| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 32, 29600-29608, August 8, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||
From the
Institute of Applied Biochemistry and
¶Institute of Materials Science, The University
of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
Received for publication, November 20, 2002 , and in revised form, April 30, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
N)
(1–5)
and isonitriles (R-NC)
(6,
7). The microbial degradation
of nitriles proceeds through two enzymatic pathways
(8–10):
(i) nitrilase catalyzes the direct cleavage of nitriles into acids
(R-C(=O)-OH) and ammonia
(11–13),
and (ii) nitrile hydratase
(NHase)1 catalyzes the
hydration of nitriles to amides (R-C(=O)-NH2)
(14–17),
which are subsequently hydrolyzed to acids and ammonia by amidase
(18–20).
These enzymes have received much attention in applied fields
(2,
8,
21) as well as academic ones
(13,
22–26).
One of the fruits of our application-oriented nitrile studies is the current
industrial production of acrylamide and nicotinamide involving NHase of an
actinomycete, Rhodococcus rhodochrous J1
(1,
9). On the other hand, NHase of
Pseudomonas chlororaphis B23
(27), which was previously
used as a catalyst for acrylamide manufacture
(9,
21), is now used for the
production of 5-cyanovaleramide, a herbicide intermediate, at the industrial
level (28). Because the amount
of NHase produced comprises more than 50% of all soluble protein in P.
chlororaphis B23 when this strain is cultured in the presence of
methacrylamide (29), there
must be a very interesting regulation mechanism for the enzyme expression
(8). We have already cloned the
NHase gene from this strain, and discovered the existence of a gene cluster
containing the NHase and amidase genes
(30). Although we have
clarified its nitrile degradation mechanism at the protein and gene levels, an
enzyme and its gene involved in the nitrile-synthetic pathway (which might be
present in this strain) have never been identified.
In the biosynthesis of nitriles, aldoxime (R-CH=N-OH) is one of the candidate intermediates (31). Indoleacetaldoxime, which is derived from tryptophan, is dehydrated to indoleacetonitrile in some higher plants. Aldoximes are also converted to cyanogenic glycosides and glucosinolates (31–34) (e.g. phenylpropionaldoxime to phenylethyl glucosinolate in Nasturtium officinale) (35). Other than these aldoximes, some naturally occurring aldoximes have so far been found in microorganisms; e.g. 2-(4-hydroxyphenyl)-2-oxoacetaldehyde oxime (which was discovered as a phosphorylated cholinesterase reactivator) in a fungus, Penicillium olsonii (36). However, there have only been two reports on purified enzymes (involved in nitrile synthesis from aldoxime) whose genes have been cloned but which belong to different superfamilies: (i) CYP71E1, a cytochrome P450 (which catalyzes the conversion of p-hydroxyphenylacetaldoxime to p-hydroxyphenylacetonitrile) in dhurrin biosynthesis of sorghum (37) and (ii) phenylacetaldoxime dehydratase (which catalyzes the dehydration of phenylacetaldoxime to phenylacetonitrile) linked with nitrilase in nitrile metabolism of Bacillus sp. strain OxB-1 (38). In particular, regarding the phenylacetaldoxime dehydratase, only one paper (38) on analyses at the protein and gene levels has been published. Although the Bacillus enzyme has been found to bind loosely to heme and requires FMN for its enzymatic activity, the function of such cofactors remains unknown. Biochemical and genetic information on the enzyme remain limited. This enzyme is also expected to be applicable to the production of nitriles from the corresponding aldoximes, because the chemical dehydration of aldoximes usually requires harsh reaction conditions (39).
In the present study, we discovered a gene (oxdA) of a novel nitrile-synthesizing enzyme linked with NHase in P. chlororaphis B23. We constructed an Escherichia coli transformant overexpressing OxdA, and we purified OxdA and determined its enzymological, physicochemical, and spectroscopic properties. We also report that aldoxime dehydratase is a unique enzyme involved in the metabolism of aldoxime in vivo in P. chlororaphis B23.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Plasmids, Strains, and Media—Plasmid pPCN4 carrying the 6.5-kb SphI-SalI fragment on pUC19 (30) was used for sequencing of the upstream region of the amidase gene in P. chlororaphis B23. E. coli DH10B (Invitrogen) was used as the host for pUC plasmids (40). E. coli BL21-Codonplus(DE3)-RIL (Novagen, Madison, WI) was used as the host for one plasmid, pET-24a(+) (Novagen), and its derivative and was also used for expression of the aldoxime dehydratase gene (oxdA). E. coli transformants were grown in 2x YT medium (41). The culture medium for P. chlororaphis B23 was composed of 1% sucrose, 0.5% (NH4)2SO4, 0.05% KH2PO4, 0.05% K2HPO4, 0.05% MgSO4·7H2O, and 0.001% of FeSO4·7H2O (pH 7.0). Unless otherwise stated, sucrose and (NH4)2SO4 were used as carbon and nitrogen sources, respectively.
DNA Manipulations—Restriction endonucleases, DNA polymerase, and T4 DNA ligase were purchased from Toyobo Co., Ltd. (Osaka, Japan). Nucleotides were sequenced by the dideoxy chain-terminating method using an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA). Unless otherwise stated, DNA manipulations were performed essentially as described by Maniatis et al. (41).
Expression and Purification of Recombinant Aldoxime Dehydratase—The coding sequence of the enzyme was amplified by PCR with pPCN4 (30) as a template. The following two oligonucleotide primers were used: sense primer (5'-CATATGGAATCTGCGATCGACACGC-3') containing an NdeI recognition site (underlined) and 22 nucleotides of oxdA starting with the ATG start codon; and antisense primer (5'-ACGCGTCGACTCAGGTGGGCGCGACAACGGC-3') containing an SalI recognition site (underlined) and 21 nucleotides that are complementary to the 3'-end sequence of oxdA ending with the TGA stop codon. The amplified DNA was subcloned into vector pUC18 and checked by DNA sequencing. The insert DNA was digested with NdeI and SalI and then inserted into the respective sites of pET-24a(+). The resultant plasmid was designated as pET-oxdA; in this construction, oxdA was under the control of the T7 promoter. E. coli BL21-CodonPlus(DE3)-RIL was transformed with pET-oxdA, and the recombinant cells were used for the overproduction and purification of aldoxime dehydratase.
The transformed cells were incubated with reciprocal shaking at 37 °C
in 240 ml of 2x YT medium containing 50 µg/ml kanamycin and 34
µg/ml chloramphenicol. After overnight cultivation, the entire culture was
inoculated into 48 liters of the same medium, followed by incubation with
shaking at 37 °C for 2 h.
Isopropyl-1-thio-
-D-galactopyranoside was then added to a
final concentration of 0.1 mM to induce the T7 promoter, and
further cultivation was carried out at 15 °C for 7 days.
All purification procedures were performed at 0–4 °C. Potassium phosphate buffer (pH 7.0) containing 5 mM 2-mercaptoethanol was used throughout the purification. Centrifugation was carried out for 30 min at 15,000 x g.
The cells were harvested by centrifugation, washed twice with 100 mM buffer containing 1 mM dithiothreitol, and then disrupted by sonication (Insonator model 201M; Kubota, Tokyo, Japan) to prepare a cell-free extract. Cell debris was removed by centrifugation. The resulting supernatant was fractionated with ammonium sulfate (40–60% saturation), followed by dialysis against 10 mM buffer. The dialyzed solution was applied to a DEAE-Sephacel column (5 x 25 cm) equilibrated with 10 mM buffer. Protein was eluted from the column with 1.2 liters of 10 mM buffer, the concentration of KCl being increased linearly from 0.1 to 0.4 M. The active fractions were collected, and then ammonium sulfate was added to give 70% saturation. After centrifugation of the suspension, the precipitate was dissolved in 0.1 M buffer and brought to 20% ammonium sulfate saturation. The enzyme solution was placed on a TSK gel Butyl-Toyopearl 650M column (5 x 22 cm) equilibrated with 10 mM buffer 20% saturated with ammonium sulfate. The enzyme was eluted by lowering the concentration of ammonium sulfate (from 20 to 5%) in 1.2 liters of the same buffer. The active fractions were combined and precipitated with ammonium sulfate at 70% saturation. The precipitate was collected by centrifugation, dissolved in 0.1 M buffer, and then dialyzed against 10 mM buffer containing 0.5% ammonium sulfate. The enzyme solution was put on a phenyl-Sepharose column (5 x 22 cm) that had been equilibrated with the same buffer. After the column had been washed fully with 10 mM buffer containing 0.5% ammonium sulfate, the enzyme was eluted with 10 mM buffer. The active fractions were combined and pooled. Solid ammonium sulfate was added to the enzyme solution to give 70% saturation. After centrifugation of the suspension, the precipitate was dissolved in 0.1 M buffer, followed by dialysis against three changes of 2 liters of 1 mM buffer (pH 6.8). After centrifugation, the enzyme solution was loaded on a Cellulofine HAp column (5 x 22 cm) equilibrated with 1 mM buffer (pH 6.8). The column was eluted with a linear gradient, 1–100 mM, of the buffer (pH 6.8). The enzyme solution was refractionated with ammonium sulfate (50–60% saturation). The precipitate was collected by centrifugation and dissolved in 0.1 M buffer. The resultant solution was dialyzed against 10 mM buffer and then centrifuged. The homogeneity of the purified protein was confirmed by SDS-PAGE.
Enzyme Assays—All of the reactions were performed under
linear conditions as to protein (
2.5 µg/ml) and time (5 min). The
standard assay A mixture comprised 100 mM potassium phosphate
buffer (pH 7.0), 5 mM butyraldoxime, and an appropriate amount of
enzyme, in a total volume of 200 µl. The reaction was started by the
addition of butyraldoxime and carried out for 5 min at 30 °C under aerobic
conditions. The reaction was stopped by the addition of 100 µl of 1
M KOH, and a supernatant was obtained by centrifugation (15,000
x g, 5 min). The reaction product was determined with a gas
chromatograph (GC-14BPF; Shimadzu, Kyoto, Japan) equipped with a flame
ionization detector and a glass column (3.2 mm x 2.1 m) packed with
Gaskuropack 56 (80/100% mesh; GL-Science, Tokyo, Japan). Standard assay A was
performed throughout the purification procedures. After the purification, the
enzyme assaying in all of the following experiments was performed by means of
standard assay B (anaerobically under reduced conditions), unless otherwise
stated. In the case of standard assay B,
Na2S2O4 (final, 5 mM) was added to
the buffer. One unit of aldoxime dehydratase activity was defined as the
amount of enzyme that catalyzed the formation of 1 µmol of
butyronitrile/min from butyraldoxime under the standard assay A and B
conditions, respectively. Specific activity is expressed as units/mg of
protein.
Determination of the Prosthetic Group—Heme staining was
carried out as described by Klatt et al.
(42). Heme was identified, and
its stoichiometry was estimated by conversion to the pyridine hemochrome
(43). This procedure consisted
of adding pyridine (final, 20%) and then KOH (final, 50 mM) and a
small amount of Na2S2O4 to the enzyme. After
15 min, the spectrum of the pyridine hemochrome was recorded. The hemochrome
was determined using the published molar absorption coefficient (
) for
the hemochrome of protoheme IX, 34.4
mM–1 cm–1,
at 557 nm (43). Acid-acetone
heme extraction was carried out
(44). The enzyme was added
with rapid stirring to 9 volumes of 15 mM HCl in acetone at 0
°C, followed by incubation for 10 min at 0 °C in the dark. After a
10-min centrifugation at 17,000 x g, the supernatant was
pooled, and the same operation was repeated to prepare a pyridine
hemochrome.
Molecular Mass Determination—The purified enzyme sample was applied to a Superose 12 HR10/30 column (Amersham Biosciences), which was attached to an ÄKTA purifier (Amersham Biosciences), and then eluted with 100 mM buffer containing 0.15 M KCl at the flow rate of 0.5 ml/min. For the enzyme in the ferrous state after reduction with Na2S2O4, on the other hand, 100 mM buffer containing 0.15 M KCl and 10 mM Na2S2O4 was used. The absorbance of the effluent was recorded at 280 and 422 nm. The molecular mass of the enzyme was calculated from the mobilities of the standard proteins, glutamate dehydrogenase (290 kDa), lactate dehydrogenase (142 kDa), enolase (67 kDa), adenylate kinase (32 kDa), and cytochrome c (12.4 kDa).
Metal Analysis—All glassware was soaked in 2 M HCl overnight and then exhaustively rinsed with distilled water before use. Prior to analysis, the enzyme was dialyzed against 1 mM potassium phosphate buffer (pH 7.0). The dialysis had no effect on the enzyme activity. The enzyme sample containing 0.94 mg of protein/ml was analyzed with an inductively coupled radiofrequency plasma spectrophotometer, Shimadzu ICPS-8000 (27.120 MHz). The metal contents of the enzyme sample were determined from the calibration curves for standard solutions.
Immunoblot Analysis—A female New Zealand White rabbit was immunized four times at 2-week intervals. The first and second injections were performed intradermally with the purified OxdA (500 µg) in Freund's complete adjuvant (Difco). The booster injection was of 500 µg of antigen homogenized in an equal volume of incomplete adjuvant (Difco).
Western blotting analysis was carried out as described below. Proteins were separated on SDS-polyacrylamide gels under reducing conditions and then electroblotted onto a polyvinylidene difluoride membrane (Sequi-BlotTM; Bio-Rad). After blocking with 1% skim milk, the blots were incubated with rabbit antisera directed against OxdA at room temperature for 2 h and then incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h. The immunoreactive proteins were detected with an ECL Western blotting detection kit (Amersham Biosciences).
Analytical Methods—The absorption spectra were recorded with
a Shimadzu UV-1700 PharmaSpec spectrophotometer. The protein concentrations
were determined with a Nakalai Tesque Co., Inc. (Kyoto, Japan) protein assay
kit with bovine serum albumin as the standard. Native PAGE and SDS-PAGE were
carried out as described by Davis
(45) and Laemmli
(46), respectively. For
SDS-PAGE, the following marker proteins were used to determine the relative
molecular mass of the enzyme: phosphorylase b (94 kDa), bovine serum
albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean
trypsin inhibitor (20.1 kDa), and 
-lactalbumin (14.4 kDa).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
and of NHase, the
presumed 47-kDa protein (P47K), and OrfE. The nucleotide sequence of the ORF
consists of 1,056 nucleotides (Fig.
1A), encoding 352 amino acid residues with a calculated
molecular mass of 40,127 Da. A possible ribosome-binding site (GAGGA) is
located 8 nucleotides upstream from the ATG. A search with the BLAST program
revealed that the deduced amino acid sequence of the ORF exhibits low
similarity (32% identity) with that of the phenylacetaldoxime dehydratase gene
(oxd) of Bacillus sp. OxB-1
(38)
(Fig. 1B). These genes
have been found to comprise a superfamily, our enzyme being the second member
of this superfamily.
|
An expression plasmid (pET-oxdA) for the ORF was constructed and introduced into E. coli BL21-CodonPlus(DE3)RIL. The recombinant protein was obtained as an insoluble form almost completely when the E. coli transformant was cultured at 37 °C. To increase the amount of the soluble enzyme form, various culture conditions were examined. Under the optimum conditions for 7 days at 15 °C, we succeeded in expressing the recombinant protein at a level corresponding to about 10% of the total amount of soluble protein (data not shown). Cell-free extracts prepared from the E. coli transformant carrying the ORF dehydrated butyraldoxime as a substrate, yielding butyronitrile. This finding demonstrated that this ORF encodes aldoxime dehydratase. Thus, the gene was designated as oxdA.
OxdA was purified through the purification steps described under
"Experimental Procedures." The purity of OxdA was confirmed by
migration of the protein as a single band corresponding to a molecular mass of
38 kDa on SDS-PAGE (Fig.
2). The Mr of the native enzyme was 76.4 kDa
according to the results of gel filtration chromatography, indicating that
OxdA consists of two identical subunits. The purified OxdA showed specific
activity of 0.758 units/mg (under aerobic conditions)
(Table I).
|
|
Cofactor of Aldoxime Dehydratase—OxdA was brownish red in
solution. The existence of heme was expected in OxdA. Thus, heme staining was
carried out after native PAGE for our aldoxime dehydratase. The gel was
stained with 3,3'-dimethoxybenzidine/H2O2
(42) and Coomassie Brilliant
Blue for heme and protein, respectively. Both methods gave a single band, and
the positions of the bands were the same (data not shown). Besides, the
absorption spectrum of this enzyme had a specific peak at 415 nm
(Fig. 3A). Thus, OxdA
was identified as a hemoprotein. To examine the properties of the heme in the
enzyme, pyridine hemochrome was prepared.
Fig. 3C shows
absorption peaks at 418.5 (Soret), 524 (-band), and 556 (-band)
nm. Furthermore, the heme prosthetic group was extracted from the enzyme on
HCl/acetone-treatment (44).
The spectrum of the pyridine hemochrome in the extract was identical with that
of the pyridine hemochrome of the enzyme (data not shown). The extractability
of the chromophore with acid-acetone and the shape of its pyridine hemochrome
spectrum confirmed that the aldoxime dehydratase carries protoheme IX as the
prosthetic group. The heme content of the enzyme was calculated to be 0.69
mol/mol of subunit, as determined from the value for at 556 nm in the
spectrum of its pyridine hemochrome. Quantitative analysis of iron in OxdA
with an inductively coupled radiofrequency plasma spectrophotometer revealed
that OxdA (as a homodimer) contains 1.62 mol of iron/mol. On the basis of
these results, it is concluded that OxdA contains iron and heme in a ratio of
1:1 and that all of the iron would be present in the heme molecule. The heme
content of the preparation is not equimolar as to the protein, suggesting that
a little of the heme in OxdA is lost during the purification procedure. It has
been reported that the Bacillus phenylacetaldoxime dehydratase loses
heme during its purification and that the purified enzyme contains only 0.35
mol of heme/mol of enzyme
(38).
|
Qualitative and quantitative analyses of the following metals in the purified OxdA solution were also performed: beryllium, boron, magnesium, aluminum, silicon, phosphorus, sulfur, calcium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, selenium, strontium, zirconium, molybdenum, palladium, silver, cadmium, tin, antimony, barium, tantalum, tungsten, platinum, gold, mercury, lead, lanthanum, and cerium. Besides iron, OxdA (as a homodimer) contained 1.58 mol of calcium/mol. On the other hand, none of the other 33 metals was detected within the limits of the assay (10 ng/ml).
Effect of the Redox Environment on the Enzyme Activity—In order to confirm the state of the iron in the heme molecule, redox spectra of the purified OxdA were examined. Under aerobic conditions, OxdA showed a strong absorption peak at 415 nm and weak absorption in a broad region (i.e. 520–580 nm) (Fig. 3A). Under the same conditions, the specific activity of the purified OxdA was not so high (0.758 units/mg; Table II), although it was not inactive. The addition of Na2S2O4 to OxdA caused an increase and shift from 415 to 428 nm of the absorption peak under anaerobic conditions (Fig. 3A). The activity increased by more than 250 times (namely from 0.758 to 197 units/mg) in the anaerobic reduced environment (Table II). Upon the subsequent addition of CO to OxdA reduced by the addition of Na2S2O4, a further increase and shift of the absorption peak from 428 to 419 nm were observed (Fig. 3A), suggesting that this peak is due to an enzyme-CO complex. The addition of an exogenous ligand of the ferrous state of the heme, CO, caused 49% inhibition of the activity (namely from 197 to 101 units/mg) (Table II); but the substrate was dehydrated even in the presence of CO. These findings suggest that CO, which would have bound beforehand at the ferrous heme of the enzyme, was replaced by butyraldoxime during the enzyme reaction. On the other hand, the absorption spectrum of OxdA was unchanged with CO under aerobic conditions (data not shown). Upon the addition of K3[Fe(CN)6] (which is an oxidizing agent), OxdA showed a shift of the absorption peak from 415 to 412 nm under aerobic conditions, whereas it showed a shift of the absorption peak from 415 to 419 nm upon the addition of 10 mM KCN under aerobic conditions (Fig. 3B). Under these conditions, enzyme activities have never been detected (Table II). These spectral features indicate that the heme iron of the purified OxdA is in the ferric state but that the enzyme functions in the ferrous state. In all of the following experiments described below, therefore, all procedures were performed after reduction by the addition of Na2S2O4; standard assay B described under "Experimental Procedures" was used as the enzyme assay unless otherwise stated.
|
For determination of the molecular mass of the ferrous enzyme, gel filtration chromatography was carried out again under reduced conditions. The reduced OxdA sample prepared by the addition of Na2S2O4 was applied to a Superose 12 HR10/30 column and then eluted with 100 mM buffer containing 0.15 M KCl and 10 mM Na2S2O4. The absorbance of the effluent was recorded at 422 nm. A single peak corresponding to 76.2 kDa was detected (data not shown). Also, the spectrum of the pooled fraction was confirmed to be identical to that of the ferrous state. These findings, together with the finding that OxdA is most active under reduced conditions, indicate that the native OxdA in the ferrous state is also a homodimer.
Stoichiometry—The stoichiometry of aldoxime consumption and nitrile formation during the dehydration of aldoxime was examined in a reaction mixture consisting of 100 mM potassium phosphate buffer (pH 7.0), 5 mM butyraldoxime, 5 mM Na2S2O4, and 31.25 nM enzyme, in a final volume of 400 µl. The reaction was carried out at 30 °C under anaerobic conditions. After a 5-min incubation, the amounts of residual butyraldoxime and butyronitrile were determined. The butyronitrile formed and the butyraldoxime remaining amounted to 1.93 and 3.12 mM, respectively. No formation of other compounds was observed. The results indicated that butyronitrile was formed stoichiometrically with the consumption of butyraldoxime.
Substrate Specificity and Kinetic Properties—The ability of OxdA to catalyze the dehydration of various aldoximes was examined. Among the tested aldoximes, only three were active as substrates for the aldoxime dehydratase, whereas aromatic aldoximes such as benzaldoxime and pyridine-4-aldoxime were inert. The kcat value for pyridine-4-aldoxime (0.090 min–1) was much lower than those for butyraldoxime and acetaldoxime (5.4 and 5.6 min–1, respectively), suggesting that OxdA prefers aliphatic aldoximes to aromatic ones as substrates (Table III). Because of their commercial unavailability, not only arylalkyland alkyl-aldoximes (e.g. phenylacetaldoxime), but also pure E-and Z-stereoisomers of aldoximes, which are not listed in Table III, could not be examined. Butyraldoxime comprised a 60:40 mixture of E/Z-stereoisomers (commercial communication). The complete consumption of E/Z-butyraldoxime in the enzyme reaction suggests that OxdA accepts both the E- and Z-forms of butyraldoxime as substrates. The specific activity of OxdA for butyraldoxime (197 µmol/min/mg; Table II) is much higher than that of the Bacillus phenylacetaldoxime dehydratase for this substrate (0.93 µmol/min/mg), although the assay conditions were not the same. Not only butyronitrile but also all of the other tested nitriles did not act as substrates for OxdA, despite the addition of a large amount of the enzyme and a long incubation period.
|
Effects of Temperature and pH on the Activity and Stability of the Enzyme—The effects of pH and temperature on the enzyme activity were examined. OxdA was active over a broad pH range (pH 5.0–10.0). Maximum activity was observed at pH 5.5; another high activity level was observed at pH 9.4 (Fig. 4A). The reason for such an interesting pH optimum pattern, which has never previously been observed for the Bacillus phenylacetaldoxime dehydratase (38), is unclear. The optimal temperature was 45 °C (Fig. 4B).
|
The stability of OxdA was examined at various temperatures. After the enzyme had been preincubated for 15 min and an aliquot of each enzyme solution was reduced by the addition of Na2S2O4, the enzyme activity was measured anaerobically. The results were as follows: 20 °C, 100%; 25 °C, 94%; 30 °C, 91%; 35 °C, 78%; 40 °C, 51%; 45 °C, 14%; 50 °C, 3.2%.
The stability of OxdA was examined at various pH values. After the enzyme had been incubated at 30 °C for 15 min in a 0.1 M concentration of the buffers citrate/sodium citrate (pH 4.4–6.5), potassium phosphate buffer (pH 6.7–7.9), Tris/HCl buffer (pH 7.9–8.8), and NH4Cl/NH4OH buffer (pH 8.9–10.4), an aliquot of each enzyme solution was reduced by the addition of Na2S2O4, and then the enzyme activity was measured anaerobically. OxdA was most stable in the pH range of 6.0–8.0, 40% of its initial activity being retained even at pH 10.0.
Inhibitors of the Enzyme Activity—Various compounds were
investigated as to their inhibitory effects on the enzyme activity
(Table IV). OxdA was very
sensitive to AgNO3, in a concentration-dependent manner. The
results were as follows: 1 mM, 14.2%; 100 µM, 31.7%;
and 10 µM, 117% of the original activity. Thiol reagents such as
iodoacetate, N-ethylmaleimide, p-chloromercuribenzoate, and
5,5'-dithiobis-2-nitrobenzoate hardly inhibited the enzyme activity. The
addition of hydroxylamine caused concentration-dependent inhibition of the
activity of OxdA (1 mM, 0%; 100 µM, 13.3%; and 10
µM, 29.5%, respectively). Another carbonyl reagent,
phenylhydrazine, also inhibited the enzyme activity, even if the concentration
was lower (1 mM, 28.9%; 100 µM, 20.1%; and 10
µM, 28.7%). OxdA was not sensitive to chelating agents, such as
,'-dipyridyl, o-phenanthroline,
8-hydroxyquinoline, and EDTA, although diethyldithiocarbamate and KCN caused
partial inhibition. Serine-modifying reagents (i.e.
phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate) did not
influence the activity.
|
Spectral Interaction of Aldoximes with Aldoxime Dehydratase—The addition of butyraldoxime (final concentration 50 mM) to OxdA (final concentration 0.23 mg/ml) reduced by Na2S2O4 under anaerobic conditions led to the rapid appearance of a peak at 416 nm; an intense difference spectrum exhibiting a peak at 412.5 nm and a trough around 430 nm has obtained (Fig. 5). Butyronitrile was unable to give the 416-nm peak when added to the Na2S2O4-reduced enzyme (data not shown). These findings suggest that the substrate (aldoxime) really approaches the ferrous heme of the enzyme. As the enzymatic reaction proceeded, the peak at 416 nm disappeared (Fig. 5). After a 10-min reaction, a spectrum close to that at the start of the reaction was observed. Under the same conditions with butyraldoxime as the substrate in the reaction mixture, the aldoxime dehydratase assay showed the complete consumption of butyraldoxime and the concomitant appearance of butyronitrile at 10 min; no substrate remained in the reaction mixture at 10 min. These findings demonstrated that the absorption peak at 416 nm is due to the intermediate in the butyronitrile synthetic reaction. On the other hand, when acetaldoxime was added as the substrate (final, 50 mM) to OxdA (final, 0.23 mg/ml) reduced by Na2S2O4, a difference spectrum similar to that in the case of butyraldoxime as the substrate was observed (data not shown), although there was a slight difference between them. Under these conditions, the addition of acetaldoxime gave a peak at 415 nm; a difference spectrum characterized by a peak at 411 nm and a trough around 430 nm was obtained. After a 30-min reaction, a spectrum close to that at the start of the reaction was observed, and the complete consumption of acetaldoxime and the concomitant appearance of acetonitrile were further confirmed. These results obtained on enzyme assaying and spectral analyses demonstrate that the substrate really approaches the ferrous heme of the enzyme during the enzymatic reaction; namely the heme molecule in OxdA under reduced conditions plays an essential part in the catalytic reaction.
|
Aldoxime Dehydratase Activity in P. chlororaphis B23—To confirm the function of OxdA in vivo, P. chlororaphis B23 was grown in several culture media. When P. chlororaphis B23 was grown in the medium containing 1% sucrose and 0.5% (NH4)2SO4 as carbon and nitrogen sources, respectively, the OxdA activity was not observed. On the other hand, P. chlororaphis B23 grew slowly when butyraldoxime (0.05%) was used as the sole carbon and nitrogen source, although the strain did not grow when butyraldoxime was used at the concentration of 0.2%. These findings suggested that butyraldoxime at a high concentration would be toxic for the strain, but butyraldoxime at a low concentration can be used as carbon and nitrogen sources. Upon replacement of sucrose and (NH4)2SO4 with butyraldoxime, the OxdA activity was detected with 0.561 units/mg under standard assay B. These findings also indicated that OxdA was an inducible enzyme.
Moreover, the formation of OxdA in P. chlororaphis B23 cells was examined by means of immunoblot analysis. Cell-free extracts were prepared from the strain grown with butyraldoxime and then subjected to SDS-PAGE. A prominent band of OxdA, migrating to a position corresponding to a molecular weight of 38,000, was detected with antiserum raised against OxdA. No OxdA was detected when the strain was grown without aldoxime. Immunoblot analysis showed the strong coincidence of the OxdA formation with the OxdA activity in vivo (Fig. 6).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Sequence analysis of the upstream region of the amidase gene in the "industrial microorganism" P. chlororaphis B23 revealed the existence of the aldoxime dehydratase gene exhibiting weak sequence similarity to the Bacillus phenylacetaldoxime dehydratase gene, whose protein was recently characterized biochemically (38), and it was found to be completely different from all of the enzymes reported above not only in sequence but also in enzymological and physicochemical properties.
In order to characterize this aldoxime dehydratase enzyme well, we attempted to express the recombinant OxdA of P. chlororaphis B23 in E. coli. Although the recombinant phenylacetaldoxime dehydratase of Bacillus sp. OxB-1 has almost never been obtained in an active form, because of its production as inclusion bodies (<10 units/liter of culture), we here succeeded in the overproduction of OxdA, which allowed the characterization of this enzyme in various ways after purification.
OxdA of P. chlororaphis B23 has been found to be a hemoprotein comprising protoheme, as judged from the isolation of the pyridine hemochrome and the results of spectroscopic analyses. Although the absorption spectrum of the Bacillus phenylacetaldoxime dehydratase is not affected by the addition of various compounds (i.e. reducing reagents, oxidizing reagents, substrates, products, and common exogenous heme ligands), a variety of spectral shifts were observed on the addition of each of various compounds to OxdA. Spectroscopic analyses and enzyme assaying have revealed that the enzyme reaction proceeds when the heme iron of OxdA is in the ferrous state. In the case of the Bacillus phenylacetaldoxime dehydratase, FMN is necessarily required, although the precise nature of the FMN requirement is unclear (38). As for the cytochrome P450s that act on aldoximes, NADPH is required for reduction of the heme to the ferrous state (47, 48). However, no enhancement of the enzyme activity has been observed on the addition of many kinds of cofactors, including FMN and NADPH, to OxdA, which would be in the ferric state under aerobic conditions. OxdA exhibits much higher activity with the addition of Na2S2O4, which is a strong reducing reagent under anaerobic conditions (Table II). These findings indicate that OxdA may not be easy to reduce. There may be a mechanism that causes the reduction of OxdA to the ferrous state. When the heme iron of OxdA is in the ferrous state under anaerobic conditions, the enzyme has been confirmed to be a dimer. This is the first report that an aldoxime-degrading enzyme acts as a dimer, although it has been reported that the Bacillus phenylacetaldoxime dehydratase is a monomer (38). Metal analysis of OxdA demonstrated that iron and calcium atoms are included in the enzyme. Both metal ions remain even during the purification procedure or dialysis, suggesting that the two metals are tightly associated with the enzyme. Whereas the iron should be derived from the heme, calcium may act as another cofactor. Further studies are required to determine the function of the calcium.
OxdA of P. chlororaphis B23 shows the most significant activity toward butyraldoxime, yielding butyronitrile through its reaction. For the NHase of this strain (27), butyronitrile is a better substrate and exhibits the highest affinity (Km = 1.03 mM) among all of the tested nitriles. Other aliphatic nitriles such as propionitrile and acrylonitrile are remarkably active as substrates for the enzyme, although aromatic nitriles are barely hydrated by the NHase (27). The amidase linked with this NHase can also act on aliphatic amides including butyramide and isobutyramide, rather than aromatic amides, as substrates (55). In this study, the kinetic parameters of OxdA indicated that aliphatic aldoximes are more effective substrates than aromatic aldoximes. We observed a similar trend in substrate specificity among the NHase, amidase and OxdA, which co-exist in the same gene cluster in P. chlororaphis B23 (Fig. 7).
|
The aldoxime dehydratase reaction is surprising, particularly in the dehydration of the substrate (aldoxime) and the formation of the carbon-nitrogen triple bond, both of which have rarely been observed for enzymes known thus far. In this reaction scheme, H2O is apparently removed from the substrate, whereas the reaction mixture contains water. Such an efficient dehydration reaction is very unusual, and the catalytic mechanism in which the heme would be involved is interesting. Although both aldoxime dehydratase and CYP71E1 (cytochrome P450) act on the same substrate, "aldoxime," and are classified as a group of aldoxime-degrading enzymes, they exhibit no sequence similarity to each other; the two enzymes thus do not share a common ancestor. Further biochemical analyses, including three-dimensional analysis, of these enzymes are required to clarify the differences in their reaction mechanisms. Such analyses will also be significant from a biotechnological perspective for the production of nitriles from the corresponding aldoximes. OxdA is an interesting target with a unique catalytic mechanism, because the enzymatic reaction proceeding even in the presence of CO is rare.
Our report is the first demonstration of a gene cluster consisting of the
structural genes of aldoxime dehydratase, amidase, and NHase, whereas aldoxime
dehydratase activity is detected in some microorganisms with NHase activity
(56–58)
(Fig. 7). This gene association
strongly supports the theory that these three enzymes are closely related in
the following metabolism: aldoxime 
nitrile amide acid and
ammonium. In Bacillus sp. strain OxB-1
(38,
56), on the contrary, there is
different aldoxime metabolism: aldoxime nitrile acid and
ammonium, through the combination of phenylacetaldoxime dehydratase and
nitrilase. In this strain, the phenylacetaldoxime dehydratase gene is coded by
the region just downstream from the nitrilase gene, which is present just
upstream of a possible regulatory gene for the expression of the nitrilase
gene (38). We have also
discovered sequence similarity (42% identity) between OxdA and an unknown ORF
(designated as Psyr0978 under GenBankTM accession number NZ_AABH01000002)
of Pseudomonas syringae pv. syringae B728a, a
phytopathogenic bacterium (data not shown). In P. syringae, the same
adjacent gene organization is observed
(Fig. 7), although the sequence
of each of the constituent ORFs in this organization is only reported in this
bacterial genome data base, and its function has never been identified. On the
other hand, we demonstrated that OxdA was in fact utilized in P.
chlororaphis B23. There were significant correlations between the
addition of aldoxime in the medium and OxdA formation, indicating that OxdA is
involved in the degradation of aldoxime in vivo
(Fig. 6). Here, we have the
following question. Do the bacteria with aldoxime-degrading activity also have
aldoxime-synthetic ability? In glucosinolate-producing plants (e.g.
Arabidopsis), aldoximes have been shown to be formed from amino acids by
cytochrome P450 belonging to the CYP79 family
(33,
59–61).
Further sequence analyses of the upstream and downstream regions of the
aldoxime-related gene cluster in the bacteria with aldoxime-degrading activity
may provide information on such aldoxime biosynthesis.
|
FOOTNOTES |
|---|
* This work was supported in part by the 21st Century COE (tara.
tsukuba.ac.jp/coe21/) Program of the Ministry of Education, Culture,
Sports, Science, and Technology; by a grant-in-aid for Scientific Research
from the Ministry of Education, Science, and Culture of Japan; by the
Industrial Technology Research Grant Program in'02 of the New Energy and
Industrial Technology Development Organization of Japan; by the National
Project on Protein Structural and Functional Analyses; and by a Research Grant
(A) of the University Research Projects. The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
Both authors contributed equally to the results of this work.
|| To whom correspondence should be addressed. Fax: 81-29-853-4605 (Institute).
1 The abbreviations used are: NHase, nitrile hydratase; ORF, open reading
frame.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
), Tris/HCl (
), and
NH4Cl/NH4OH (
). B, the reactions were
carried out for 5 min at various temperatures. Relative activity is expressed
as the percentage of the maximum activity attained under the experimental
conditions used.
