From the Department of Plant Physiology, Ruhr-Universität, Universitätsstrasse 150, D-44801 Bochum, Germany
Received for publication, August 29, 2000, and in revised form, October 24, 2000
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ABSTRACT |
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Nitrilases (nitrile aminohydrolases, EC 3.5.5.1)
are enzymes that catalyze the hydrolysis of nitriles to the
corresponding carbon acids. Among the four known nitrilases of
Arabidopsis thaliana, the isoform NIT4 is the most
divergent one, and homologs of NIT4 are also known from species not
belonging to the Brassicaceae like Nicotiana tabacum and
Oryza sativa. We expressed A. thaliana NIT4 as
hexahistidine tag fusion protein in Escherichia coli. The
purified enzyme showed a strong substrate specificity for Among the nitrilases of Arabidopsis thaliana, the first
higher plant nitrilases that have been cloned (1-3), isoform 4 is clearly divergent. The members of the NIT1 group (NIT1, NIT2, and NIT3)
are highly similar and share a minimum of 82% identical amino acids.
In A. thaliana, they occur clustered on chromosome 3, and
although the patterns of expression are distinctly different for each
isoform (2-5), their enzymatic characteristics are very similar,
albeit not identical (4). As shown previously (4), a major role for
these nitrilases appears to be in the metabolism of nitriles released
by breakdown of glucosinolates. A further common feature is that all
three isoenzymes can convert indole-3-acetonitrile (IAN)1 to indole-3-acetic
acid (IAA), the plant growth hormone (1-3). A nitrilase (which
isoform, is at present unknown) may be part of the IAA synthase enzyme
complex of A. thaliana, a soluble 160-kDa complex catalyzing
the conversion of L-tryptophan to IAA in vitro (6).
Nitrilase 4 is peculiar in that it occurs as a single gene at a
different chromosomal location (chromosome 5) (3), does not accept IAN
as a substrate (Ref. 7 and this paper), and occurs in plants of
different taxonomic position, such as tobacco (8) and rice
(GenBankTM accession number AB027054). Nitrilases belonging
to the NIT4 family thus may have a different and more general function
and will likely not be associated with auxin production.
A nitrile of widespread occurrence in higher plants is
In a previous study we reported about the enzymatic characterization of
the A. thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3 gene cluster (4).
During this work, we notified that Arabidopsis NIT4 has a
quite different substrate specificity compared with the NIT1/NIT2/NIT3
group. Here, we report about the elucidation of the enzymatic function
of the NIT4 enzyme family.
Plant Material--
A. thaliana ecotype C24 and
Nicotiana tabacum W38 were grown in a greenhouse in standard
soil at 20 °C, 70% relative humidity, and 210 µmol photons
m General Procedures--
The following general procedures have
been described elsewhere (4): sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and protein determination.
Vector Construction and Cloning of Nitrilase 4 cDNAs--
All basic molecular techniques were adapted from
Ausubel et al. (12) or Sambrook et al. (13).
Sequences of polymerase chain reaction-amplified or mutated cDNAs
were verified by sequencing. Cloning of NIT4 cDNA was described
previously (4). The cDNAs for the N. tabacum nitrilases
TNIT4A and TNIT4B were kindly provided by Dr. Kazuo Yamaguchi
(Institute for Gene Research, Kanazawa University, Kanazawa,
Japan), and cloning into pET-21b(+) (Novagen, Madison, WI) was done
as described for A. thaliana NIT4 (4). Mutations were
introduced using the GeneEditor in vitro Site-directed Mutagenesis System (Promega, Mannheim, Germany).
Expression and Purification of NIT4 Enzymes--
The
Escherichia coli strain BL21 (DE3) was used for expression
of plant nitrilases. Bacteria grown overnight (600 ml) were collected
by centrifugation (5000 × g, 5 min, 4 °C) and
resuspended in 60 ml of lysis buffer (50 mM sodium
phosphate buffer, pH 8.0, 300 mM NaCl, 10 mM
imidazole, 5 mM 2-mercaptoethanol, 1 mg
ml Preparation of Plant Extracts--
One gram of plant material
was ground to a fine powder in a mortar with liquid nitrogen and thawed
with continuing grinding in 3 ml of 100 mM potassium
phosphate buffer, pH 8.0, 1 mM EDTA, 1 mM DTT.
The homogenate was centrifuged (15 min, 10,000 × g, 4 °C), and the supernatant was again centrifuged (20 min,
100,000 × g, 4 °C). The resulting supernatant
(soluble proteins) was brought to 40% saturation of
(NH4)2SO4 by adding a 100%
saturated (NH4)2SO4 solution
dropwise. After stirring on ice for 20 min, precipitated proteins were
collected by centrifugation (15 min, 10,000 × g, 4 °C) and resuspended in a small volume (0.5-2 ml, depending on the
size of the pellet) of 100 mM potassium phosphate buffer, pH 8.0, 1 mM EDTA, 1 mM DTT. Because extracts
of blue lupine seedling have a high Asn content, they were desalted in
the same buffer using PD-10 columns (Amersham Pharmacia Biotech) before
and after the (NH4)2SO4 precipitation.
Colorimetric Determination of Nitrilase and Nitrile Hydratase
Activity--
Nitrilase activity was determined by analyzing the
released ammonia using the Bertholet reaction as described previously
(4). In brief, the substrate (3 mM) was incubated with
400-600 ng of purified protein or 50-100 µg of protein (crude
extracts) in 0.05 M potassium phosphate buffer, pH 8.0, at
30 °C in a total volume of 1 ml. For background control,
heat-denatured enzyme (10 min, 100 °C) was used. After the indicated
times (10 min to 4 h) aliquots of 0.1 ml were taken, and the
reaction was stopped by adding 0.1 ml each of 0.33 M sodium
phenolate, 0.02 M sodium hypochlorite, and 0.01% (w/v)
sodium pentacyanonitrosyl ferrate(III) (sodium nitroprusside). After
heating for 2 min at 95 °C, the sample was diluted with 0.6 ml of
water, and the absorbance was read at 640 nm. Each experiment was
calibrated with NH4Cl solutions of known concentrations.
For parallel determination of both nitrilase and nitrile hydratase
(NHase) activity, two 0.1-ml samples were taken and boiled for 10 min
to stop the reaction. Both aliquots were diluted to 1 ml with water,
and 0.1 ml of the first sample was used for determination of ammonia
(resulting from nitrilase activity). To the second sample, 0.25 units
of asparaginase (from Erwinia chrysanthemi, Sigma) was added
and incubated for 30 min at 37 °C. Subsequently, ammonia was
determined using a 0.1-ml aliquot as described above (representing both
nitrilase and NHase activity). NHase activity was then calculated from
the difference of both samples. The 1:10 dilution was necessary for two
reasons. (i) The concentration of nitrilase- and asparaginase-released
ammonia was usually outside the linear range of the calibration curve (which was between 0.01 and 0.5 mM). (ii) The asparaginase
used showed Ala(CN) hydratase activity when incubated at Ala(CN)
concentrations higher 1 mM resulting in overestimated
background values because Ala(CN) does not decrease below 1 mM in the background samples. By diluting the sample (and
therefore the Ala(CN)) before asparaginase was added, this effect was negligible.
Determination of Nitrilase and Nitrile Hydratase Activity by
LC-MS--
For some experiments, nitrilase and NHase activity were
determined by liquid chromatography coupled to electrospray-ionization mass spectrometry (LC-ESI-MS). After the indicated times, 0.1-ml aliquots were withdrawn from the reaction vessels, and 100% (v/v) ethanol was added to reach a final concentration of 80% (v/v) ethanol.
The samples were boiled for 10 min and subsequently centrifuged (15 min
at 13,000 rpm in a tabletop centrifuge) to collect insoluble material.
The supernatant was evaporated to dryness and subsequently resuspended
in 0.5 ml of 0.5 mM pentadecafluorooctanoic acid (PDFOA). After a second centrifugation, the supernatant was transferred to a
fresh reaction tube. Reverse phase liquid chromatography of the
underivatized amino acids was carried out according to Chaimbault
et al. (14) using a Luna C18(2) column (0.5 µm, 1 × 150 mm, Phenomenex, Aschaffenburg, Germany) on a Waters Cap-LC (Waters,
Milford, MA). The injection volume was 5 µl, and the chromatography
proceeded isocratically in 0.5 mM PDFOA at a flow rate of
40 µl min Nitrilase 4 Isoforms Are not Restricted to
Brassicaceae--
Partial or complete cDNA or genomic sequences of
nitrilases are known from several plant species like A. thaliana (1-3), Brassica campestris (Chinese cabbage)
(15, 16), Lotus japonicus (GenBankTM accession
number AW720658), N. tabacum (tobacco) (8), and Oryza
sativa (rice) (GenBankTM accession number AB027054).
By comparing the homologies between these nitrilases
(Fig. 1), they can be divided into two
groups. The first group, referred to as NIT1 group, seemed to be
specific for nitrilases from Brassicaceae. Because many species of the Brassicaceae are characterized by their high glucosinolate content and
glucosinolate-derived nitriles are among the best substrates for
NIT1-NIT3, a function of these enzymes in glucosinolate metabolism has
been proposed (4). Arabidopsis NIT4 belongs to the second group, further referred to as NIT4 group to which, in addition to the
NIT4s of the Brassicaceae, all known nitrilases of other plants belong.
This clustering of the NIT4 sequences could also be observed by
phylogenetic analysis using the PHYLIP software package (Dr. J. Felsenstein, Department of Genetics, University of Washington, Seattle,
WA) (data not shown). The NIT4 homologs therefore have to be
considered as orthologs which means that the present NIT4
genes share a common ancestor. It therefore seems likely that members
of the NIT4 group may also have a conserved function.
Expression of Enzymatically Active NIT4 of A. thaliana in E. coli--
By using exactly the same strategy as described previously
for NIT1, NIT2, and NIT3 (4), NIT4 could be expressed in E. coli from its genuine start codon as a fusion protein with a
hexahistidine tag joined to the C-terminal amino acid of the enzyme via
a Val-Glu dipeptide spacer. Although, as in the case of the other three nitrilases from A. thaliana, most of the bacterially
expressed protein was found in the 10,000 × g
sediment, a fraction of the recombinant nitrilase could be purified
from the soluble protein lysate by metal-chelate affinity
chromatography on nickel-nitrilotriacetate columns. The resulting
fraction was purified at least to 95% homogeneity as judged by
Coomassie Blue-stained SDS gels. We tested more than 25 selected
substrates using the purified enzyme because in preliminary experiments
we observed striking and qualitatively different results when the
purified enzyme or a crude extract of NIT4-expressing E. coli was used. The results show that NIT4 is highly specific for
NIT4 Is Both a Nitrilase and a Nitrile Hydratase--
Since
the enzymatic assay used so far was based on the analysis of released
ammonia, it was necessary to show that this ammonia represented the
nitrile nitrogen and not the amino nitrogen. Thus, we analyzed the
reaction products by thin layer chromatography (data not shown) and
liquid chromatography coupled to nanospray-ionization mass spectrometry
(LC-ESI-MS) (Fig. 2). The production of
aspartic acid (Asp) could unequivocally be shown by co-chromatography
with authentic Asp during TLC (data not shown) as well as by LC-ESI-MS (Fig. 2A) and by its collision-induced decomposition
spectrum (Fig. 2B). Unexpectedly, Asn could also be detected
and occurred in amounts about 1.5 times higher than Asp. Therefore,
NIT4 not only has a nitrilase activity (converting Ala(CN) to Asp) but also a nitrile hydratase (NHase) activity (converting Ala(CN) to Asn).
Nitrilases hydrolyze nitriles by the successive addition of two
molecules of water, whereas the substrate remains covalently bound to
the enzyme's catalytic-site cysteine (18). Our results suggested that Asn may occur as a free intermediate of the nitrilase reaction rather than enzyme-bound. However, this could be ruled out by
the observation that Asn is only marginally converted to Asp by NIT4
(Table I). This negligible asparaginase activity of NIT4 (~6 nkat (mg
protein) Is the Catalytic Site for Nitrilase and NHase Activity the
Same?--
The known reaction mechanisms of nitrilases and NHases are
quite different (for review see Ref. 19). Although the first bind their
substrate covalently to a cysteine residue, the latter use a nonheme
iron for their activity. The formation of Asn from Ala(CN) by NIT4 may
be the result of a "premature" release of Asn during the nitrilase
reaction or it may occur at a second active site with NHase activity.
The substrate concentration dependence of the two reactions
(Fig. 4 and Table
II) revealed that the
Km of Ala(CN) for both reactions is very similar if
not identical, whereas the maximum velocity
(Vmax) is higher for the Asn formation. This
result suggests that the catalytic center for both reactions might be
the same. Both reactions showed the same temperature and pH dependence
(Table II), and they were both inhibited by N-ethylmaleimide
(100% inhibition at 2 mM). The involvement of a cysteine
residue in both reactions was further indicated by their inhibition at
higher concentrations of DTT (~50% inhibition at 10 mM).
To pinpoint this residue(s), the proposed catalytically active cysteine
of NIT4 for the nitrilase reaction (Cys-197) was mutated to alanine.
The mutant protein (NIT4C197A) showed no nitrilase activity, and a
strongly inhibited NHase activity (~5% of the wild type protein)
(Fig. 2) demonstrating (i) the importance of Cys-197 for both
activities but also (ii) that the NHase activity does not completely
depend on this residue.
Is the NHase Activity of NIT4 Genuine?--
In 1995, Dufour
et al. (20) showed that a single amino acid substitution
(Gln to Glu) in the cysteine protease papain resulted in a novel NHase
activity of the mutated protein. The observed NHase activity of
A. thaliana NIT4 could therefore be the result of an
artificial mutation. Sequence errors could be ruled out because the
reported genomic and cDNA sequences of NIT4 from A. thaliana encode the same polypeptide. Additionally, NHase activity was seen with three different NIT4 homologs (see below). A critical factor may be the intentional "mutation" of the C terminus of the
enzyme introduced with the His tag (EVHHHHHH) that was used in all
tested recombinant NIT4 proteins. To study the influence of the His
tag, we expressed NIT4 using its genuine stop codon in E. coli and enriched the protein by
(NH4)2SO4 precipitation and gel
filtration. NIT4 activity was found in the void volume of a Superdex
200 HiLoad 16/60 column (Amersham Pharmacia Biotech) indicating that
the native molecular mass is greater than 600 kDa. The active fractions
showed no asparaginase activity under the conditions applied, but both
nitrilase and NHase activities could be detected, displaying a ratio of
1:1.25 like the His-tagged protein (data not shown). The NHase activity
is therefore an intrinsic property of the NIT4 protein.
In a recent report, the activation of the NIT4 gene of
A. thaliana during leaf senescence was described (21). We
therefore compared Ala(CN) hydrolysis in extracts from leaves of plants beginning to shoot and from leaves of flowering plants showing signs of
senescence (Fig. 5). Both enzymatic activities were severalfold higher
in extracts from senescent leaves. Interestingly, the ratios of the two
activities (NHase/nitrilase) decreased from 3.3 in nonsenescent leaves
to 1.2 in senescent leaves.
The occurrence of nitrilases in higher plants is known since 1958 (22), but until now the main interest was directed to their ability to
convert indole-3-acetonitrile to the plant hormone indole-3-acetic acid
(1, 3, 22). Genomic or cDNA sequences of nitrilases are now known
from A. thaliana, two Brassica species (B. campestris and Brassica oleracea) (15, 16), tobacco
(N. tabacum) (17), and rice (O. sativa,
GenBankTM accession number AB027054). In addition several
expressed sequence tag clones exist, which encode nitrilase-like
proteins. The nitrilases of A. thaliana were characterized
in most detail. A. thaliana possesses four different
nitrilase genes that are differentially expressed (2-4). While
nitrilases homologous to Arabidopsis NIT1, NIT2, and NIT3
are only known from Brassicaceae, NIT4 homologs are also found in
tobacco, rice, and lotus (Fig. 1). Phylogenetic analysis indicates that
the NIT4 enzymes are orthologs (data not shown), and we would therefore
expect them to catalyze the same reaction.
Arabidopsis NIT4 has a high substrate specificity for
The expected product of nitrilase-catalyzed hydrolysis of
Ala(CN) would be aspartic acid (Asp). However, we found that NIT4 not
only produced Asp but also, to an even higher amount, Asn (Fig. 2). The
formation of Asn from Ala(CN) would formally be an NHase reaction, and
Asn may be further hydrolyzed to Asp by an asparaginase activity. Does
NIT4 possess an asparaginase activity and is Asn therefore a free
intermediate of NIT4-catalyzed Asp formation from Ala(CN)? Although we
detected a minor asparaginase activity in some of our NIT4 preparations
(Table I) this, in all probability, resulted from minor contaminations
of these preparations with E. coli asparaginase (see above).
Additionally, this activity is so low that it could never account for
the formation of the amounts of Asp observed. Importantly, time course
studies neither showed a lag phase in Asp formation nor a turnover of
Asn (Fig. 3) which would both be expected if Asn were an intermediate
of Asp formation. Clearly, Asn is not an intermediate but one of two
true end products of NIT4-catalyzed Ala(CN) hydrolysis.
NIT4 therefore displays a new type of NHase activity differing from the
known mechanism of "classical" NHases. Elucidating this mechanism
will be of value for the design of new nitrile-degrading enzymes that
could be of use for environmental and industrial applications. A
possible explanation for this NHase activity may be a "premature"
release of the enzyme-bound substrate after the addition of the first
molecule of water. If the second molecule of water is not delivered
fast enough, the amide may be released, whereas, if the water is
present, the substrate will be processed further to the acid.
Interestingly, an enzyme showing the same enzymatic characteristics as
NIT4 was purified from Pseudomonas sp. 13 in 1983 (23), but
no sequence of this protein was reported until now. The reported biochemical data of this enzyme are very similar to NIT4, but its
Km for Ala(CN) is about 1 order of magnitude higher compared with Arabidopsis NIT4.
Ala(CN) is a product of cyanide detoxification of plants. It is
produced from cyanide and cysteine by cyanoalanine synthase (9). Recent
results indicate that cyanoalanine synthase is an enzyme homologous or
identical to mitochondrial cysteine synthase (24, 25). In most species
analyzed, Ala(CN) is then converted to Asn, although in some species it
is converted to the dipeptide One possible source of cyanide in higher plants is the biosynthesis of
the plant hormone ethylene from 1-aminocyclopropane-1-carboxylic acid
(27). During this reaction cyanoformic acid is produced which then
spontaneously degrades to carbon dioxide and cyanide. Interestingly,
the Arabidopsis NIT4 promoter was found to be activated during leaf senescence as shown by Northern blot analysis (20). We
detected severalfold higher NIT4 activity in extracts from senescent
leaves of A. thaliana compared with extracts from
nonsenescent leaves, and the ratio of NHase to nitrilase activity
decreased (Fig. 5). This change may indicate that different enzymes are involved in Ala(CN) metabolism at different developmental stages, but
it may also indicate that NHase and nitrilase activity of NIT4 could be
regulated independently, e.g. by posttranslational modifications. Whether NIT4 activity or NIT4 expression is connected to
ethylene biosynthesis or cyanide production during leaf senescence will
be addressed in future studies.
The results presented in this paper clearly show that NIT4 enzymes from
A. thaliana and N. tabacum are Ala(CN)
hydratases/nitrilases. NIT4 orthologs are known from several
different species of quite different taxonomical position, and it is
likely that NIT4 homologs may be present in all higher
plants. NIT4 is proposed to take part in cyanide detoxification
in vivo in cooperation with Ala(CN) synthase. We propose to
reserve the gene name NIT4 for Ala(CN) hydratases/nitrilases. Nitrilases from other plants belonging to the
NIT4 family should therefore also be called NIT4 independent of the
total number of NIT genes present, as previously done for B. campestris (15), tobacco (8), and rice.
-cyano-L-alanine (Ala(CN)), an intermediate product of
cyanide detoxification in higher plants. Interestingly, not only
aspartic acid but also asparagine were identified as products of
NIT4-catalyzed Ala(CN) hydrolysis. Asn itself was no substrate for
NIT4, indicating that it is not an intermediate but one of two reaction
products. NIT4 therefore has both nitrilase and nitrile hydratase
activity. Several lines of evidence indicate that the catalytic center
for both reactions is the same. The NIT4 homologs of N. tabacum were found to catalyze the same reactions and protein
extracts of A. thaliana, N. tabacum and
Lupinus angustifolius also converted Ala(CN) to Asp and Asn
in vitro. NIT4 may play a role in cyanide detoxification
during ethylene biosynthesis because extracts from senescent leaves of
A. thaliana showed higher Ala(CN) hydratase/nitrilase activities than extracts from nonsenescent tissue.
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-cyano-L-alanine (Ala(CN)), which is enzymatically
produced by cyanoalanine synthase from cyanide and cysteine as
substrates (9). Depending on the plant species observed, Ala(CN) is
subsequently converted to asparagine or to the dipeptide
-Glu-(Ala(CN)). An enzyme converting Ala(CN) to Asp (cyanoalanine
hydratase, EC 4.2.1.65) was characterized biochemically from lupine
(Lupinus angustifolius) (10, 11), but the gene encoding this
enzyme has not yet been cloned.
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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2 s
1 for a 16-h
photoperiod. Seeds of L. angustifolius were sown on Vermiculite and grown in a growth chamber under the following climatic
conditions: 16-h photoperiod, 120 µmol photons
m
2 s
1, 24 °C
during photoperiod, 20 °C during night, 70% relative humidity.
1 lysozyme). Lysis was carried out on ice
for 30 min and was completed with six bursts of ultrasound (1 min, 40 watts) using an ultrasound tip (Sonifier B-17, Branson). The
10,000 × g supernatant (10 min, 4 °C), containing
soluble nitrilase protein, was used for
(NH4)2SO4 precipitation (40%
saturation), and the precipitate was resuspended in 12 ml of lysis
buffer omitting lysozyme. This fraction was used for purification of
the hexahistidine-tagged nitrilases using a 0.5-ml column of
Ni2+-nitrilotriacetic acid-agarose (Qiagen, Hilden,
Germany). Nitrilase bound to the column was eluted with 50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 250 mM imidazole. One ml of the peak fraction was collected and
desalted using a NAP-10 column (Amersham Pharmacia Biotech) which was
equilibrated in 50 mM potassium phosphate, pH 8.0, 1 mM DTT. The resulting nitrilase fraction was purified at
least to 95% homogeneity as judged by Coomassie Blue-stained SDS gels.
Protein concentration varied between 80 and 120 µg
ml
1, and total volume was 1.5 ml. The protein
was shock-frozen in liquid nitrogen and stored at
80 °C for a
maximum duration of 8 weeks.
1. The identification of eluting
Ala(CN), aspartic acid, and asparagine was achieved by ESI-MS on a
Q-TOF2 (Micromass, Manchester, UK) operated in positive ion mode with
the following settings: capillary, 3000 V; cone voltage, 16 V;
collision energy, 5 eV; collision gas off; MS profile, 113, 113, 113. Expected m/z values were 115 [Ala(CN) + H]+,
133 [Asn + H]+, and 134 [Asp + H]+.
Quantification was done using external standards and was assisted by
the Masslynx software (version 3.4, Micromass, Manchester, UK). To
obtain collision-induced decomposition spectra of the amino acids, the
automatic MS/MS switching option of the Masslynx software was used
with collision gas on and collision energy raised to 15 eV in the MS/MS
mode. Signals for the spectra were recorded from m/z values
of 30-140.
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ABSTRACT
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Fig. 1.
Homology tree of nitrilase proteins from
plants. Amino acid sequences of nitrilases from A. thaliana (A.t.), B. campestris
(B.c.), L. japonicus (L.j.), N. tabacum (N.t.), and O. sativa
(O.s.) were aligned using the optimal alignment method of
the DNAMAN software (Lynnon BioSoft, Vaudreuil, Canada) with default
parameters. To use the only partially known sequences of B. campestris, all sequences were truncated to 148 amino acids. The
numbers at the branches indicate the percentage of
homology.
-cyano-L-alanine (Ala(CN)) (Table
I). The activity of NIT4 against
3-phenylpropionitrile (PPN) or allylcyanide, which are the best
substrates for NIT1-NIT3 (4), was very low, and indole-3-acetonitrile (IAN), a precursor of the plant hormone indole-3-acetic acid (IAA), was
not detectably converted by NIT4. This is in agreement with in
planta data of Schmidt et al. (7) and Dohmoto
et al. (17) who were unable to elicit an auxin response with
IAN in wild type tobacco (which expresses at least two NIT4 homologs)
as well as in NIT4-overexpressing tobacco (17). Transgenic tobacco
plants expressing either NIT2 (7), NIT1
(17),2 or NIT3 (17) of
A. thaliana converted this substrate to IAA and developed
strong phenotypic symptoms of auxin overproduction.
Substrate specificity of NIT4 of A. thaliana
1. Activity not
detectable (in alphabetical order): 2-aminobenzonitrile,
benzonitrile, 2-chloroacetamide, cyanoacetamide,
-cyanocinnamic
acid, 1-cyano-1-cyclopropane carboxylic acid, 6-cyanopurine,
2-cyanopyridine, 3-cyanopyridine, 4-cyanopyridine,
cyclopropanecarbonitrile, glutamine, 4-hydroxybenzonitrile,
3-hydroxypropionitrile, indole-3-acetonitrile, indole-3-carbonitrile,
mandelonitrile, naphthalene-1-carbonitrile, (phenylthio)acetonitrile,
and propionitrile.
1) cannot account for the observed
levels of Asp that is formed at a rate of ~500 nkat (mg
protein)
1. Furthermore, asparaginase activity
was not observed in every preparation and results therefore most likely
from small contaminations of the preparations with E. coli
asparaginase. In addition, time course studies showed that Asp and Asn
are formed at the same time with a constant ratio with no detectable
turnover of Asn (Fig. 3). If Asn would
indeed be an intermediate of the reaction, we would expect it to
accumulate before Asp synthesis starts, whereas with the onset of Asp
formation its level should decrease. Taken together, these results
prove that NIT4 converts Ala(CN) to either Asp or Asn. Asn is no
substrate of the enzyme, and thus, Asn is no free intermediate of the
nitrilase reaction of this bifunctional enzyme.
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Fig. 2.
Hydrolysis of Ala(CN) by different nitrilases
and identification of the reaction products by mass spectrometry.
Aliquots of 400 ng of purified protein (NIT4, TNIT4A, TNIT4B, and
NIT4C197A) or 100 µg of a crude extract of E. coli were
incubated with 3 mM Ala(CN) for 20 min at 30 °C.
A, the reaction products were subsequently separated by high
pressure liquid chromatography followed by ESI-MS detection. The
retention times of the amino acids Ala(CN), Asp, and Asn are indicated.
B, collision-induced decomposition spectra were derived from
a 2nd aliquot of the NIT4 sample shown in A. Mass-to-charge
ratios (m/z) and structures of the characteristic fragments
for Asp and Asn are indicated.
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Fig. 3.
Time course of Ala(CN) hydrolysis by NIT4 of
A. thaliana. Arabidopsis NIT4 (400 ng)
was incubated with 3 mM Ala(CN) at 30 °C for 1 h.
For determination of the amino acid concentrations, aliquots of 100 µl were withdrawn and subjected to LC-ESI-MS as described under
"Experimental Procedures." The data shown are representative of
three separate experiments.
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Fig. 4.
Determination of Km
and Vmax of Arabidopsis
NIT4 for Ala(CN). A, Arabidopsis NIT4 (400 ng) was incubated at different concentrations of Ala(CN) for 20 min at
30 °C. Subsequently, nitrilase (circles) and NHase
activities (squares) were determined by the colorimetric
assay as described under "Experimental Procedures." The data shown
are means ± S.D. from four experiments. B shows the
Michaelis-Menten transformation of data from a separate experiment
using lower concentrations of Ala(CN).
Parameters of nitrilase and NHase activity of NIT4 from A. thaliana
-Cyanoalanine Hydrolysis Is a Common Feature of NIT4
Proteins--
As mentioned above, proteins homologous to NIT4 are also
known from tobacco (8) and rice. The cDNAs of the tobacco
nitrilases TNIT4A and TNIT4B were kindly provided to us by Dr. Kazuo
Yamaguchi (Institute for Gene Research, Kanazawa University, Kanazawa,
Japan). By using the same cloning strategy as used for A. thaliana NIT4, both cDNAs could be expressed in E. coli, and the proteins were purified using
Ni2+-chelate affinity chromatography (data not shown). Both
enzymes hydrolyzed Ala(CN) to Asp and Asn (Fig. 2) showing, however, a lower ratio of NHase/nitrilase activity (~1) (Table
III) compared with the
Arabidopsis enzyme, thus producing relatively more Asp. Interestingly, PPN was a better substrate for the tobacco enzymes than
for Arabidopsis NIT4, the ratio of Ala(CN) to PPN
consumption was ~5-6 times higher with the tobacco enzymes (Table
III). As already mentioned, A. thaliana has a high content
of glucosinolates, and glucosinolate-derived nitriles are substrates
for the Arabidopsis isoenzymes NIT1, NIT2, and NIT3. It is
therefore possible that Arabidopsis NIT4 lost PPN hydrolase
activity during evolution to avoid uncontrolled hydrolysis of
glucosinolate-derived nitriles.
Comparison of Arabidopsis NIT4 with tobacco TNIT4A and TNIT4B
-Cyanoalanine Hydrolysis in Plant Extracts--
In 1972, Castric et al. (10) reported the characterization of a
-cyanoalanine hydratase enriched from blue lupine seedlings (L. angustifolius), which formed Asn from Ala(CN). It is not
entirely clear from this paper if the authors also looked for Asp
production from Ala(CN) by their enzyme. We prepared extracts from
leaves of A. thaliana and tobacco and from seedlings of blue
lupine. Enzymatic activity was enriched from the crude extracts by
(NH4)2SO4 precipitation (40%
saturation). All extracts showed Ala(CN) NHase as well as nitrilase
activity (Fig. 5). Under the conditions
tested, no asparaginase activity could be detected, indicating that the observed Asp is formed by a nitrilase reaction. The tobacco extract produced Asp and Asn from Ala(CN) in similar amounts, whereas the
extract from blue lupine showed an ~2 times higher NHase activity. This activity (100 pkat (mg protein)
1) was
comparable to the activity described earlier (140 pkat (mg protein)
1 of a 0-30% (saturation)
(NH4)2SO4 extract) (10). We also
detected Ala(CN) hydratase/nitrilase activities ranging from 50 to 400 pkat (mg protein)
1 in extracts of
Bryonia dioica (Cucurbitaceae), Lactuca sativa (Asteraceae), and Lycopersicon esculentum (Solanaceae),
displaying NHase/nitrilase ratios from 1 to 1.8 (data not shown).
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Fig. 5.
NIT4 activity in extracts of different
plants. Protein was extracted from 1 g of tissue (L. angustifolius, 5-day-old light-grown seedlings; N. tabacum, the oldest leaf of a 3-month-old plant; A. thaliana, nonsenescent (ns) rosette leaves of
nonbolting plants or senescent (s) rosette leaves of
flowering plants) and enzymatic activity enriched by
(NH4)2SO4 precipitation (40%
saturation). 50-100 µg of protein were incubated with 3 mM Ala(CN) at 37 °C for 3 h. Nitrilase and NHase
activity were determined by LC-ESI-MS as described under
"Experimental Procedures." The data shown are means ± S.D.
from three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyano-L-alanine (Ala(CN)) (Table I), which is also
effectively converted by the NIT4 homologs of tobacco (Fig. 2) but is
no substrate for Arabidopsis NIT1-NIT3 (4). In contrast to
the report by Bartel and Fink (3), we observed no hydrolysis of IAN to
IAA by Arabidopsis NIT4. Because we used a relatively low
amount of purified enzyme (a maximum of 2400 ng), the minimal activity
observable under the conditions applied is about 150 pkat (mg
protein)
1, which may be not sensitive enough
to detect very weak IAN hydrolysis. The detection limit, however,
represents 0.02% of the activity measured with Ala(CN) as substrate.
-glutamyl-cyanoalanine
(
-Glu-Ala(CN)). The enzyme(s) catalyzing the formation of Asn from
Ala(CN) (cyanoalanine hydratase = Ala(CN) NHase) were
biochemically studied from lupine seedlings in the laboratory of E. Conn in the early 70s (10) and later by Galoyan et al. (11),
but until now, genes encoding such enzymes have not been cloned, and
characterizations of corresponding enzymes from other plant species are
lacking. Is Conn's cyanoalanine hydratase and NIT4 the same enzyme? In
several feeding experiments using H14CN, radioactive label
could be detected in Asn but Asp was not significantly labeled (10, 26)
arguing against a NIT4-catalyzed reaction in which similar amounts of
Asp and Asn would be expected. We therefore tested the ability of plant
extracts from A. thaliana, tobacco, tomato, L. sativa, B. dioica, and blue lupine to hydrolyze Ala(CN)
and could unequivocally detect Ala(CN) NHase as well as Ala(CN)
nitrilase activity in all tested extracts. In extracts of blue lupine
the Ala(CN) NHase activity was dominant; nevertheless, nitrilase
activity was clearly detectable. It is therefore likely that the
Ala(CN) NHase from blue lupine is a NIT4 homolog. This topic is
currently under investigation in our laboratory. Our in
vitro data are, however, in contrast to the data obtained in vivo from the H14CN labeling experiments mentioned
above. A possible explanation could be that the turnover rate of Asp is
higher than that of Asn in vivo, as observed in cotton roots
(26). In this case Asp would not accumulate.
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ACKNOWLEDGEMENT |
---|
We are grateful to Dr. Kazuo Yamaguchi for kindly providing the TNIT4A and TNIT4B cDNAs.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany, and by Fonds der Chemischen Industrie, Frankfurt, Germany (literature provision).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Plant
Physiology, Ruhr-Universität, Universitätsstr. 150, D-44801
Bochum, Germany. Tel.: 49-234-3224290; Fax: 49-234-3214187;
E-mail: Markus.Piotrowski@ruhr-uni-bochum.de.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M007890200
2 R.-C. Schmidt, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
IAN, indole-3-acetonitrile;
Ala(CN), -cyano-L-alanine;
IAA, indole-3-acetic acid;
LC-ESI-MS, liquid chromatography coupled to
electrospray-ionization mass spectrometry;
NHase, nitrile hydratase;
PDFOA, pentadecafluorooctanoic acid;
PPN, 3-phenylpropionitrile;
DTT, dithiothreitol.
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REFERENCES |
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