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Originally published In Press as doi:10.1074/jbc.M407223200 on August 31, 2004

J. Biol. Chem., Vol. 279, Issue 46, 47619-47625, November 12, 2004
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Identification of Crucial Histidines Involved in Carbon-Nitrogen Triple Bond Synthesis by Aldoxime Dehydratase*

Kazunobu Konishi{ddagger}§, Kyoko Ishida{ddagger}§, Ken-Ichi Oinuma{ddagger}§, Takehiro Ohta¶, Yoshiteru Hashimoto{ddagger}, Hiroki Higashibata{ddagger}, Teizo Kitagawa¶, and Michihiko Kobayashi{ddagger}||

From the {ddagger}Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Myodaiji, Okazaki, Aichi 444-8787, Japan

Received for publication, June 28, 2004 , and in revised form, August 9, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Aldoxime dehydratase (OxdA), which is a novel heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. The combination of site-directed mutagenesis of OxdA (mutation of all conserved histidines in the aldoxime dehydratase superfamily), estimation of the heme contents and specific activities of the mutants, and CD and resonance Raman spectroscopic analyses led to the identification of the proximal and distal histidines in this unique enzyme. The heme contents and CD spectra in the far-UV region of all mutants except for the H299A one were almost identical to those of the wild-type OxdA, whereas the H299A mutant lost the ability of binding heme, demonstrating that His299 is the proximal histidine. On the other hand, substitution of alanine for His320 did not affect the overall structure of OxdA but caused loss of its ability of carbon-nitrogen triple bond synthesis and a lower shift of the Fe-C stretching band in the resonance Raman spectrum for the CO-bound form. Furthermore, the pH dependence of the wild-type OxdA closely followed the His protonation curves observed for other proteins. These findings suggest that His320 is located in the distal heme pocket of OxdA and would donate a proton to the substrate in the aldoxime dehydration mechanism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have extensively studied the biological metabolism of toxic compounds that have a triple bond between carbon and nitrogen, such as nitriles (R–C{equiv}N) (1–5) and isonitriles (R–N{equiv}C) (6, 7). The microbial degradation of nitriles proceeds through two different enzymatic pathways (8–10): (i) nitrilase catalyzes the hydrolysis of nitriles into acids (R–C(=O)–OH) and ammonia (11–13); and (ii) nitrile hydratase 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). We recently discovered a gene (oxdA) encoding a nitrile-synthesizing enzyme (aldoxime dehydratase) upstream of nitrile hydratase and amidase genes in Pseudomonas chlororaphis B23, which was used and is being used for the industrial production of acrylamide (9, 21, 27) and 5-cyanovaleramide (28), respectively. Very recently, we succeeded in overexpressing OxdA in Escherichia coli, obtaining purified OxdA (29).

OxdA is a novel heme protein including protoheme IX as the prosthetic group, and it catalyzes a unique and intriguing reaction: formation of a carbon-nitrogen triple bond and dehydration of a substrate (R–CH=N–OH) even in the presence of water in the reaction mixture (29). The nitrile formed through this reaction is subsequently hydrated into an amide by the coupled nitrile hydratase during nitrile metabolism of P. chlororaphis B23. The enzymatic reaction from aldoxime to nitrile is not only academically interesting but is also expected to be applicable to the practical production of nitriles, because it is performed under mild conditions in contrast with the chemical dehydration of aldoxime under harsh conditions (30). It was previously reported that rat liver microsomal cytochromes P450 and P450 3A4 (31) (the major P450 isozyme in human liver), whose sequence exhibits no similarity to that of the aldoxime dehydratase family (comprising OxdA (29) and phenylacetaldoxime dehydratase (32)), also catalyze the dehydration of aldoximes; but their catalytic activities are not so high.

The OxdA reaction has been thought to involve the direct binding of a substrate to the heme iron without other exogenous compounds (e.g. O2 or H2O2) (29, 31), in contrast with various other reactions (e.g. monooxygenation) of general hemoproteins, and therefore it is strongly attracting the attention of not only biochemists but also biophysicists. However, information on the structure and function of aldoxime dehydratase has been quite limited. As we previously reported (33), resonance Raman spectroscopic studies on OxdA initially demonstrated that the OxdA heme contains a proximal histidine ligand. In other hemoproteins, histidine residues are known to have an important function in the binding of the heme moiety (34–37). Here, we have attempted to identify the proximal histidine residue in OxdA by means of site-directed mutagenesis and spectroscopic analyses. Furthermore, another histidine was identified as the distal amino acid residue involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Site-directed Mutagenesis—Site-directed mutagenesis (His169, His296, His299, His320, and His338 being converted to Ala, respectively) was carried out on oxdA by means of an overlap extension PCR protocol (38, 39). To construct the H169A mutant, two PCRs, with plasmid pET-oxdA as the template, were performed with primer pairs H169A-S plus T7T and T7P plus H169A-AS (Table I). These reactions produced 3' and 5' fragments of oxdA, respectively, whose sequences overlapped by 20 base pairs at the mutation. A second round of PCR was performed by mixing equimolar amounts of the first round products, followed by amplification between primers T7P and T7T to produce the full-length oxdA. The second-round product was digested with NdeI and SalI, ligated into expression vector pET-24a(+), and then sequenced. A clone with the sequence for the desired H169A mutation was chosen and transformed into E. coli BL21-CodonPlus(DE3)-RIL. The recombinant cells were used for the overproduction and purification of the H169A mutant enzyme.


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  		TABLE I
Oligonucleotide primers used for the preparation of OxdA mutants

Boldface letters show the nucleotides changed for the desired mutations. Each position number indicates the position of the nucleotide sequence of oxdA (GenBankTM accession number AB093544 [GenBank] ).

 
Each of the H296A, H299A, H320A, and H338A mutations was carried out in the same manner as the H169A mutation, with internal primer pairs H296A-S plus H296A-AS, H299A-S plus H299A-AS, H320A-S plus H320A-AS, and H338A-S plus H338A-AS, respectively (Table I).

Expression and Purification of Recombinant OxdA and Its Mutants— Recombinant OxdA and its mutants were overexpressed according to the same procedure described previously (33). All of the mutants except for H299A were isolated as holoproteins, the H299A mutant being isolated as an apoprotein (see "Results"). The wild-type OxdA and its mutants were purified in the same manner as described previously (33) with some modifications. All steps were performed at 0–4 °C. Potassium phosphate buffer (pH 7.0) 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, 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 (30–60% saturation), followed by dialysis against 10 mM buffer. The dialyzed solution was applied to a DEAE-Sephacel column (4 x 20 cm) (Amersham Biosciences) equilibrated with 10 mM buffer. Protein was eluted from the column with 1.0 liter of 10 mM buffer, the concentration of KCl being increased linearly from 0.1 to 0.5 M. The active fractions were collected, and then ammonium sulfate was added to give 20% saturation. The enzyme solution was placed on a TSK gel Butyl-Toyopearl 650M column (4 x 15 cm) (Tosoh Co., Tokyo, Japan) equilibrated with 10 mM buffer 20% saturated with ammonium sulfate. The enzyme was eluted by lowering the concentration of ammonium sulfate (from 20 to 0%) in 1.0 liter of the same buffer. The active fractions were combined and then precipitated with ammonium sulfate at 70% saturation. The precipitate was collected by centrifugation, dissolved in 0.1 M buffer, and then dialyzed against three changes of 5 liters of 1 mM buffer (pH 6.8). After centrifugation, the enzyme solution was loaded on a Cellulofine HAp column (4 x 5 cm) (Seikagaku Kogyo Co., Tokyo, Japan) 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 resultant solution was dialyzed against 10 mM buffer and then centrifuged. The active fractions were collected and concentrated by ultrafiltration using an Amicon YM-30 membrane (Millipore Corp., Bedford, MA) and a Vivaspin 30,000 molecular weight cut-off PES membrane (Sartorius K.K., Tokyo, Japan). The enzyme solution was loaded on a Superdex 200 column (1 x 30 cm) (Amersham Biosciences) equilibrated with 50 mM buffer including 0.15 M KCl. The active fractions were collected. The homogeneity of the purified recombinant OxdA and its mutants was confirmed by SDS-PAGE.

SDS-PAGE was performed in a 12% polyacrylamide slab gel according to Laemmli (40). The gel was stained with Coomassie Brilliant Blue R-250. The molecular mass of the subunit of each mutant was determined from the relative mobilities of marker proteins, phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydratase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and {alpha}-lactalbumin (14.4 kDa).

The heme content was estimated by the pyridine ferrohemochrome method as previously reported (29). Protein concentrations were determined with a Nakalai Tesque Co., Inc. (Kyoto, Japan) protein assay kit, with bovine serum albumin as the standard. The reduction of the wild-type OxdA and its mutants was carried out by adding a few grains of sodium dithionite to the purified OxdAs.

Spectral Measurements—CD spectra of the isolated wild-type OxdA and its mutants were obtained using a spectrometer (J-720; Jasco, Tokyo, Japan) at room temperature. Proteins at a concentration of 0.3 mg/ml in 100 mM potassium phosphate buffer, pH 7.0, were each placed in a cuvette (1-mm cell length). The ellipticity in the CD spectra was normalized as to the protein concentration. The {alpha}-helical content can be evaluated from the ellipticity at 222 nm by the following equation (41, 42),

(Eq. 1)

Resonance Raman spectra were excited at 413.1 nm with a Kr+ ion laser (Spectra Physics, model 2060) or at 430 nm with a diode laser (58-BTLR010; Hitachi Metal, Tokyo, Japan). The excitation light was focused into the cell, the laser power being 3 milliwatts at the cell for the reduced form of OxdAs, but 0.005 milliwatts for CO-bound OxdAs. The sample solutions for the Raman measurements were sealed in quartz cells, which were rotated at 1,500 rpm at room temperature. Typically, 50-µl aliquots of 0.4 mg/ml protein in 100 mM potassium phosphate buffer, pH 7.0, were put into the cell. The scattered light at right angle was dispersed with a single polychromator (DG-1000; Ritsu) equipped with a liquid nitrogen-cooled charge-coupled device camera. The spectral slit width was 6 cm–1. Raman shifts were calibrated using indene and CCl4 as frequency standards, providing an accuracy of ±1 cm–1 for intense isolated lines.

Enzyme Assay—The standard reaction mixture (under anaerobic conditions) comprised 100 mM potassium phosphate buffer (pH 7.0), 5 mM butyraldoxime, 5 mM Na2S2O4, and an appropriate amount of enzyme, in a total volume of 400 µl. The reaction was started by the addition of butyraldoxime and carried out for 5 min at 30 °C. The reaction was stopped by the addition of 200 µl of 10 mM NH2OH·HCl. 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). 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 conditions. Specific activity is expressed as units/mg of protein.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Amino Acid Sequence Alignment of the Aldoxime Dehydratase Superfamily—In our previous study (29), we determined the nucleotide sequence of aldoxime dehydratase from P. chlororaphis B23, which consists of 1,056 nucleotides encoding 352 amino acid residues. A search with the BLAST program revealed that the deduced amino acid sequence of aldoxime dehydratase from P. chlororaphis B23 exhibits similarity with those from Bacillus sp. OxB-1 (32% identity) (32), Rhodococcus globerulus A-4 (77% identity) (43), and Pseudomonas syringae (42% identity) (29), respectively. These enzymes are considered to comprise a superfamily (aldoxime dehydratase superfamily), and each of the genes forms a gene cluster for aldoxime metabolism with the corresponding nitrile hydratase and amidase genes or the nitrilase gene (29). Alignment of the deduced amino acid sequences of the above four enzymes in the aldoxime dehydratase superfamily (Fig. 1) revealed the existence of five conserved histidines in this superfamily: His169, His296, His299, His320, and His338, where the amino acid residue numbers refer to the sequence of aldoxime dehydratase from P. chlororaphis B23. We prepared point mutants by substituting alanine for each of the five histidines conserved in OxdA, as shown below.



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  		FIG. 1.
Alignment of the aldoxime dehydratase superfamily. B23, P. chlororaphis B23; A-4, R. globerulus A-4; Psyr, P. syringae; OxB-1, Bacillus sp. OxB-1. Identical residues are denoted by asterisks in the four members of the superfamily. Histidines that are conserved in the members' sequences are numbered and highlighted in white on black.

 
Expression and Purification of Five His -> Ala Mutants— Site-directed mutagenesis has been applied to determine which residue serves as an axial ligand in hemoproteins and to elucidate the functional roles of key residues in the active site (35, 44–50). Since OxdA from P. chlororaphis B23 has a histidine as the proximal ligand (33), we focused on the conserved histidines in the aldoxime dehydratase superfamily, all of the conserved histidines being individually mutated to alanine. The H169A, H296A, H299A, H320A, and H338A mutant enzymes of OxdA were expressed in E. coli and purified by the purification procedure described under "Experimental Procedures." The purity of each OxdA mutant was confirmed by migration of the protein as a single band corresponding to a molecular mass of ~38 kDa on SDS-PAGE (Fig. 2).



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  		FIG. 2.
SDS-PAGE of the wild-type OxdA and its mutants. Lane M, marker proteins; lane 1, wild type; lane 2, H169A mutant; lane 3, H296A mutant; lane 4, H299A mutant; lane 5, H320A mutant; lane 6, H338A mutant. The samples purified from each transformant by the procedure described under "Experimental Procedures" were loaded onto the SDS-polyacrylamide gel.

 
Basic Physicochemical Properties of the Five His -> Ala Mutants—In order to determine the effect of mutation of all conserved histidines on heme binding, at first, we estimated the heme contents of the histidine to alanine mutants of OxdA. Table II presents the heme contents of the wild-type OxdA and all of the His mutants of OxdA. All of the histidine to alanine mutants except H299A contained a stoichiometric equivalent of heme like the wild-type OxdA, suggesting that the H299A mutant was unable to bind heme because it had lost His299 from the peptide chain of OxdA.


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  		TABLE II
Heme contents, mean residue molar ellipticities at 222 nm, {alpha}-helical contents, and specific activities of the wild-type OxdA and its mutants

 
A CD spectrum in the far-UV region provides us with information on the secondary structure in a protein (51). Fig. 3 depicts CD spectra for the isolated forms of the wild-type OxdA and its mutants. The spectral shape and {alpha}-helical contents of the mutants except H299A were similar to those of the wild-type OxdA (Fig. 3 and Table II). These results indicate that all of the mutants except H299A are properly folded like the wild-type OxdA.



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  		FIG. 3.
CD spectra for the wild-type OxdA and its mutants. The wild-type OxdA ({diamond}) and the H169A ({square}), H296A ({triangleup}), H299A (x), H320A (+), and H338A ({circ}) mutants were examined. All spectra were measured as described under "Experimental Procedures."

 
Since we previously clarified that the ferrous form of OxdA is the active state (29), the enzyme activities of the five histidine mutants of OxdA were measured in an anaerobic reduced environment. As shown in Table II, the alanine substitution of neither His169, His296, nor His338 reduced the enzyme activity. On the other hand, the purified enzymes and cell-free extracts of the H299A and H320A mutants had exhibited no enzyme activity at all. Although the replacement of His320 with alanine did not affect the heme binding or the secondary structure of OxdA, the H320A mutant exhibited no activity.

Effects of pH on the Enzyme Activity of the Wild-type OxdA— Fig. 4 illustrates the pH-dependent profile of the enzyme activity of the wild-type OxdA. OxdA allows the study of the ability of carbon-nitrogen triple bond synthesis over a wide pH range (pH 5–9.5). The pH dependence of the enzyme activity of the wild-type OxdA shown in Fig. 4 was slightly different from that we previously reported (29) because of the different purification procedure for OxdA (using the buffer without 2-mercaptoethanol), which enabled us to isolate the enzyme containing a significant level of heme (i.e. ~1 mol/mol of subunit). The enzyme activity of the wild-type OxdA broadly displayed the sigmoidal pH dependence, with a maximum change at about pH 6. This pH dependence of the wild-type OxdA closely followed the His protonation curves observed for other proteins like {alpha}-hemolysin, horseradish peroxidase, and so on (52, 53).



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  		FIG. 4.
pH dependence of the activity of the wild type. The reactions were carried out in the following buffers: 100 mM citrate/K2HPO4 ({diamondsuit}) and 50 mM H3BO3 plus 50 mM KCl/NaOH ({blacksquare}).

 
Resonance Raman Spectroscopy of the H320A Mutant and Wild-type OxdA—In order to determine the reason for the loss of enzyme activity upon the mutation of His320 to alanine, we obtained resonance Raman spectra for the ferrous and CO-bound forms of the H320A mutant and the wild-type OxdA (Figs. 5,6,7). It has been established that resonance Raman spectra in the high frequency region contain a few marker bands sensitive to the oxidation state ({nu}4) and the spin and coordination states ({nu}2 and {nu}3) of the heme iron in so far known hemoproteins (54). The ferrous form of the wild-type OxdA gave {nu}2, {nu}3, and {nu}4 bands at 1557, 1471, and 1358 cm–1, respectively, at typical frequencies of the five-coordinate high spin ferrous heme. As shown in Fig. 5, the frequencies of the marker bands of the ferrous H320A mutant are very similar to those of the ferrous wild-type OxdA, suggesting that the oxidation, spin, and coordination states of the heme in the H320A mutant are identical to those of wild-type OxdA. Moreover, the coordination states of the proximal His to the heme iron in the ferrous H320A mutant and the wild-type OxdA were examined by means of low frequency resonance Raman spectra (Fig. 6). An intense band around 226 cm–1, which is assigned to the Fe–His stretching mode, was observed for the wild-type OxdA and the H320A mutant. Therefore, the property of the proximal His was found not to be affected by the mutation at His320.



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  		FIG. 5.
High frequency regions of resonance Raman spectra of the ferrous wild-type OxdA (a) and ferrous H320A mutant (b).

 



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  		FIG. 6.
Low frequency regions of resonance Raman spectra of the ferrous wild-type OxdA (a) and ferrous H320A mutant (b).

 



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  		FIG. 7.
The resonance Raman spectra in the Fe-CO stretching and Fe-C-O bending regions of the wild-type OxdA(12C16O), wild-type OxdA(13C18O), H320A mutant(12C16O), and H320A mutant(13C18O) and their difference spectra: wild-type OxdA-(12C16O) (a); wild-type OxdA(13C18O) (b); a–b difference spectrum (c); H320A mutant(12C16O) (d); H320A mutant(13C18O) (e); and d–e difference spectrum (f).

 
It had been established that the Fe-CO stretching mode, {nu}Fe-CO, exhibits a wide range of frequencies (450–550 cm–1), reflecting the environment of the distal heme pocket (55). The CO isotope dependence of resonance Raman spectra of the wild-type OxdA and the H320A mutant is shown in Fig. 7. The isotope difference spectra demonstrate that the Fe-CO stretching bands of the wild-type OxdA(CO) and the H320A mutant(CO) were observed at 512 and 494 cm–1, respectively. The Fe-CO stretching band of the H320A mutant(CO) was shifted to a lower frequency than that of the wild-type OxdA(CO). The downward frequency shift means that the environment around CO is more hydrophobic in the H320A mutant than in the wild-type OxdA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
OxdA catalyzes the conversion of an aldoxime to a nitrile when the heme iron of OxdA is in the ferrous state. Although we previously demonstrated that the ferrous OxdA has a free distal heme pocket (similar to those of horseradish peroxidase (56) and hemoglobin (57)), which enables an aldoxime to easily gain access to the heme iron and that the proximal ligand is a histidine residue (33), there has been no report of determination of crucial histidines for carbon-nitrogen triple bond synthesis by OxdA. The combination of site-directed mutagenesis, estimation of the heme contents and specific activities of the mutants, and CD and resonance Raman spectroscopic approaches employed in this study for the first time has led to the identification of the proximal and distal histidines in this unique enzyme with the tremendous ability to catalyze the dehydration of an aldoxime even in the presence of water in the reaction mixture to form a C-N triple bond.

Identification of the Proximal Histidine in OxdA—It is commonly assumed that the amino acid residue that serves as the proximal ligand in hemoproteins belonging to a superfamily is conserved in its members. For example, the proximal histidine located at the 8th position of the F helix of hemoglobin, which transports oxygen in most vertebrates and some invertebrates, is conserved in the hemoglobins of various organisms (58). In order to identify the proximal histidine of OxdA, we prepared point mutants, substituting Ala for all conserved His residues (i.e. His169, His296, His299, His320, and His338), respectively. All of the histidine mutants except for the H299A one were shown to have the abilities to bind heme and to be properly folded (Table II and Fig. 3). Since the replacement of a proximal ligand with another amino acid (i.e. alanine), which cannot bind to the heme iron, markedly decreases the heme binding affinity of the protein (35), these results obtained in the mutant experiments suggest that His299 is the proximal histidine in OxdA. Furthermore, a 3.6% decrease of {alpha}-helical content by replacement of His299 with alanine is not so large, demonstrating that an overall conformational change of the H299A mutant would be small. Thus, the heme deficiency of the H299A mutant would be due to the lack of an appropriate heme ligand but not due to an overall conformational change of the H299A mutant.

Although replacement of His169, His296, and His338 by alanine in OxdA did not affect the reduction of the enzyme activity for carbon-nitrogen triple bond synthesis, the H299A and H320A mutants completely lacked this enzyme activity. Whereas these findings do not rule out our suggestion that His299 is the proximal histidine in OxdA, they indicate another possibility that His299 is not the proximal histidine but simply plays a structural role in OxdA, His320 being the proximal histidine. However, the resonance Raman spectra of the H320A mutant in the low and high frequency region revealed that this mutant has the same five-coordinate high spin heme and Fe–His bond as the wild-type OxdA, demonstrating that His320 is not the proximal histidine of OxdA. Thus, we here conclude that His299 is the proximal histidine of OxdA.

Identification of the Distal Histidine in OxdA—Identification of the amino acid residues that are located in the distal pocket of hemoproteins and that directly interact with a substrate is important for elucidation of the reaction mechanism. Although we previously reported that there would be a positively charged or proton-donating residue in the distal pocket (33), information on the distal side of OxdA is quite limited.

As described above, like the wild-type OxdA, the ferrous H320A mutant has a five-coordinate high spin ferrous heme and a proximal histidine, whereas it has no ability to catalyze the dehydration of an aldoxime to a nitrile. These results suggest that His320 is a crucial amino acid residue for carbon-nitrogen triple bond synthesis by OxdA. In order to obtain further information on the H320A mutant, we examined the Fe-C stretching mode (which is sensitive to the distal environment of hemoproteins) of the H320A mutant. The Fe-C stretching mode of the H320A mutant (494 cm–1) was remarkably lower compared with that of the wild-type OxdA (512 cm–1). The replacement of His320 with alanine seems to cause a change in the distal site environment of OxdA. These findings for the H320A mutant clearly indicate that His320 is the distal histidine of OxdA and plays a key role in carbon-nitrogen triple bond synthesis by OxdA.

Role of Distal His320 in Carbon-Nitrogen Triple Bond Synthesis—Although steric hindrance was previously thought to play an important role in determination of the Fe-C stretching frequency, the most recent work has suggested that polarity is the key determinant (55). Positive charges near the oxygen atom of CO enhance {pi}-back bonding from Fe2+ to CO, resulting in a decrease in {nu}C-O and an increase in {nu}Fe-CO. On the other hand, negative charges inhibit this {pi}-back bonding and thereby increase {nu}C-O and decrease {nu}Fe-CO. It was previously reported that the Fe–C stretching band of the CO-bound form of sperm whale wild-type myoglobin, which has a hydrogen bond between the oxygen atom of the Fe{delta}(+)=C=O{delta}(–) resonance structure and N{epsilon}–H of the distal histidine, was observed at 507 cm–1, whereas that of the H64L myoglobin mutant was observed at 490 cm–1 by the lack of a hydrogen bond between His64 and the bound CO (55, 59). These results for the H64L myoglobin mutant are very similar to those for the H320A OxdA mutant. The Fe{delta}(+) = C = O{delta}(–) resonance structure of OxdA (CO) would be also stabilized by a hydrogen bond to His320, as seen in that of myoglobin. Consequently, we here propose that His320 of OxdA donates a proton to the substrate, which binds to the heme iron, in the aldoxime dehydration mechanism (which will be discussed below). This proposal is also supported by the results of pH-dependent experiments on the wild-type OxdA (Fig. 4). The pH dependence on the enzymatic activity of the wild-type OxdA shows the existence of a crucial histidine for the activity of OxdA. On the other hand, the Fe–His stretching mode of OxdA was not affected by pH in the pH region of 5.5–9.5 (data not shown), as previously found in pH-dependent experiments on myoglobin, which has a similar Fe–His linkage (53). The activity of the wild-type OxdA was greatly affected by pH and decreased with an increase in pH. These pH-dependent profiles of the wild-type OxdA imply that the rate-determining step for the aldoxime dehydration of OxdA is the proton donation step of a histidine located in the distal heme pocket.

Possible Reaction Mechanism for Carbon-Nitrogen Triple Bond Synthesis by OxdA—There has been no report on the aldoxime dehydration mechanism of the aldoxime dehydratase superfamily. However, from results obtained with various iron porphyrin systems and rat liver microsomal cytochromes P450, respectively (31, 60), it is suggested that the first step of aldoxime dehydration comprises binding of the aldoxime nitrogen atom to the heme iron, the following step (the rate-determining step) comprises abstraction of the OH group of aldoxime, which is due to protonation of the OH group by an acidic amino acid residue, and the final step comprises elimination of a hydrogen atom on the carbon neighboring the nitrogen atom of aldoxime.

As aforementioned, the H320A mutant studies and the pH-dependent experiments on the wild-type OxdA suggested that the rate-determining step in the aldoxime dehydration of OxdA is the proton donation step of His320. Thus, these findings have led to the proposed enzymatic reaction mechanism of OxdA illustrated in Fig. 8, but further studies are required to determine the detailed reaction mechanism of OxdA.



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  		FIG. 8.
A possible reaction mechanism for the dehydration of aldoxime by OxdA. B, a basic amino acid residue of the OxdA active site.

 

   FOOTNOTES
 
* This work was supported in part by the 21st Century COE Program (www.tara.tsukuba.ac.jp/~coe21/) from 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 an Industrial Technology Research Grant Program in 2002 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, and by the National Project on Protein Structural and Functional Analyses, by a Research Grant (A) for University Research Projects. This work was also supported by JSPS Research Fellowships for Young Scientists (to T. O.) and by Grant-in-aid for Specifically Promoted Research 14001004 (to T. K.). 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. Back

§ These authors contributed equally to this work. Back

|| To whom correspondence should be addressed. Fax: 81-29-853-4605.


   ACKNOWLEDGMENTS
 
We thank Haruo Iizuka (University of Tsukuba) for help in the purification of the mutant OxdAs. Special thanks are also due to Associate Professor Koichiro Ishimori and Takanori Uzawa (Kyoto University) for the use of the CD spectrometer and the helpful technical advice.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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