Isonitrile Hydratase from Pseudomonas putida N19 –2 CLONING, SEQUENCING, GENE EXPRESSION, AND IDENTIFICATION OF ITS ACTIVE AMINO ACID RESIDUE*

Isonitrile hydratase is a novel enzyme in Pseudomonas putida N19–2 that catalyzes the conversion of isonitriles to N -substituted formamides. Based on N-terminal and internal amino acid sequences, a 535-bp DNA fragment corresponding to a portion of the isonitrile hydratase gene was amplified, which was used as a probe to clone a 6.4-kb DNA fragment containing the whole gene. Sequence analysis of the 6.4-kb fragment revealed that the isonitrile hydratase gene ( inhA ) was 684 nucleotides long and encoded a protein with a molecular mass of 24,211 Da. Overexpression of inhA in Escherichia coli gave a large amount of soluble isonitrile hydratase ex-hibiting the same molecular and catalytic properties as the native enzyme from the Pseudomonas strain. The predicted amino acid sequence of inhA showed low similarity to that of an intracellular protease in Pyrococcus horikoshii (PH1704), and an active cysteine residue in the protease was conserved in the isonitrile hydratase at the

An isonitrile (more generally called an isocyanide) is a highly toxic compound with an isocyano group (-NϵC).The isonitriles form the only class of organic compounds that contain a stable, formally mono-coordinated carbon, cf.carbon monoxide (1).The isocyano group exhibits the unusual characteristic that it reacts with nucleophiles and electrophiles on the same carbon atom of the functional group, which has been exploited for synthetic applications (1); in particular, isocyanide-based multicomponent reactions are a powerful tool for the one-pot synthesis of diverse and complex compounds and are being increasingly used for the discovery of new drugs and agrochemicals (2)(3)(4).
On the other hand, naturally occurring isonitriles have so far been discovered in various organisms, including bacteria, fungi, marine sponges, etc.The first report of an isocyanide metabolite, xanthocillin, which was isolated from Penicillium notatum, was published in 1957 (5).An indoleacryloisocyanide was isolated from Pseudomonas NCIB 11237 through screening for antibiotic compounds produced by bacteria (6).The isonitriles that are elaborated by marine organisms, such as axisonitrile-1 (7) and 9-isocyanopupukeanane (8), form the largest group of naturally occurring isonitriles.A lot of other reports on the structures and biological activities of natural isocyanides have also been published (for reviews, see Refs. 1 and 9).However, information on their metabolism is quite limited.Although parts of the metabolic intermediates of some isonitriles have been elucidated through incorporation experiments (10 -13), the entire pathways remained undetermined.Moreover, none of the enzymes involved in isonitrile metabolism, except for our enzyme described below, has yet been identified, and no analyses at the gene level have been performed.
We have extensively studied (14 -19) the enzymes (i.e.nitrilase, nitrile hydratase, and amidase) involved in the metabolism of nitriles, which are isomers of isonitriles, and therefore are interested in the differences between the metabolism of nitriles and that of isonitriles.Recently, we isolated a microorganism that is able to degrade isonitriles, sp.N19 -2, from soil and identified it as Pseudomonas putida.In this strain, we discovered a novel enzyme that catalyzes the hydration of an isonitrile to the corresponding N-substituted formamide and named it isonitrile hydratase (20).It has been approved as a new enzyme by NC-IUBMB; EC 4.2.1.103.Among known enzymes, only nitrogenase has been reported to act on an isonitrile; it converts methyl isocyanide to the corresponding amine (21,22).However, isonitriles are not physiological substrates of the enzyme, and there is no evidence that it is involved in the metabolism of isonitriles in vivo.Therefore, our work on the isonitrile hydratase is the first on an enzyme involved in the metabolism of isocyano compounds.
In the present study, for the first time, we cloned an isonitrile hydratase gene (inhA) from P. putida N19 -2 and constructed an Escherichia coli transformant overexpressing InhA.We report the interesting sequence similarity between the isonitrile hydratase and an intracellular protease in Pyrococcus horikoshii (PH1704).We also attempted to identify its active amino acid by site-directed mutagenesis and obtained evidence that a cysteine residue (Cys-101) plays an important role in the catalytic mechanism of isonitrile hydratase.
Amino Acid Sequencing of Isonitrile Hydratase-The isonitrile hydratase was purified from P. putida N19 -2 as described previously (20), and its N-terminal amino acid sequence was determined by automated Edman degradation with an Applied Biosystems model 470A gas-phase amino acid sequencer (Foster City, CA).To determine its internal sequences, the purified enzyme was digested with lysyl endopeptidase (Wako Pure Chemicals, Tokyo, Japan) in 20 mM Tris-HCl buffer (pH 9.0) at 37 °C for 6 h.The reaction mixture was applied to a Smart System (reversed-phase chromatography; Amersham Biosciences) on a RPC C2.1/10 column and eluted with a linear gradient of acetonitrile (0 -80%) (v/v) in the presence of 0.1% (v/v) trifluoroacetic acid at a flow rate of 0.1 ml/min.The peptide fragments isolated were sequenced by automated Edman degradation.
Preparation of Genomic DNA from P. putida N19 -2-Genomic DNA was prepared from P. putida N19 -2 as follows: P. putida N19 -2 was cultured at 28 °C for 12 h in 20 ml of LB medium (23) with reciprocal shaking.The cells were harvested by centrifugation, washed with Tris/ EDTA buffer (50 mM Tris/50 mM EDTA (pH 8.0)), and then suspended in 1.5 ml of Tris/EDTA buffer containing 15% (w/v) sucrose.Then 1.5 ml of Tris/EDTA buffer containing 1% (w/v) sodium N-lauroylsarcosine was added to the cell suspension, and the mixture was incubated at 37 °C for 30 min.The solution was subjected to equilibrium centrifugation in a CsCl-ethidium bromide gradient, and the fraction containing genomic DNA was pooled, extracted with n-butanol to remove ethidium bromide, and then dialyzed against 10 mM Tris/1 mM EDTA (pH 8.0).
Southern hybridization was carried out using an Alkphos direct labeling and detection system with CDP-Star (Amersham Biosciences) according to the procedure recommended by the supplier.Colony hybridization was carried out as follows: the recombinant colonies were transferred to a nylon membrane, lysed with denaturing buffer (0.5 M NaOH, 1.5 M NaCl, 0.1% SDS) for 15 min, and then treated with neutralizing buffer (1 M Tris/HCl, 1.5 M NaCl (pH 7.5)) for 5 min and 2ϫ SSC (1ϫ SSC ϭ 0.15 M NaCl, 15 mM sodium citrate) for 15 min, successively.After DNA fixation by UV cross-linking, the membrane was washed in 3ϫ SSC containing 0.1% SDS at 68 °C for 3 h and then hybridization was carried out with the same system as used for Southern hybridization.Nucleotides were sequenced by the dideoxy-chain terminating method using an ABI Prism 310 genetic analyzer (Applied Biosystems).
Expression and Purification of Recombinant Isonitrile Hydratase-The coding sequence of the enzyme (inhA) was amplified by PCR with pINH10 as a template.The following two oligonucleotide primers were used: sense primer, 5Ј-CATATGGCGTTGCAGATCGGTTTTC-3Ј containing a NdeI recognition site (underlined) and 22 nucleotides of inhA starting with the ATG start codon (nucleotides 29 -50 in Fig. 1); antisense primer, 5Ј-GAATTCTCAGCGCAGATTGAGCTTCGC-3Ј containing an EcoRI recognition site (underlined) and 21 nucleotides that are complementary to the 3Ј-end sequence of inhA ending with the TGA stop codon (nucleotides 695-715 in Fig. 1).The amplified DNA was subcloned into vector pUC18 and checked by DNA sequencing.The insert DNA was digested with NdeI and EcoRI and then inserted into the respective sites of pET-21a(ϩ).The resultant plasmid was designated as pET-inhA; in this construction, inhA was under the control of the T7 promoter.E. coli BL21-CodonPlus(DE3)-RIL was transformed with pET-inhA, and the recombinant cells were used for the overproduction and purification of isonitrile hydratase.
The transformed cells were incubated with reciprocal shaking at 37 °C in 20 ml of 2ϫ YT medium containing 50 g/ml ampicillin and 34 g/ml chloramphenicol.After overnight cultivation, the entire culture was inoculated into 2 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, 0.01, 0.1, or 1 mM to induce the T7 promoter, and further cultivation was carried out at 37 °C for 4 or 12 h.The cells were harvested by centrifugation, washed twice with 10 mM potassium phosphate buffer (pH 7.5) containing 10% (v/v) glycerol, and then disrupted by sonication (Insonator Model 201 M; Kubota, Tokyo, Japan) to prepare a cell-free extract.The recombinant enzyme was partially purified from this extract through ammonium sulfate fractionation and DEAE-Sephacel column chromatography, by the same procedure as used for the purification of isonitrile hydratase from P. putida N19 -2 (20).Then the enzyme solution was applied to a Resource Q column (6.4 ϫ 30 mm) equilibrated with 10 mM buffer, which was attached to an A ¨KTA purifier (Amersham Biosciences) and eluted by increasing the ionic strength of KCl in a linear manner from 0 to 0.3 M. The active fractions were pooled, precipitated with 70% saturated ammonium sulfate, and then dialyzed against 10 mM buffer.
Site-directed Mutagenesis-Site-directed mutagenesis (converting Cys-101 to Ala, Thr-102 to Ala, Glu-79 to Gln, and Glu-81 to Gln, respectively) was carried out on inhA by means of an overlap extension PCR protocol (24,25).To construct the C101A mutant, two PCRs, with plasmid pET-inhA as the template, were performed with primer pairs C101A-S plus T7T and T7P plus C101A-AS (Table I).These reactions produced 3Ј and 5Ј fragments of inhA, respectively, whose sequences overlapped by 24 bp at the mutation.The second round of PCR was performed by mixing equimolar amounts of the first-round products and amplifying between primers T7P and T7T to produce the fulllength inhA.The second-round product was digested with NdeI and EcoRI, ligated into expression vector pET-21a(ϩ), and then sequenced.A clone with the sequence for the desired C101A mutation was chosen and transformed into E. coli BL21-CodonPlus(DE3)-RIL.The recombinant cells were used for the overproduction and purification of the C101A mutant enzyme.The T102A, E79Q, and E81Q mutations were constructed in the same manner as the C101A mutation, with the internal primer pairs T102A-S plus T102A-AS, E79Q-S plus E79Q-AS, and E81Q-S plus E81Q-AS, respectively (Table I).
Enzyme Assay-Isonitrile hydratase activity was assayed by the method described previously (20).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 standard experimental conditions.The protein concentration was determined according to Bradford (26).The specific activity is expressed as units/mg of protein.
Circular Dichroism Analysis-CD measurements were carried out with an Aviv model 62A DS spectrometer (Aviv Instrument, Lakewood, NJ) at 20 °C with a 1-mm lightpath cell.The CD spectra were obtained at the protein concentration of 0.2 mg/ml in the far UV region (200 -260 nm).

RESULTS
Cloning and Nucleotide Sequencing of the Isonitrile Hydratase Gene-Isonitrile hydratase was purified to homogeneity from P. putida N19 -2, and the amino acid sequences of peptides were determined by digesting the enzyme with lysyl endopeptidase.Two oligonucleotide primers were synthesized based on the N-terminal and internal sequences (corresponding to amino acids 10 -17 and 180 -188 in Fig. 1, respectively) and used for PCR amplification with genomic DNA of P. putida N19 -2 as a template, resulting in the generation of a 535-bp fragment.The deduced amino acid sequence of the amplified fragment was consistent with the internal sequences of the enzyme determined by Edman degradation, indicating that the fragment was a portion of the enzyme gene.
To obtain the entire isonitrile hydratase gene, after digestion of the genomic DNA with several restriction enzymes, Southern hybridization was performed using the 535-bp fragment as a probe.A single 6.4-kb SalI fragment was positively detected, and this fragment was recovered and ligated with SalI-digested pUC18 to transform E. coli DH10B.After screening of the recombinant plasmids by colony hybridization, a positive clone, designated as pINH10, was obtained.
The nucleotide sequencing of pINH10 revealed a 684-bp open reading frame encoding 228 amino acids (Fig. 1), which corresponded precisely to those determined with the purified isonitrile hydratase.The molecular mass of the protein encoded by this gene (inhA) was calculated to be 24,211 Da, which was a little bit different from that of the enzyme subunit (molecular mass ϭ 29 kDa) determined on SDS-PAGE (20).This discrepancy may be explained by the unusual mobility of the enzyme protein on SDS-PAGE, which was caused by its small SDS binding capacity, because some proteins exhibit greater resistance to binding than others (28).A typical Shine-Dalgarno sequence was present 6 bp upstream from the initiation codon, but none of the consensus promoter sequences were observed in the upstream region.There was a palindromic sequence (⌬G ϭ Ϫ41.9 kcal/mol) just downstream the termination codon, which seemed to be a putative transcriptional termination signal.
Expression and Purification of Recombinant Isonitrile Hydratase-To overproduce isonitrile hydratase in E. coli, the coding sequence (inhA) was inserted between the NdeI and EcoRI sites of pET-21a(ϩ), resulting in pET-inhA, in which the isonitrile hydratase gene was under the control of the T7 promoter.When E. coli harboring pET-inhA was cultured in the presence of isopropyl-1-thio-␤-D-galactopyranoside, isonitrile hydratase activity was observed in the cell-free extract.The maximum level of isonitrile hydratase activity was 5.23 units/mg (Table II); this value corresponded to 31.7% of the specific activity (16.5 units/mg: 100%) of the isonitrile hydratase purified from P. putida N19 -2 (20).We analyzed the cell-free extract by SDS-PAGE and detected a 29-kDa protein band corresponding to the subunit of the P. putida N19 -2 enzyme.Therefore, hyperproduction of isonitrile hydratase in the active form was attained.We also examined other hostvector systems (such as pUC plasmids and E. coli JM109) to increase the production of isonitrile hydratase but with none of the transformants was the enzyme activity more than 5.23 units/mg in the cell-free extract (data not shown).Thus, we used the combination of pET-inhA and E. coli BL21-Codon-Plus(DE3)-RIL in the following studies.
The isonitrile hydratase produced in the recombinant cells was purified to homogeneity through ammonium sulfate fractionation and two-step column chromatography procedures (Fig. 3, lane 1).The purified enzyme exhibited almost the same specific activity (17.3 units/mg; Table III) as the P. putida N19 -2 enzyme.

TABLE II Isonitrile hydratase activities of E. coli transformants under various culture conditions
Isopropyl-1-thio-␤-D-galactopyranoside (IPTG) was added to 2ϫ YT medium at the same time as the culture was started.be involved in the catalytic mechanism, and Glu-79 is the glutamate residue closest to Gly-75 (Fig. 2).Glu-81 was also selected, because it is located near Gly-75 and is the only conserved glutamate residue in the overall sequence.Each of the mutant enzymes was expressed in E. coli, purified to homogeneity (Fig. 3, lanes 2-5) according to the same procedure as used for the wild-type enzyme, and then characterized.
The specific activities of the mutant enzymes are shown in Table III.The C101A mutant exhibited no detectable isonitrile hydratase activity at all, even when a large amount of enzyme (over 200 times as much as usually used for assaying of the wild-type enzyme) was added to the reaction mixture.The T102A mutant exhibited a reduction of Ͼ90% in activity.On the other hand, the E79Q and E81Q mutants exhibited almost the same specific activity as the wild-type enzyme.The circular dichroism spectra of these four mutants were very similar to that of the parental enzyme; particularly those of the C101A mutant and the T102A mutant were almost identical to that of the wild-type enzyme, and those of the E79Q mutant and the E81Q mutant were a little bit above that of the wild-type enzyme (Fig. 4).These findings indicate that essentially no major change in the overall conformation of the enzyme protein was induced by the mutations.The native molecular mass of each mutant determined by gel-permeation chromatography (data not shown) was also similar to that of the wild-type enzyme, suggesting that the subunit composition was not altered (i.e.homodimer (20)).These findings demonstrate that Cys-101 and Thr-102 are important for catalytic activity, whereas Glu-79 and Glu-81 are not.

DISCUSSION
An isonitrile is a compound with an NC functional group that possesses an unusual valence structure and reactivity.Based on its unique reactivity, i.e. the ␣-addition of nucleophiles and electrophiles at the same isocyanide carbon, many synthetically useful methods have been developed (1).Among them, isocyanide-based multicomponent reactions are by far the most versatile and are receiving increasing attention, particularly in the pharmaceutical industry; they have become popular reactions for the preparation of a drug-like compound library (4).
On the other hand, many natural isonitriles have been isolated from various organisms.Most of them show a strong antibiotic effect and have potential as possible agents of practical use, e.g. a series of isocyanoterpenes, isolated from marine sponges, exhibit antimalarial activities (32,33), and other terpenoid isocyanides have antifouling properties similar to those of copper sulfate (34).However, no investigation of isonitrile metabolism at the protein or gene level was performed.Therefore, we embarked on the field of isonitrile biochemistry.In the previous paper (20), we reported the discovery of a novel enzyme involved in isonitrile metabolism, i.e. isonitrile hydratase.
A Hymeniacidon species (marine sponge) producing an isocyano-compound, 2-isocyanopupukeanane, was found to transform it into the corresponding formamide (10), which suggests the existence of isonitrile hydratase in the species.Because hydration is one of the simplest reactions that could occur in isonitrile metabolism, it is possible that isonitrile hydratase is not rarely present in isonitrile-producing organisms.There-fore, structural and functional analysis of it would be a good first step for elucidating the metabolic pathways for naturally occurring isonitriles.In the present work, we conducted the cloning and sequencing of the isonitrile hydratase gene and searched for homologs of it in public databases.We also attempted the expression of isonitrile hydratase in E. coli and succeeded in hyperproduction of the enzyme in the active form.Because the enzymatic hydration of isonitrile is quite a novel reaction, we are interested in the catalytic mechanism of isonitrile hydratase, which remains undetermined.Thus, we attempted the identification of its active amino acid residues by site-directed mutagenesis.
The amino acid sequence of isonitrile hydratase exhibited significant similarity to those of several proteins belonging to the ThiJ/PfpI family.This family includes proteins involved in RNA-protein interaction regulation, thiamine biosynthesis, and protease activity, which do not appear to be related to one another.The role of this family remains unclear, and most of the members are predicted proteins that have, to our knowledge, not been functionally analyzed except for ThiJ, which catalyzes hydroxymethylpyrimidine phosphorylation in the thiamine biosynthetic pathway (35,36), and for intracellular proteases (PfpI and PH1704) in hyperthermophilic archaea (30,31).Here, we discovered that the members of the ThiJ/PfpI family and isonitrile hydratase (which is an enzyme playing a novel physiological role in isonitrile degradation) comprise a novel superfamily.
Our previous inhibition studies on isonitrile hydratase suggested that the enzyme may have an active cysteine residue (20).Isonitrile hydratase exhibits low sequence similarity with PH1704, and a cysteine residue that corresponds to the putative nucleophile (Cys-100) in PH1704 is conserved in isonitrile hydratase (at amino acid position 101).Mutational analysis clearly demonstrated that Cys-101 is crucial for isonitrile hydratase activity.A carbon atom of the NC functional group is negatively charged (R-N ϩ ϵC Ϫ ) and therefore seems to be inert as to nucleophilic attack.In fact, an isonitrile under acidic conditions is easily and spontaneously transformed to the corresponding formamide through a chemical reaction, where the initial attack comprises protonation of the isocyano carbon, i.e. electrophilic addition (37).However, an aromatic isonitrile has been found to undergo nucleophilic attack of hydroxide in alkaline aqueous dioxane or in aqueous dimethyl sulfoxide, which was concerted with or followed by proton transfer from water, leading to the formation of the corresponding formamide (38,39).This finding suggests that the nucleophilic attack of the active cysteine residue is not so unreasonable in the isonitrile hydratase reaction.The specific activity of isonitrile hydratase decreases as the pH decreases below 6.0 (20).This finding is also consistent with the postulation that the initial attack on the NC functional group comprises nucleophilic addition, because the nucleophile becomes less active under acidic conditions because of protonation.A possible reaction mechanism is shown in Fig. 5.The nucleophilic attack of Cys-101 is assumed to lead to the formation of an enzyme-thioimidate intermediate, which is subsequently hydrolyzed (via a tetrahedral intermediate; see [I] in Fig. 5) to yield the N-substituted formamide.In the Pyrococcus protease PH1704, His-101 and Glu-74 are supposed to form a catalytic triad with Cys-100, although the reaction mechanism has never been determined by biochemical analysis.In the isonitrile hydratase, on the other hand, Thr-102 (which corresponds to His-101 in PH1704) appears to be an important component of the enzyme activity, whereas Glu-79 and Glu-81 (which were selected in place of Gly-75, actually corresponding to Glu-74 in PH1704) are not.Judging from a generally high pK a value (ϳ13) of the hydroxyl group in a threonine residue (40), it is not likely that Thr-102 forms a catalytic diad (or triad) with Cys-101 (and another amino acid residue) and functions as a catalytic base to activate the nucleophilic cysteine.Thus, the catalytic role of Thr-102 would be different from that of His-101 in PH1704.Although there is sequence similarity between the isonitrile hydratase and protease, their reaction schemes are completely different from each other; the former reaction comprises the hydration of an isonitrile (-NϵC) to an N-substituted formamide, and the latter one comprises the hydrolysis of a peptide bond (-C(ϭO)-NH-).To elucidate the catalytic mechanism of isonitrile hydratase, further investigations involving determination of the role of Thr-102 and identification of another active amino acid (that functions as a general base abstracting the proton from the active Cys-101) are required.
We are interested in the evolutionary relationship between the isonitrile hydratase involved in NϵC triple bond hydration and the C-N hydrolases such as nitrilase (which catalyzes the hydrolysis of a nitrile to an acid and ammonium) (17,(41)(42)(43), nitrile hydratase (which catalyzes the hydration of a nitrile to an amide) (18,19,44,45), amidase (which catalyzes the hydrolysis of an amide to an acid and ammonium) (46 -48), and protease (which catalyzes the hydrolytic cleavage of a peptide bond).Although nitrile hydratase acts on the CϵN triple bond and apparently catalyzes the same hydration reaction as that in the case of isonitrile hydratase, there is no sequence similarity between these enzymes.The present work suggests that isonitrile hydratase is more closely related to the proteases than the nitrile-metabolizing enzymes.To understand the meaning of the evolutionary relationship, further analyses of isonitrile hydratase, such as more extensive mutagenesis studies and crystallographic analysis, are in progress.These investigations could lead to new developments in the research fields of the enzymology and physiology of C-N hydrolases.

FIG. 1 .
FIG. 1.Nucleotide and amino acid sequences of the isonitrile hydratase gene.The underlined amino acid sequences were determined by Edman degradation.A potential ribosome-binding sequence is indicated by S.D. (Shine-Dalgarno), and a relevant stop codon is indicated by an asterisk.An inverted repeat sequence downstream from the isonitrile hydratase gene is indicated by opposing arrows.The nucleotide sequence has been deposited in the DNA Data Bank Japan under DDBJ accession number AB088117.

FIG. 2 .
FIG. 2. Alignment of the amino acid sequences of isonitrile hydratase (InhA) and the intracellular proteases from P. horikoshii (PH1704) and P. furiosus (PfpI).Residues that are conserved between InhA and either of the intracellular proteases are highlighted in reverse type.Arrows indicate the amino acid residues (i.e.Glu-79, Glu-81, Cys-101, and Thr-102 in InhA, from left to right, respectively) that were mutated in this work.

FIG. 5 .
FIG. 5. A mechanistic proposal for the isonitrile hydratase reaction.The active sulfhydryl group is denoted by ESH.The tetrahedral intermediate is denoted by [I].

TABLE I
Oligonucleotide primers used for the preparation of mutant isonitrile hydratasesBold letters indicate the nucleotides changed for the desired mutations.