Discovery of a Novel Enzyme, Isonitrile Hydratase, Involved in Nitrogen-Carbon Triple Bond Cleavage*

Isonitrile containing an N≡C triple bond was degraded by microorganism sp. N19-2, which was isolated from soil through a 2-month acclimatization culture in the presence of this compound. The isonitrile-degrading microorganism was identified asPseudomonas putida. The microbial degradation was found to proceed through an enzymatic reaction, the isonitrile being hydrated to the corresponding N-substituted formamide. The enzyme, named isonitrile hydratase, was purified and characterized. The native enzyme had a molecular mass of about 59 kDa and consisted of two identical subunits. The enzyme stoichiometrically catalyzed the hydration of cyclohexyl isocyanide (an isonitrile) to N-cyclohexylformamide, but no formation of other compounds was detected. The apparent K m value for cyclohexyl isocyanide was 16.2 mm. Although the enzyme acted on various isonitriles, no nitriles or amides were accepted as substrates.

Nitriles are very toxic and are generally organic compounds containing a C-N moiety that are not biodegradable. We have studied nitrile metabolism (1-3); we clarified the structures and functions of enzymes involved (i.e. nitrilase (4 -7), nitrile hydratase (8 -11), and amidase (12)(13)(14)) and their genes and their regulation mechanisms in metabolism.
On the other hand, no reports have appeared on an enzyme involved in the metabolism of isonitrile (more generally called isocyanide), which is an isomer of nitrile containing an isocyano group.
Like nitrile, isonitrile is generally highly toxic, and some organisms produce isocyano compounds that probably have a self-defensive function (15,16). For example, Penicillium notatum synthesizes an isocyanide metabolite, xanthocillin, that exhibits a wide spectrum of antibiotic activity (17). A marine sponge, Axinella cannabina, produces a sesquiterpenoid compound containing the isocyanide structure, axisonitrile-1 (18). 9-Isocyanopupukeanane, an unusual smelling substance lethal to fish and crustaceans, is found in the mucus of a nudibranch, Phyllidia varicosa, and its prey, a sponge, Hymeniacidon sp. (19). There have been many other reports about such naturally occurring isocyanides (for reviews, see Refs. 15 and 16). However, the details of their synthetic and degradative pathways remain unknown, and no enzyme involved in the metabolism of such isocyano-compounds has yet been reported. Among so far known enzymes, only nitrogenase has been reported to act on an isonitrile; it reduces methyl isocyanide to give dimethylamine (by four electrons transfer) or methane plus methylamine (by six electrons transfer) (20,21). However, methyl isocyanide is just an alternative substrate for nitrogenase. The enzymes metabolizing isonitriles as its physiological substrate remain unknown.
We are interested in how C-N hydrolases evolved. Because isonitrile contains a nitrogen-carbon bond in its structure, an isonitrile-hydrating enzyme (which has not been discovered) would also belong to the category of C-N hydrolases. A search for such an enzyme and its functional analysis would probably provide us with new knowledge about C-N hydrolases, which might serve as clues for elucidating the pathways of their functional and structural evolution. In the present paper, we describe the isolation of an isonitrile-degrading microorganism, Pseudomonas putida N19-2, from soil and the purification and characterization of an enzyme named isonitrile hydratase that catalyzes the hydration of isonitrile to the corresponding N-substituted formamide.

Materials
Cyclohexyl isocyanide and N-cyclohexylformamide were purchased from Fluka Chemical Co. (Buchs, Switzerland) and Tokyo Kasei Kogyo Co. (Tokyo, Japan), respectively. DEAE-Sephacel and a low molecular weight standard kit were obtained from Amersham Pharmacia Biotech. TSK gel Butyl-Toyopearl 650M was purchased from Tosoh Co. (Tokyo, Japan). Cellulofine GCL-2000 superfine was purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Marker proteins for molecular mass determination by high performance liquid chromatography (HPLC) 1 were purchased from Oriental Yeast Co. (Tokyo, Japan). All other chemicals used were from commercial sources and were of reagent grade.

Isolation of Isonitrile-degrading Bacteria
Isonitrile-degrading bacteria were isolated from soil samples as follows. A spoonful of a soil sample was added to a test tube containing 10 ml of medium (pH 7.0) consisting of 10 g of glycerol, 0.5 g of K 2 HPO 4 , 0.5 g of KH 2 PO 4 , 0.5 g of MgSO 4 ⅐7H 2 O, 0.005 g of FeSO 4 ⅐7H 2 O, 1 ml of a vitamin mixture (0.4 g of thiamine hydrochloride, 0.2 g of riboflavin, 0.4 g of pyridoxime hydrochloride, 0.4 g of nicotinic acid, 10 mg of folic acid, 0.4 g of calcium pantothenate, 2 mg of biotin, 2 g of inositol, and 0.2 g of p-aminobenzoic acid in 1 liter of distilled water), and cyclohexyl isocyanide at a final concentration of 0.01% (v/v)/liter of tap water. * This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. 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.
Cultivation was performed with shaking at 28°C for 1 week. Once a week, 1 ml of the culture was inoculated into 10 ml of fresh medium. After 3 weeks of cultivation, the concentration of cyclohexyl isocyanide was increased to 0.02% (v/v). After a month of further cultivation, the microorganisms were spread on agar plates and isolated.

Assaying of the Isonitrile-degrading Abilities of the Isolated Strains
Each of the isolated strains was inoculated into a test tube containing 10 ml of 2ϫ YT medium (16 g of tryptone, 10 g of yeast extract, and 5 g of NaCl/liter of distilled water) supplemented with cyclohexyl isocyanide at a final concentration of 0.02% (v/v) and then incubated at 28°C for 48 h with reciprocal shaking. Then the cells were harvested by centrifugation, washed twice with 0.01 M potassium phosphate buffer (pH 7.5), and suspended in 0.1 M potassium phosphate buffer (pH 7.5).
The isonitrile-degrading abilities of the isolated strains were assayed by means of the resting cell reaction. The reaction mixture (400 l) was composed of 40 mol of potassium phosphate buffer (pH 7.5), 4 mol of cyclohexyl isocyanide, 20 l of methanol, and an appropriate amount of cell suspension. Methanol was added to enhance the solubility of the substrate. The reaction was carried out at 20°C for 30 min and stopped by placing the reaction mixture on ice water and then rapidly removing the cells by centrifugation at 0 -4°C. The residual amount of cyclohexyl isocyanide in the reaction mixture was determined by HPLC with a Shimadzu LC-6A system (Kyoto, Japan) equipped with a Cosmosil 5C 18 -AR-II column (reversed-phase; 4.6 by 150 mm: Nacalai Tesque, Kyoto, Japan) and a refractive index detector (RID-6A, Shimadzu), instead of the UV spectrophotometric detector (SPD-6A) of the original system. The following solvent system was used: 5 mM KH 2 PO 4 -H 3 PO 4 buffer (pH 2.9), acetonitrile (1:1 v/v) at a flow rate of 1.0 ml/min.

Identification of the Compound Produced from
Cyclohexyl Isocyanide by P. putida N19-2 The product in the reaction mixture with cells of P. putida (which was identified as described under "Results") was extracted with chloroform/methanol (2:1 v/v), concentrated, and then analyzed by gas chromatography-mass spectrometry (GC-MS) analysis. GC-MS was performed with a Shimadzu GCMS-QP5050 equipped with an FFS ULBON HR-1 capillary column (0.25 ϫ 50 m; Shinwa Chemical Industries, Ltd., Kyoto, Japan). The initial column temperature of 50°C was raised at 5°C/min to 120°C. The injection and detector temperatures were 250°C. The carrier gas was helium, at a flow rate of 28 ml/min. Culture Conditions for P. putida N19-2 P. putida N19-2 was collected from an agar plate and then inoculated for the first subculture. The first subculture was carried out at 28°C for 24 h with reciprocal shaking in a test tube containing 10 ml of 2ϫ YT medium supplemented with cyclohexyl isocyanide at a final concentration of 0.02% (v/v). Then 1 ml of the first subculture was inoculated into a 500-ml shaking flask containing 90 ml of the second subculture medium (pH 7.0) consisting of 10 g of glycerol, 1 g of (NH 4 ) 2 SO 4 , 13.4 g of K 2 HPO 4 , 6.5 g of KH 2 PO 4 , 1 g of NaCl, 0.2 g of MgSO 4 ⅐7H 2 O, 0.01 g of FeSO 4 ⅐7H 2 O, 0.01 g of ZnSO 4 ⅐7H 2 O, 1 ml of a vitamin mixture, and cyclohexyl isocyanide at a final concentration of 0.02% (v/v)/liter of tap water. The second subculture was also performed at 28°C for 24 h with reciprocal shaking. Then 5 ml of the culture was inoculated into a 2-liter shaking flask containing 500 ml of the same medium as used for the second subculture, followed by incubation at 28°C with reciprocal shaking. After 24 h of incubation, the cells were harvested by continuous flow centrifugation at 18,000 ϫ g at 4°C and then washed twice with 0.01 M potassium phosphate buffer (pH 7.5) containing 10% (v/v) glycerol.

Purification of Isonitrile Hydratase
All purification steps were performed at 0 -4°C. Potassium phosphate buffer (pH 7.5) containing 10% (v/v) glycerol was used throughout the purification steps. Centrifugation was carried out for 30 min at 18,000 ϫ g.
Step 1: Preparation of a Cell-free Extract-Washed cells (40 g) from 12 liters of culture broth were suspended in 150 ml of 0.1 M buffer and then disrupted by sonication at 9 kHz for 30 min with an Insonator model 201M (Kubota, Tokyo, Japan). The cell debris was removed by centrifugation.
Step 3: DEAE-Sephacel Column Chromatography-The dialyzed so-lution was applied to a DEAE-Sephacel column (5 ϫ 20 cm) equilibrated with 10 mM buffer. Protein was eluted from the column with 1 liter of the same buffer by increasing the concentration of KCl linearly from 0 to 1 M. The active fractions were pooled, 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 then dialyzed against 10 mM buffer.
Step 4: TSK Gel Butyl-Toyopearl 650M Column Chromatography-The enzyme solution from step 3 was mixed with an equal amount of 50 mM buffer containing 40% saturated ammonium sulfate and then placed on a TSK gel Butyl-Toyopearl 650M column (2.6 ϫ 22 cm) equilibrated with 50 mM buffer containing 20% saturated ammonium sulfate. The enzyme was eluted by lowering the concentration of ammonium sulfate (20 to 0% saturation) in 1 liter of the same buffer. The active fractions were combined and precipitated with ammonium sulfate to bring 70% saturation. The precipitate was collected by centrifugation, dissolved in 0.1 M buffer, and then dialyzed against 10 mM buffer.
Step 5: Cellulofine GCL-2000sf Column Chromatography-The enzyme solution from step 4 was concentrated with a Centricon-10 microconcentrator (Amicon Inc., Beverly, MA) to 0.7 ml, and then placed on a Cellulofine GCL-2000sf column (2.6 ϫ 108 cm) equilibrated with 10 mM buffer. The rates of sample loading and column elution were kept at 15 ml/h. The active fractions were combined and then precipitated with ammonium sulfate at a final concentration of 70%. The precipitate was collected by centrifugation and then dissolved in 0.1 M buffer. The enzyme solution was dialyzed against 10 mM buffer and then preserved in 10 mM buffer containing 50% (v/v) glycerol at Ϫ20°C.

Enzyme Assay
Isonitrile hydratase activity was assayed in a reaction mixture (400 l) consisting of 40 mol of potassium phosphate buffer (pH 7.5), 4 mol of cyclohexyl isocyanide, 20 l of methanol, and an appropriate amount of enzyme. Methanol was added to enhance the solubility of the substrate. The enzyme activity was not inhibited even in the presence of 10% (v/v) methanol (data not shown). The reaction was carried out at 20°C for 30 min and stopped by adding 400 l of cold acetonitrile to the reaction mixture. The amount of N-cyclohexylformamide formed was determined by HPLC, which was performed with the same system as used for the measurement of cyclohexyl isocyanide under "Assaying of the Isonitrile-degrading Abilities of the Isolated Strains," except that a UV spectrophotometric detector (SPD-6A) was used. The wavelength of 198 nm was used for the monitoring.
One unit of isonitrile hydratase activity was defined as the amount of enzyme that catalyzed the formation of 1 mol N-cyclohexylformamide/ min from cyclohexyl isocyanide under the above conditions. Protein was determined by the Coomassie Brilliant Blue G-250 dye binding method of Bradford (22), using dye reagent supplied by Bio-Rad. The specific activity is expressed as units/mg protein.

Molecular Mass Determination
The enzyme sample (20.4 g) was subjected to HPLC on a TSK G-3000SW column (0.75 ϫ 60 cm; Tosoh Co., Tokyo, Japan) and then eluted with 50 mM potassium phosphate buffer (pH 7.5) at a flow rate of 0.5 ml/min. The absorbance of the effluent was recorded at 280 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 10 mM potassium phosphate buffer (pH 7.5). The dialysis caused no effect on the enzyme activity (16.5 units/mg). The enzyme sample containing 0.9 mg protein/ml was analyzed with an inductively coupled radiofrequency plasma spectrophotometer, Shimadzu ICPS-8000 (27.120 MHz; Kyoto, Japan). The metal contents of the enzyme sample were determined from the calibration curves for standard solutions.

NH 2 -terminal Sequence Analysis
The purified enzyme sample (20.4 g) was subjected to an Applied Biosystems model 476A protein sequencer (Foster City, CA), and the NH 2 -terminal sequence was analyzed by automated Edman degradation.
The assaying of substrate specificity was carried out in a reaction mixture (400 l) consisting of 40 mol of potassium phosphate buffer (pH 7.5), 4 mol of substrate, 20 l of methanol, and an appropriate amount of enzyme. However, when propionitrile, n-butyronitrile, isobutyronitrile, n-valeronitrile, propionamide, n-butyramide, isobutyramide, and n-valeramide were used as substrates, their final concentrations were 100 mM. Methanol was added to enhance the solubility of the substrate. The reaction was carried out at 20°C for 30 min (unless otherwise specified) and stopped by adding 400 l of cold acetonitrile (when isonitriles were used as substrates) or 100 l of 1 M HCl (when nitriles or amides were used as substrates) to the reaction mixture.
The levels of substrate consumption were determined with the same HPLC system as used under "Enzyme Assay." The following wavelengths were used for monitoring: 194 nm for benzyl isocyanide, 195 nm for methyl isocyanoacetate and ethyl isocyanoacetate, 198 nm for cyclohexanecarboxamide, 200 nm for methacrylonitrile, 205 nm for crotononitrile and benzyl cyanide, 220 nm for acrylonitrile, and 230 nm for the others. Isocyanomethyl phosphonic acid diethyl ether, propionitrile, n-butyronitrile, isobutyronitrile, n-valeronitrile, and cyclohexyl cyanide were measured by HPLC with a refractive index detector (RID-6A) instead of a UV spectrophotometric detector (SPD-6A).

RESULTS
Isolation and Identification of P. putida N19-2-After about 2 months from the start of the study, using the acclimatization culture method as described under "Experimental Procedures," we finally isolated one microorganism, sp. N19-2, that is able to degrade cyclohexyl isocyanide.
Then we examined the isonitrile-degrading ability of the strain N19-2. Cyclohexyl isocyanide does not exhibit strong UV absorption, and it is difficult to measure the residual amount of it in a resting cell reaction mixture with a HPLC system equipped with a UV spectrophotometric detector (which is generally used for HPLC). Therefore, we developed a method for the measurement of cyclohexyl isocyanide involving a refractive index detector instead of a UV spectrophotometric detector. Because the compound was spontaneously decomposed at low pH, the reaction was stopped by the addition of an organic solvent (cold acetonitrile) and not a mineral acid (such as hydrochloric acid), mineral acids being generally used as reaction-stopping reagents. Using these techniques, we have successfully assayed the isonitrile-degrading enzyme.
As the result of the resting cell reaction, strain N19-2 degraded cyclohexyl isocyanide and produced an unknown compound concomitantly with the isonitrile degradation. The strain showed an activity level of 0.32 mol isonitrile degradation/min/mg of dry cells.
Morphologically, strain N19-2 is a Gram-negative short rod, nonendospore forming, and motile. Its physiological characteristics are as follows: oxidase, positive; catalase, positive; oxidative/fermentative dissimilation of glucose, oxidative; reduction of nitrate and indole, negative; acid production from glucose and maltose, negative; decomposition of arginine, positive; de-composition of urea, aesculin, gelatin, tyrosine, and casein, negative; ␤-galactosidase, negative; growth on sole carbon sources, positive with glucose, mannnose, gluconate, caprate, malate, citrate, glycine, phenylalanine, benzylamine, and betaine and negative with arabinose, mannitol, N-acetylglucosamine, maltose, adipate, and inositol; cytochrome oxidase, positive; residual nitrate, positive; and production of pigment in King's B broth, negative. The GC content of the genomic DNA of this strain was determined as 62.4% with a DNA-GC kit (Yamasa Shoyu Co., Choshi, Japan) according to the method developed by three groups independently (24 -26). Based on these characteristics, strain N19-2 was identified as P. putida.
Identification of the Reaction Product-The unknown compound produced from cyclohexyl isocyanide through the cell reaction of P. putida N19-2 was analyzed by GC-MS, as described under "Experimental Procedures." As a result, it was found that both the retention time (10.4 min under the experimental conditions) and the MS spectrum (Fig. 1A) of the reaction product agreed with those of authentic N-cyclohexylformamide (Fig. 1B). Furthermore, their retention times on HPLC chromatography were the same (data not shown). No other compounds (which may be produced from cyclohexyl isocyanide), such as cyclohexylamine, cyclohexanol, cyclohexanone, formate, and ammonia, exhibited similarity to the reaction product on HPLC or GC-MS analysis (data not shown). Thus, the reaction product was identified as N-cyclohexylformamide.
The amounts of residual cyclohexyl isocyanide and formed N-cyclohexylformamide in the reaction mixture were determined to be 1.24 and 0.75 mol, respectively, when the initial amount of cyclohexyl isocyanide as the substrate was 2.00 mol. No other compounds were detected in the reaction mixture on HPLC or GC-MS analysis. The results demonstrated that N-cyclohexylformamide was formed stoichiometrically with the consumption of cyclohexyl isocyanide. When the cells of P. putida N19-2 were heat-treated by boiling for 5 min before the resting cell reaction, no formation of N-cyclohexylformamide was observed. This finding demonstrated that this conversion of cyclohexyl isocyanide to N-cyclohexylformamide is not a chemical but an enzymatic reaction.
These findings indicated that P. putida N19-2 contains an enzyme that catalyzes the hydration of isonitrile to the corresponding N-substituted formamide. Therefore, we named this enzyme "isonitrile hydratase." Purification of Isonitrile Hydratase-We found that the isonitrile hydratase activity of P. putida N19-2 was observed only when cyclohexyl isocyanide or another isonitrile was added to the culture medium. 2 The highest activity was obtained by the addition of cyclohexyl isocyanide. Therefore, the purification of the enzyme was carried out from an extract of the cells cultured in the presence of cyclohexyl isocyanide. We observed on SDS-PAGE that the crude extract contained a protein that was not synthesized without the addition of the isonitrile to the culture medium (Fig. 2, lane C). The ratio of the protein band in active fractions became greater as the purification procedure proceeded, demonstrating that it was isonitrile hydratase (data not shown).
Through the purification steps described under "Experimental Procedures," the enzyme was purified 38.2-fold with a yield of 16.7% (Table I). The purified enzyme gave only one band on SDS-PAGE (Fig. 3), corresponding to a molecular mass of 29 kDa. Further evidence of the purity of the enzyme preparation was provided by the results of HPLC on a TSK G-3000SW column, which gave a single symmetrical protein peak. The molecular mass of the native enzyme was determined to be 59 kDa by gel permeation HPLC. Thus, the enzyme probably consists of two identical subunits.
The absorption spectrum of the purified enzyme in 0.01 M potassium phosphate buffer (pH 7.5) showed the maximum absorbance at 278 nm. No other peak absorption or shoulder was observed, suggesting that no co-factor would be bound to the enzyme. The NH 2 -terminal sequence was determined as ALQIGFLLFP. It exhibited no homology with the amino acid sequences of the reported proteins.
Stoichiometry-The stoichiometry of isonitrile consumption and N-substituted formamide formation during the hydration of isonitriles was examined in a reaction mixture consisting of 40 mol of potassium phosphate buffer (pH 7.5), 4 mol of cyclohexyl isocyanide, 20 l of methanol, and 0.42 nmol of the enzyme in a final volume of 400 l. The reaction was carried out at 20°C in an airtight tube. After 30 min of incubation, the amounts of residual cyclohexyl isocyanide and N-cyclohexylformamide were determined. The N-cyclohexylformamide formed and the cyclohexyl isocyanide remaining were 1.96 and 1.98 mol, respectively. No formation of other compounds was noted. The results indicated that N-cyclohexylformamide was formed stoichiometrically with the consumption of cyclohexyl isocyanide.
Effects of pH and Temperature-The effects of pH and temperature on the enzyme activity were examined. The enzyme exhibited maximum activity at pH 6.0 -6.5, as shown in Fig.  4A. The optimal temperature was 35°C, and the enzyme activity was rapidly lost above 40°C (Fig. 4B).
The substrate is very stable under 60°C. As to the effect of pH, it is partly decomposed to N-cyclohexylformamide by spontaneous and chemical (nonenzymatic) reaction in the acidic conditions under pH 5.0. Thus, the enzyme activities under pH 5.0 shown in Fig. 4A were calculated from the modified amount of product; we subtracted the amount of N-cyclohexylformamide formed in control (the reaction mixture without the enzyme) from that of each sample before the calculation. The substrate is stable over pH 5.5.
Substrate Specificity-The ability of the enzyme to catalyze the hydration or hydrolysis of various isonitriles, nitriles, and amides was examined. Table II shows that all of the tested isonitriles were active as substrates for the isonitrile hy-  dratase. However, the relative activities toward the isonitriles other than cyclohexyl isocyanide and benzyl isocyanide were rather low. The hydration of cyclohexyl isocyanide followed Michaelis-Menten-type kinetics, and the K m and V max values were 16.2 mM and 39.6 mol/min/mg, respectively. Because of their commercial unavailability, aliphatic or aromatic isonitriles, which are not listed in Table II, such as methyl isocyanide and phenyl isocyanide, could not be examined. Not only N-cyclohexylformamide but also all of the tested nitriles and amides did not act as substrates for our enzyme, despite the addition of a large amount of the enzyme (1.

units) and a long incubation period (24 h).
Inhibitors-Various compounds were investigated as to their inhibitory effects on the enzyme activity (Table III). The enzyme was very sensitive to CdCl 2 , HgCl 2 , CuSO 4 , AgNO 3 , CoCl 2 , NiSO 4 , and iodoacetate. Other thiol reagents such as N-ethylmaleimide and p-chloromercuribenzoate also inhibited the enzyme activity. Carbonyl reagents, e.g. hydroxylamine, phenylhydrazine, semicarbazide, and aminoguanidine, hardly inhibited the enzyme. Chelating agents, such as ␣,␣Ј-dipyridyl, o-phenanthroline, 8-hydroxyquinoline, and EDTA, did not influence the activity at all, but diethyldithiocarbamate and KCN caused partial inhibition. The enzyme was unaffected by reducing reagents, e.g. dithiothreitol, 2-mercaptoethanol, and Na 2 S 2 O 4 . However, oxidizing reagents such as H 2 O 2 and ammonium persulfate caused appreciable inhibition. The enzyme was sensitive to serine-modifying reagents, i.e. phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. DISCUSSION Isonitriles are unique in that they form the only class of organic compounds that contain a stable, formally mono-coordinated carbon (15). The isocyano group exhibits a dual nucleophilic/electrophilic character, which is often exploited for synthetic applications: e.g. ␣-addition reactions, multi-component condensation reactions, in the synthesis of peptides, in coordination chemistry, organometallic reactions, and carbohydrate chemistry (15).
On the other hand, there have been many reports of naturally occurring isonitriles (15,16). To our knowledge, however, no enzyme involved in the metabolism of isonitriles has been found. Among so far known enzymes, only nitrogenase is known to act on an isonitrile; it converts methyl isocyanide to the corresponding amine (20,21). But isonitriles are not physiological substrates of the enzyme. Although it might be possible that isonitrile-metabolizing organisms produce and utilize nitrogenase to degrade isonitriles, it remains unclear whether it is the case or not. In this work, we isolated an isonitriledegrading bacterium, which has been identified as P. putida, from soil and purified isonitrile hydratase from the cells. This enzyme has been found to act only on isonitriles, leading to formation of the corresponding N-substituted formamides. The reaction catalyzed by this enzyme is quite distinct from that of nitrogenase; the former is hydration, and the latter is reduction.
The enzyme consists of two identical subunits and contains no metal. Isonitrile hydratase is different from nitrilase (which catalyzes the direct hydrolysis of nitrile to the corresponding carboxylic acid and ammonia; Refs. 32 and 33) and nitrile The reaction was carried out at 20°C in the standard reaction mixture except that various isonitriles were used as substrates in place of cyclohexyl isocyanide. The enzyme activities towards the substrates other than cyclohexyl isocyanide were assayed as the consumption of the substrate, whereas the activity towards cyclohexyl isocyanide was determined as the formation of the product, as described under "Enzyme Assay." The synthesis of N-cyclohexylformamide, corresponding to 16.5 mol/min/mg of protein, was taken as 100%.  hydratase (which catalyzes the hydration of nitrile to the corresponding amide; Refs. 1, 11, and 34). With respect to the noninvolvement of a metal in isonitrile hydratase, however, the enzyme is similar to nitrilase of R. rhodochrous J1 (5,35) rather than to the metal-containing nitrile hydratases of Brevibacterium, Pseudomonas, and R. rhodochrous J1 (1,2,(27)(28)(29)(30), although the reaction catalyzed by this enzyme is hydration and not hydrolysis. The enzyme is also like the R. rhodochrous J1 nitrilase as to the sensitivity to various compounds. It is sensitive to thiol reagents and oxidizing reagents, whereas it is not influenced by chelators or reducing reagents. This suggests that isonitrile hydratase may have an active cysteine residue in its catalytic center, like the R. rhodochrous J1 nitrilase (5). However, experiments on the substrate specificity of this enzyme have revealed that it does not attack nitriles or amides as substrates. On the other hand, both the purified nitrilase from R. rhodochrous J1 and the purified high molecular mass nitrile hydratase from R. rhodochrous J1 did not act on isonitriles, which were substrates for isonitrile hydratase (data not shown). Further studies are required to clarify the reaction mechanism of isonitrile hydratase.
The main physiological role of isonitrile hydratase for P. putida N19-2 seems to be detoxification. We intended to isolate an isonitrile-assimilating microorganism through acclimatization culture utilizing cyclohexyl isocyanide as a nitrogen source in the culture medium. However, the resting cells of P. putida N19-2 released almost all of the consumed cyclohexyl isocyanide to the reaction mixture as N-cyclohexylformamide, suggesting that P. putida N19-2 has little or no ability to assimilate the formamide produced from cyclohexyl isocyanide. Therefore, isonitrile hydratase would likely be a detoxification enzyme rather than the first participant in the pathway for isonitrile assimilation.
Some organisms produce isocyano-compounds that are probably used to protect them from their enemies (15)(16)(17)(18)(19). To elucidate the metabolic pathways for such isonitriles, a few incorporation experiments have been carried out in vivo (36 -38), e.g. the compounds that were supposed to be involved in the metabolism of naturally occurring isonitriles were labeled with a radioisotope and then fed to the isonitrile-producing organisms, and then the metabolites were isolated and assayed for radioactivity. These experiments revealed some of the isonitrile metabolic pathways. For example, it has clearly been demonstrated that 2-isocyanopupukeanane is transformed to the corresponding N-substituted formamide and isothiocyanate in a Hymeniacidon species, whereas the reverse reaction does not occur (36). The experiments involving P. notatum have indicated that tyrosine could act as the precursor of xanthocillin; both the nitrogen atoms of the isocyano groups and the carbon framework were derived from tyrosine (37,38). Although the origin of the carbon atoms of the isocyano groups remains a mystery, there has been one report suggesting that xanthocillin might be synthesized from the corresponding diformamide (39); this is contrary to the case of 2-isocyanopupukeanane. However, all of the experiments were in vivo and not in vitro. As described above, no research at the protein or gene level has been done. Our work on the isonitrile degradation in P. putida N19-2 is the first at the protein level, and now molecular cloning of the gene is in progress. It is expected that isonitrile-producing organisms may also have isonitrile-degrading enzymes, as indicated for Hymeniacidon sp. If such enzymes exhibit similarity to our isonitrile hydratase in the primary structure, the cloned gene of isonitrile hydratase could possibly serve as a probe for obtaining these enzyme genes. Moreover, it might enable one to clone the genes of the isonitrile-synthetic enzymes if the genes involved in the metabolism of isonitrile are closely located, forming gene clusters.
In conclusion, this report is about an enzyme responsible for the degradation of isonitrile; we discovered an enzyme involved in the NϵC triple bond hydration reaction. Further characterization of this enzyme could provide new clues for elucidating the synthetic and metabolic pathways for naturally occurring isonitriles. Moreover, it would also provide us with novel knowledge about C-N hydrolases and lead to a better understanding of their evolutionary pathways.