Isonitrile Hydratase from Pseudomonas putidaN19–2
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m208571200
ISSN1083-351X
AutoresMasahiko Goda, Yoshiteru Hashimoto, Masanori Takase, Sachio Herai, Yasuhito Iwahara, Hiroki Higashibata, Michihiko Kobayashi,
Tópico(s)Enzyme Structure and Function
ResumoIsonitrile hydratase is a novel enzyme inPseudomonas 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 coligave a large amount of soluble isonitrile hydratase exhibiting the same molecular and catalytic properties as the native enzyme from thePseudomonas strain. The predicted amino acid sequence ofinhA 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 corresponding position (Cys-101). A mutant enzyme containing Ala instead of Cys-101 did not exhibit isonitrile hydratase activity at all, demonstrating the essential role of this residue in the catalytic function. Isonitrile hydratase is a novel enzyme inPseudomonas 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 coligave a large amount of soluble isonitrile hydratase exhibiting the same molecular and catalytic properties as the native enzyme from thePseudomonas strain. The predicted amino acid sequence ofinhA 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 corresponding position (Cys-101). A mutant enzyme containing Ala instead of Cys-101 did not exhibit isonitrile hydratase activity at all, demonstrating the essential role of this residue in the catalytic function. An isonitrile (more generally called an isocyanide) is a highly toxic compound with an isocyano group (-N≡C). The isonitriles form the only class of organic compounds that contain a stable, formally mono-coordinated carbon, cf. carbon monoxide (1Edenborough M.S. Herbert R.B. Natl. Prod. Rep. 1988; 5: 229-245Crossref PubMed Google Scholar). 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 (1Edenborough M.S. Herbert R.B. Natl. Prod. Rep. 1988; 5: 229-245Crossref PubMed Google Scholar); 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 (2Dömling A. Curr. Opin. Chem. Biol. 2000; 4: 318-323Crossref PubMed Scopus (194) Google Scholar, 3Dömling A. Curr. Opin. Chem. Biol. 2002; 6: 306-313Crossref PubMed Scopus (507) Google Scholar, 4Dömling A. Ugi I. Angew. Chem. Int. Ed. 2000; 39: 3168-3210Crossref PubMed Google Scholar). 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 (5Hagedorn I. Tönjes H. Pharmazie. 1957; 12: 567-580PubMed Google Scholar). An indoleacryloisocyanide was isolated fromPseudomonas NCIB 11237 through screening for antibiotic compounds produced by bacteria (6Evans J.R. Napier E.J. Yates P. J. Antibiot. 1976; 29: 850-852Crossref PubMed Scopus (29) Google Scholar). The isonitriles that are elaborated by marine organisms, such as axisonitrile-1 (7Cafieri F. Fattorusso E. Magno S. Santacroce C. Sica D. Tetrahedron. 1973; 29: 4259-4262Crossref Scopus (81) Google Scholar) and 9-isocyanopupukeanane (8Burreson B.J. Scheuer P.J. Finer J. Clardy J. J. Am. Chem. Soc. 1975; 97: 4763-4764Crossref Scopus (149) Google Scholar), 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. 1Edenborough M.S. Herbert R.B. Natl. Prod. Rep. 1988; 5: 229-245Crossref PubMed Google Scholar and 9Scheuer P.J. Acc. Chem. Res. 1992; 25: 433-439Crossref Scopus (93) Google Scholar). However, information on their metabolism is quite limited. Although parts of the metabolic intermediates of some isonitriles have been elucidated through incorporation experiments (10Hagadone M.R. Scheuer P.J. Holm A. J. Am. Chem. Soc. 1984; 106: 2447-2448Crossref Scopus (62) Google Scholar, 11Achenbach H. Grisebach H. Z. Naturforsch. 1965; 20B: 137-140Crossref Scopus (36) Google Scholar, 12Achenbach H. König F. Chem. Ber. 1972; 105: 784-793Crossref Scopus (21) Google Scholar, 13Pfeifer S. Bär H. Zarnack J. Pharmazie. 1972; 27: 536-542PubMed Google Scholar), 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 (14Kobayashi M. Shimizu S. Nature Biotechnol. 1998; 16: 733-736Crossref PubMed Scopus (274) Google Scholar, 15Yamada H. Kobayashi M. Biosci. Biotechnol. Biochem. 1996; 60: 1391-1400Crossref PubMed Scopus (410) Google Scholar, 16Kobayashi M. Nagasawa T. Yamada H. Trends Biotechnol. 1992; 10: 402-408Abstract Full Text PDF PubMed Scopus (309) Google Scholar, 17Kobayashi M. Izui H. Nagasawa T. Yamada H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 247-251Crossref PubMed Scopus (135) Google Scholar, 18Komeda H. Kobayashi M. Shimizu S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4267-4272Crossref PubMed Scopus (108) Google Scholar, 19Kobayashi M. Shimizu S. Eur. J. Biochem. 1999; 261: 1-9Crossref PubMed Scopus (374) Google Scholar) 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 asPseudomonas 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 (20Goda M. Hashimoto Y. Shimizu S. Kobayashi M. J. Biol. Chem. 2001; 276: 23480-23485Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). 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 (21Kelly M. Postgate J.R. Richards R.L. Biochem. J. 1967; 102: 1-3Crossref PubMed Scopus (43) Google Scholar, 22Rubinson J.F. Corbin J.L. Burgess B.K. Biochemistry. 1983; 22: 6260-6268Crossref PubMed Scopus (30) Google Scholar). 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. P. putida N19–2 was isolated previously from soil (20Goda M. Hashimoto Y. Shimizu S. Kobayashi M. J. Biol. Chem. 2001; 276: 23480-23485Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). E. coli DH10B (Invitrogen) was used as the host for pUC plasmids (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar).E. coli BL21-CodonPlus(DE3)-RIL (Novagen) was used as the host for a plasmid, pET-21a(+) (Novagen), and its derivative and was also used for expression of the isonitrile hydratase gene (inhA). E. coli transformants were grown in 2× YT medium (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The isonitrile hydratase was purified from P. putida N19–2 as described previously (20Goda M. Hashimoto Y. Shimizu S. Kobayashi M. J. Biol. Chem. 2001; 276: 23480-23485Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), 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 μRPC 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. 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 (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) 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) sodiumN-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). An oligonucleotide sense primer (23-mer, 512 variants, 5′TTYCCICARGTNCARCARYTNGA-3′; I = deoxyinosine) and an antisense primer (26-mer, 512 variants, 5′-TCRAAIGGIGGNGCNGGNGCRTAYTC-3′) were synthesized based on the N-terminal (FPQVQQLD) and internal amino acid (EYAPAPPFD) sequences of the enzyme, respectively. A reaction mixture (50 μl) comprising 10 ng of genomic DNA, 300 pmol of each primer, and Ex Taqpolymerase (Takara Bio Inc., Otsu, Japan) was subjected to PCR (94 °C for 30 s, 45 °C for 30 s, 72 °C for 60 s; 30 cycles), and the amplified DNA fragment (535 bp) was gel-purified. The DNA fragment was then used as a probe for Southern hybridization and colony hybridization to clone the full-length isonitrile hydratase gene (inhA). 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 (0.5m NaOH, 1.5 m NaCl, 0.1% SDS) for 15 min, and then treated with neutralizing buffer (1 m Tris/HCl, 1.5m NaCl (pH 7.5)) for 5 min and 2× SSC (1× SSC = 0.15m NaCl, 15 mm sodium citrate) for 15 min, successively. After DNA fixation by UV cross-linking, the membrane was washed in 3× 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). 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, 5′-CATATGGCGTTGCAGATCGGTTTTC-3′ containing aNdeI recognition site (underlined) and 22 nucleotides ofinhA starting with the ATG start codon (nucleotides 29–50 in Fig. 1); antisense primer, 5′-GAATTCTCAGCGCAGATTGAGCTTCGC-3′ containing anEcoRI recognition site (underlined) and 21 nucleotides that are complementary to the 3′-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(+). 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 2× YT medium containing 50 μg/ml ampicillin and 34 μg/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-β-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. putidaN19–2 (20Goda M. Hashimoto Y. Shimizu S. Kobayashi M. J. Biol. Chem. 2001; 276: 23480-23485Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Then the enzyme solution was applied to a Resource Q column (6.4 × 30 mm) equilibrated with 10 mm buffer, which was attached to an ÄKTA 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 (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 (24Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar, 25Pogulis R.J. Yallejo A.N. Pease L.R. Methods Mol. Biol. 1996; 57: 167-176PubMed Google Scholar). 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 3′ and 5′ 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 full-length inhA. The second-round product was digested with NdeI and EcoRI, ligated into expression vector pET-21a(+), and then sequenced. A clone with the sequence for the desired C101A mutation was chosen and transformed intoE. 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).Table IOligonucleotide primers used for the preparation of mutant isonitrile hydratasesNameSequencePositionT7P :5′-TAATACGACTCACTATAGGG-3′T7T :5′-GCTAGTTATTGCTCAGCGG-3′C101A-S :5′-GTTACGTCGGTTGCGACCGGTTCGCTTGTG-3′317–346C101A-AS:5′-CGAACCGGTCGCAACCGACGTAACGTACCG-3′311–340T102A-S :5′-TCGGTTTGCGCCGGTTCGCTTGTGCTTGGC-3′323–352T102A-AS:5′-CACAAGCGAACCGGCGCAAACCGACGTAAC-3′317–346E79Q-S :5′-GGGCCGTTGATGCAGGATGAGCAGACGCTG-3′251–280E79Q-AS :5′-CTGCTCATCCTGCATCAACGGCCCGACCCC-3′245–274E81Q-S :5′-TTGATGGAGGATCAGCAGACGCTGGACTTC-3′257–286E81Q-AS :5′-GTCCAGCGTCTGCTGATCCTCCATCAACGG-3′254–283Bold letters indicate the nucleotides changed for the desired mutations. Open table in a new tab Bold letters indicate the nucleotides changed for the desired mutations. Isonitrile hydratase activity was assayed by the method described previously (20Goda M. Hashimoto Y. Shimizu S. Kobayashi M. J. Biol. Chem. 2001; 276: 23480-23485Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). One unit of isonitrile hydratase activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol N-cyclohexylformamide/min from cyclohexyl isocyanide under the standard experimental conditions. The protein concentration was determined according to Bradford (26Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217508) Google Scholar). The specific activity is expressed as units/mg of protein. SDS-PAGE was performed in a 12.5% polyacrylamide slab gel according to Laemmli (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207523) Google Scholar). The gel was stained with Coomassie Brilliant Blue R-250. The molecular mass of the enzyme subunit was determined from the relative mobilities of marker proteins, phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa). 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). Isonitrile hydratase was purified to homogeneity fromP. 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-kbSalI fragment was positively detected, and this fragment was recovered and ligated with SalI-digested pUC18 to transformE. 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 = 29 kDa) determined on SDS-PAGE (20Goda M. Hashimoto Y. Shimizu S. Kobayashi M. J. Biol. Chem. 2001; 276: 23480-23485Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). 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 (28Nelson C.A. J. Biol. Chem. 1971; 246: 3895-3901Abstract Full Text PDF PubMed Google Scholar). 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 (ΔG = −41.9 kcal/mol) just downstream the termination codon, which seemed to be a putative transcriptional termination signal. A search of protein sequence databases with the BLAST server revealed that isonitrile hydratase exhibits significant similarity to several ThiJ/PfpI family proteins, e.g. a putative protein inNostoc sp. PCC 7120 (DDBJ BAB771641; 54% identity), two putative proteins in Caulobacter crescentus CB15 (GenBankTMAAK24921.1 and AAK23756.1; 51 and 46% identity, respectively), a putative AraC-type transcriptional regulator in Methanosarcina mazei Goe1 (GenBankTMAAM33006.1; 40% identity), and a putative 4-methyl-5-(β-hydroxyethyl)-thiazole monophosphate biosynthesis enzyme in Mycobacterium tuberculosis CDC1551 (GenBankTMAAK44280.1; 40% identity). Isonitrile hydratase also showed sequence similarity to the intracellular proteases fromP. horikoshii (PH1704) (Swiss-Prot O59413; 24% identity) (29Kawarabayasi Y. Sawada M. Horikawa H. Haikawa Y. Hino Y. Yamamoto S. Sekine M. Baba S. Kosugi H. Hosoyama A. Nagai Y. Sakai M. Ogura K. Otsuka R. Nakazawa H. Takamiya M. Ohfuku Y. Funahashi T. Tanaka T. Kudoh Y. Yamazaki J. Kushida N. Oguchi A. Aoki K. Yoshizawa T. Nakamura Y. Robb F.T. Horikoshi K. Masuchi Y. Shizuya H. Kikuchi H. DNA Res. 1998; 5: 55-76Crossref PubMed Scopus (555) Google Scholar) and Pyrococcus furiosus (PfpI) (Swiss-ProtQ51732; 24% identity) (30Halio S.B. Blumentals I.I. Short S.A. Merrill B.M. Kelly R.M. J. Bacteriol. 1996; 178: 2605-2612Crossref PubMed Google Scholar), although the similarities were very low. Recently, the crystal structure of PH1704 was solved (31Du X. Choi I.G. Kim R. Wang W. Jancarik J. Yokota H. Kim S.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14079-14084Crossref PubMed Scopus (98) Google Scholar). Judging from the three-dimensional structural information, Cys-100 must be the active site nucleophile, which comprises a catalytic triad with His-101 and Glu-74 (of an adjacent monomer) in PH1704. The active Cys-100 in PH1704 was conserved at the corresponding position in isonitrile hydratase (Cys-101) (Fig. 2). Therefore, we investigated the catalytic role of Cys-101 in isonitrile hydratase by means of site-directed mutagenesis. The residues (i.e.Thr-102 and Glu-79/Glu-81 in isonitrile hydratase) that correspond to His-101 and Glu-74 in PH1704 were also investigated (see below). To overproduce isonitrile hydratase in E. coli, the coding sequence (inhA) was inserted between the NdeI and EcoRI sites of pET-21a(+), 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-β-d-galactopyranoside, isonitrile hydratase activity was observed in the cell-free extract. The maximum level of isonitrile hydratase activity was 5.23 units/mg (TableII); 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 (20Goda M. Hashimoto Y. Shimizu S. Kobayashi M. J. Biol. Chem. 2001; 276: 23480-23485Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). 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 host-vector 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 andE. coli BL21-CodonPlus(DE3)-RIL in the following studies.Table IIIsonitrile hydratase activities of E. coli transformants under various culture conditionsCultivation timeIPTG concentrationSpecific activityhmmunits/mg401.301200.5640.011.41120.013.3840.13.96120.15.23411.561210.91Isopropyl-1-thio-β-d-galactopyranoside (IPTG) was added to 2× YT medium at the same time as the culture was started. Open table in a new tab Isopropyl-1-thio-β-d-galactopyranoside (IPTG) was added to 2× YT medium at the same time as the culture was started. 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; TableIII) as the P. putida N19–2 enzyme.Table IIISpecific activities of the wild-type and mutant isonitrile hydratasesEnzymeSpecific activityRelative activityunits/mg%Wild type17.3100E79Q18.7108E81Q15.489C101ANDaNot detected.0T102A1.418.2a Not detected. Open table in a new tab We constructed four mutants of isonitrile hydratase by site-directed mutagenesis, converting Cys-101 to Ala (C101A), Thr-102 to Ala (T102A), Glu-79 to Gln (E79Q), and Glu-81 to Gln (E81Q), respectively. Cys-101 is the amino acid residue that corresponds to Cys-100 in PH1704, whereas Thr-102 corresponds to His-101 in PH1704 (Fig. 2). Instead of Gly-75 (which actually corresponds to Glu-74 in PH1704), Glu-79 was selected as a candidate for the active amino acid residue, because the glycine residue did not seem to 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 >90% 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 (20Goda M. Hashimoto Y. Shimizu S. Kobayashi M. J. Biol. Chem. 2001; 276: 23480-23485Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar)). These findings demonstrate that Cys-101 and Thr-102 are important for catalytic activity, whereas Glu-79 and Glu-81 are not. 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 α-addition of nucleophiles and electrophiles at the same isocyanide carbon, many synthetically useful methods have been developed (1Edenborough M.S. Herbert R.B. Natl. Prod. Rep. 1988; 5: 229-245Crossref PubMed Google Scholar). 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 (4Dömling A. Ugi I. Angew. Chem. Int. Ed. 2000; 39: 3168-3210Crossref PubMed Google Scholar). 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 (32Angerhofer C.K. Pezzuto J.M. König G.M. Wright A.D. Sticher O. J. Nat. Prod. 1992; 55: 1787-1789Crossref PubMed Scopus (96) Google Scholar, 33Wright A.D. Wang H. Gurrath M. König G.M. Kocak G. Neumann G. Loria P. Foley M. Tilley L. J. Med. Chem. 2001; 44: 873-885Crossref PubMed Scopus (111) Google Scholar), and other terpenoid isocyanides have antifouling properties similar to those of copper sulfate (34Fusetani N. Curr. Org. Chem. 1997; 1: 127-152Google Scholar). However, no investigation of isonitrile metabolism at the protein or gene level was performed. Therefore, we em
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