilase gene family in Arabidopsis thaliana Proc Natl Acad Sci USA 91, 6649±6653.
6 Schmidt, R.C., MuÈller, A., Hain, R., Bartling, D & Weiler, E.W (1996) Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme Plant J 9, 683±691.
7 Piotrowski, M., SchoÈnfelder, S & Weiler, E.W (2001) The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode b-cyano- L -alanine hydratase/nitrilase J Biol Chem 276, 2616±2621.
8 Tsunoda, H & Yamaguchi, K (1995) The cDNA sequence of an auxin-producing nitrilase homolog in tobacco (GenBank D63331) (PGR 95-058) Plant Physiol 109, 339.
9 Vorwerk, S., Biernacki, S., Hillebrand, H., Janzik, I., MuÈller, A., Weiler, E.W & Piotrowski, M (2001) Enzymatic characterization
of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster Planta 212, 508±516.
10 Kende, H (1989) Enzymes of ethylene biosynthesis Plant Physiol.
91, 1±4.
11 Nagasawa, T., Shimizu, H & Yamada, H (1993) The superiority
of the third-generation catalyst, Rhodococcus rhodochrous J1 nitrile hydratase, for industrial production of acrylamide Appl Microbiol Biotechnol 40, 189±195.
12 Kobayashi, M., Nagasawa, T & Yamada, H (1992) Enzymic synthesis of acrylamide: a success story not yet over Trends Biotechnol 10, 402±408.
13 Layh, N., Stolz, A., BoÈhme, J., Eenberger, F & Knackmuss, H.-J (1994) Enantioselective hydrolysis of racemic naproxen nitrile and naproxen amide to S-naproxen by new bacterial isolates.
J Biotechnol 33, 175±182.
14 Eenberger, F & Graef, B.W (1998) Chemo- and enantioselective hydrolysis of nitriles and acid amides, respectively, with resting cells of Rhodococcus sp C3II and Rhodococcus erythropolis MP50.
J Biotechnol 60, 165±174.
15 Eenberger, F & Oûwald, S (2001) Enantioselective hydrolysis of (RS)-2-¯uoroarylacetonitriles using nitrilase from Arabidopsis thaliana Tetrahedron: Asymmetry 12, 279±285.
16 Eenberger, F & Oûwald, S (2001) Selective hydrolysis of aliphatic dinitriles to monocarboxylic acids by a nitrilase from Arabidopsis thaliana Synthesis 1866±1872.
17 Harper, D.B (1977) Microbial metabolism of aromatic nitriles Enzymology of C±N cleavage by Nocardia sp (Rhodococcus group) N.C.I.B 11216 Biochem J 165, 309±319.
18 Bhalla, T.C., Miura, A., Wakamoto, A., Ohba, Y & Furuhashi,
K (1992) Asymmetric hydrolysis of a-aminonitriles to optically active amino acids by a nitrilase of Rhodococcus rhodochrous PA-34 Appl Microbiol Biotechnol 37, 184±190.
19 Stevenson, D.E., Feng, R., Dumas, F., Groleau, D., Mihoc, A & Storer, A.C (1992) Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase Biotechnol Appl Biochem 15, 283±302.
20 Kobayashi, M., Yanaka, N., Nagasawa, T & Yamada, H (1990) Puri®cation and characterization of a novel nitrilase of
Trang 8Rhodococcus rhodochrous K22 that acts on aliphatic nitriles J.
Bacteriol 172, 4807±4815.
21 Bestwick, L.A., Gronning, L.M., James, D.C., Bones, A &
Rossiter, J.T (1993) Puri®cation and characterization of a nitrilase
from Brassica napus Physiol Plant 89, 811±816.
22 Nagasawa, T., Mauger, J & Yamada, H (1990) A novel nitrilase,
arylacetonitrilase, of Alcaligenes faecalis JM3: puri®cation and
characterization Eur J Biochem 194, 765±772.
23 Kobayashi, M., Nagasawa, T & Yamada, H (1989) Nitrilase of
Rhodococcus rhodochrous J1 Puri®cation and characterization.
Eur J Biochem 182, 349±356.
24 Bandyopadhyay, A.K., Nagasawa, T., Asano, Y., Fujishiro, K.,
Tani, Y & Yamada, H (1986) Puri®cation and characterization
of benzonitrilases from Arthrobacter sp strain J-1 Appl Environ.
Microbiol 51, 302±306.
25 Harper, D.B (1977) Fungal degradation of aromatic nitriles.
Enzymology of C-N cleavage by Fusarium solani Biochem J 167,
685±692.
26 Stalker, D.M., Malyj, L.D & McBride, K.F (1988) Puri®cation
and properties of a nitrilase speci®c for the herbicide bromoxynil
and corresponding nucleotide sequence analysis of the bxn gene.
J Biol Chem 263, 6310±6314.
27 Robinson, W & Hook, R (1964) Ricinine nitrilase I Reaction
product and substrate speci®city J Biol Chem 239, 4257±4262.
28 Hook, R & Robinson, W (1964) Ricine nitrilase II Puri®cation
and properties J Biol Chem 239, 4263±4267.
29 Thimann, K.V & Mahadevan, S (1964) Nitrilase I Occurrence,
preparation, and general properties of the enzyme Arch Biochem.
Biophys 105, 133±141.
30 Layh,N.,Stolz,A., FoÈrster,S.,Eenberger, F &Knackmuss,H.-J.
(1992) Enantioselective hydrolysis of O-acetylmandelonitrile to
O-acetylmandelic acid by bacterial nitrilases Arch Microbiol 158,
405±411.
31 Gavagan, J.E., Fager, S.K., Fallon, R.D., Folsom, P.W., Herkes, F.E., Eisenberg, A., Hann, E.C & DiCosimo, R (1998) Chemo-enzymic production of lactams from aliphatic a,x-dinitriles.
J Org Chem 63, 4792±4801.
32 Goldlust, A & Bohak, Z (1989) Induction, puri®cation, and characterization of the nitrilase of Fusarium oxysporum f sp melonis Biotechnol Appl Biochem 11, 581±601.
33 Dufour, E., Storer, A.C & MeÂnard, R (1995) Engineering nitrile hydratase activity into a cysteine protease by a single mutation Biochemistry 34, 16382±16388.
34 Chaturvedi, R.K., MacMahon, A.E & Schmir, G.L (1967) The hydrolysis of thioimidate esters Tetrahedral intermediates and general acid catalysis J Am Chem Soc 89, 6984±6993.
35 Chaturvedi, R.K & Schmir, G.L (1969) The hydrolysis of thioimidate esters II Evidence for the formation of three species
of the tetrahedral intermediate J Am Chem Soc 91, 737±746.
36 Wieser, M & Nagasawa, T (2000) Stereoselective nitrile-con-verting enzymes In Stereoselective Biocatalysis (Patel, R.N., ed.),
pp 463±465 Marcel Dekker, New York.
37 Bengis-Garber, C & Gutman, A.L (1988) Bacteria in organic synthesis: selective conversion of 1,3-dicyanobenzene into 3-cyanobenzoic acid Tetrahedron Lett 29, 2589±2590.
38 Eenberger, F & Oûwald, S (2001) E-Selective hydrolysis of E,Z a,b-unsaturated nitriles by the recombinant nitrilase AtNIT1 from Arabidopsis thaliana Tetrahedron: Asymmetry 13, 2581± 2587.
39 QuiroÂs, M., Astorga, C., Rebolledo, F & Gotor, V (1995) Enzymic selective transformations of diethyl fumarate Tetra-hedron 51, 7715±7720.
40 Klibanov, A.M & Siegel, E.H (1982) Geometric speci®city of porcine liver carboxylesterase and its application for the produc-tion of cis-arylacrylic esters Enzyme Microb Technol 4, 172±175.