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The Desymmetrization of Bicyclic β-Diketones by an Enzymatic Retro-Claisen Reaction

2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês

10.1074/jbc.m011538200

1083-351X

Gideon Grogan, Gareth A. Roberts, Despina J. Bougioukou, Nicholas J. Turner, Sabine L. Flitsch,

Microbial bioremediation and biosurfactants

The enzyme 6-oxocamphor hydrolase, which catalyzes the desymmetrization of 6-oxocamphor to yield (2R,4S)-α-campholinic acid, has been purified with a factor of 35.7 from a wild type strain ofRhodococcus sp. NCIMB 9784 grown on (1R)-(+)-camphor as the sole carbon source. The enzyme has a subunit molecular mass of 28,488 Da by electrospray mass spectrometry and a native molecular mass of ∼83,000 Da indicating that the active protein is trimeric. The specific activity was determined to be 357.5 units mg−1, and theKm was determined to be 0.05 mm for the natural substrate. The N-terminal amino acid sequence was obtained from the purified protein, and using this information, the gene encoding the enzyme was cloned. The translation of the gene was found to bear significant homology to the crotonase superfamily of enzymes. The gene is closely associated with an open reading frame encoding a ferredoxin reductase that may be involved in the initial step in the biodegradation of camphor. A mechanism for 6-oxocamphor hydrolase based on sequence homology and the known mechanism of the crotonase enzymes is proposed. The enzyme 6-oxocamphor hydrolase, which catalyzes the desymmetrization of 6-oxocamphor to yield (2R,4S)-α-campholinic acid, has been purified with a factor of 35.7 from a wild type strain ofRhodococcus sp. NCIMB 9784 grown on (1R)-(+)-camphor as the sole carbon source. The enzyme has a subunit molecular mass of 28,488 Da by electrospray mass spectrometry and a native molecular mass of ∼83,000 Da indicating that the active protein is trimeric. The specific activity was determined to be 357.5 units mg−1, and theKm was determined to be 0.05 mm for the natural substrate. The N-terminal amino acid sequence was obtained from the purified protein, and using this information, the gene encoding the enzyme was cloned. The translation of the gene was found to bear significant homology to the crotonase superfamily of enzymes. The gene is closely associated with an open reading frame encoding a ferredoxin reductase that may be involved in the initial step in the biodegradation of camphor. A mechanism for 6-oxocamphor hydrolase based on sequence homology and the known mechanism of the crotonase enzymes is proposed. The desymmetrization of prochiral substrates in organic synthesis remains a powerful technique for the generation of chiral intermediates with, in principle, 100% yield with absolute optical purity. In addition to chemical processes that have been reviewed recently (1Willis M.C. J. Chem. Soc. Perkin Trans. 1999; 1: 1765-1784Crossref Google Scholar), enzyme-catalyzed methods have assumed an important role in desymmetrization (2Schoffers E. Golebiowski A. Johnson C.R. Tetrahedron. 1996; 52: 3769-3826Crossref Scopus (340) Google Scholar) owing to the well documented advantages of biocatalysis in general. Such methods have usually exploited the regioselectivity of a hydrolytic enzyme, such as a lipase or nitrilase, to effect transformation of one of two identical functions in a molecule (3Yang Y.-F. Sih C.J. Tetrahedron Lett. 1984; 25: 4999-5002Crossref Scopus (66) Google Scholar, 34Yokoyama M. Sugai T. Ohta H. Tetrahedron: Asymmetry. 1993; 6: 1081-1084Crossref Scopus (47) Google Scholar). Enzymatic desymmetrizations have for the most part been performed using carbon-heteroatom bond hydrolases of this type, although Taschner and Black (4Taschner M.J. Black D.J. J. Am. Chem. Soc. 1988; 110: 6892-6893Crossref Scopus (138) Google Scholar) were successful in desymmetrizing a series of prochiral cyclic ketones using an enzymatic Baeyer-Villiger reaction. The metabolism of (1R)-(+)-camphor byCorynebacterium sp. T1 (now taxonomically reclassified and deposited as Rhodococcus sp. NCIMB 9784) was described in the 1960s by Gunsalus and co-workers (5Chapman P.J. Meerman G. Gunsalus I.C. Srinivasan R. Rinehart K. J. Am. Chem. Soc. 1966; 88: 618-619Crossref Scopus (35) Google Scholar). The pathway is distinct from that found in Pseudomonas putida (ATCC 17453; NCIMB 10007) in that initial hydroxylation occurs in the 6-endo position of the camphor skeleton (Fig. 1). 6-endo-Hydroxy camphor 2 is oxidized to a symmetrical diketone 3, which is then cleaved by a retro-Claisen reaction to yield a keto acid 4, which was reported by Gunsalus and co-workers (5Chapman P.J. Meerman G. Gunsalus I.C. Srinivasan R. Rinehart K. J. Am. Chem. Soc. 1966; 88: 618-619Crossref Scopus (35) Google Scholar) to have a negative optical rotation. This last enzymatic reaction is in fact a desymmetrization, and is interesting in that it apparently proceeds by an unusual enzyme-catalyzed retro-Claisen reaction. Enzymes that hydrolyze 1,3-diketo functionality, β-diketone hydrolases (6Pokorny D. Steiner W. Ribbons D.W. Trends Biotechnol. 1997; 15: 291-297Abstract Full Text PDF Scopus (29) Google Scholar) are rare, and only three reports describe their purification to homogeneity and characterization (7Hsiang H.H. Sim S.S. Mahuran D.J. Schmidt Jr., D.E. Biochemistry. 1972; 11: 2098-2102Crossref PubMed Scopus (24) Google Scholar, 8Davey J.F. Ribbons D.W. J. Biol. Chem. 1975; 250: 3826-3830Abstract Full Text PDF PubMed Google Scholar, 15Kawagoshi Y. Fujita M. World J. Microbiol. Biotechnol. 1998; 14: 95-100Crossref Scopus (30) Google Scholar, 35Pokorny D. Brecker L. Pogorevc M. Steiner W. Griengl H. Kappe T. Ribbons D.W. J. Bacteriol. 1999; 181: 5051-5059Crossref PubMed Google Scholar). In the first two cases, the substrate specificity of the enzymes extends only to 3,5-diketo acids. This specificity has been recently explained by elucidation of the x-ray crystal structure of fumarylacetoacetate hydrolase (9Timm D.E. Mueller H.A. Bhanumoorthy P. Harp J.M. Bunick G.J. Structure. 1999; 7: 1023-1033Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), which shows a dependence on a divalent calcium ion for substrate recognition and enolate stabilization. In view of the ongoing interest in the biocatalytic generation of chiral intermediates and desymmetrization in particular, we were interested in studying the potential of the enzyme, which we have named 6-oxocamphor hydrolase, for the desymmetrization of other cyclic and bicyclic β-diketones. In this paper, we present the purification and initial characterization of the enzyme from the wild type strain ofRhodococcus and the cloning of the gene encoding its activity. Our findings suggest that the enzyme does not share homology with other β-diketone hydrolases but rather is related to a different class of enzyme, the crotonase superfamily. Restriction endonucleases were from New England Biolabs. T4 DNA ligase, T4 polynucleotide kinase, and calf intestinal alkaline phosphatase were from Roche Molecular Biochemicals. RNase was purchased from Sigma-Adrich (Poole, United Kingdom). Protein and DNA size markers were obtained from (Amersham, United Kingdom) Pharmacia Biotech. [γ-32P]ATP (3000 Ci/mmol) was purchased from Amersham Pharmacia Biotech. Isopropyl-β-d-thiogalactopyranoside was obtained from U. S. Biochemical Corp. All other chemicals were purchased from Sigma-Aldrich. 6-Oxocamphor 3 was synthesized by pyridinium chlorochromate oxidation (10Corey E.J. Suggs J.W. Tetrahedron Lett. 1975; 31: 2647-2650Crossref Scopus (2697) Google Scholar) of 6-endo-hydroxy camphor derived from ethyl acetate extractions of the mother liquor of fermentations ofRhodococcus sp. NCIMB 9784 grown on (1R)-(+)-camphor performed as described below. Escherichia coli XL1 Blue supercompetent cells were obtained from Stratagene (La Jolla, CA) and grown on 1% yeast extract, 1% Tryptone, and 0.5% NaCl at 37 °C. Rhodococcus sp. NCIMB 9784 was obtained from the National Culture of Industrial and Marine Bacteria (Aberdeen, United Kingdom). The bacterium was maintained on nutrient agar slopes at room temperature. Ten 250-ml shake flasks containing 50 ml of basal salts medium supplemented with 35 mm sodium pyruvate were inoculated from slope and grown on an orbital shaker at 220 rpm at 30 °C for 3 days. This combined inoculum was used to seed 10 liters of basal salts medium in a 12-liter fermentation vessel (BioFlo 1000 fermenter, New Brunswick Scientific) supplemented with 1 g liter−1 (R)-(+)-camphor (Aldrich). Following 2 days of growth at 30 °C with an impeller speed of 250 rpm and an air flow of 4 liters min−1, a further 1 g liter−1 was added. After an additional 1 day of growth, the bacteria were harvested by centrifugation to yield a typical wet weight of 4 g liter−1. Activity of 6-oxocamphor hydrolase was monitored using a Hewlett Packard 8453 UV-visible spectrophotometer. The assay was performed as follows: to a 3-ml stirred cuvette containing 2990 μl of 50 mm Tris/HCl buffer, pH 7.0 (henceforth referred to as “buffer”) and the appropriate concentration of substrate (for standard assays, 100 μm) was added 10 μl of enzyme solution, and the disappearance of substrate was measured using a decrease in absorption at 294 nm. Calculations were made using an ε value of 258 mol dm−3 cm−3 for 6-oxocamphor. For inhibition/activation studies, 2990 μl of buffer containing 3 units of 6-oxocamphor hydrolase and the appropriate concentration of additive were preincubated at 25 °C prior to assay. The assay was initiated by addition of 10 μl of a 30 mm ethanolic solution of the substrate. 40 g of cell paste was suspended in 150 ml of buffer, and to this suspension was added 150 ml of 5-mm glass beads. The mixture was homogenized twice for 15 min each at 4500 rpm in a Type KDL Dynomill. The resulting homogenate was centrifuged at 11,000 rpm using a GSA rotor to yield a cell extract (320 ml) from which a 40–80% ammonium sulfate cut was derived. After dissolution in buffer and dialysis against 6 liters of the same buffer (with one change), the protein solution (100 ml) was taken to a concentration of 1.7 m ammonium sulfate and loaded onto a 2.5 × 10-cm phenyl Sepharose column that was eluted with a decreasing gradient of ammonium sulfate. Fractions exhibiting 6-oxocamphor hydrolase activity were pooled and precipitated by the addition of 80% ammonium sulfate. After overnight dialysis against 6 liters of buffer at 4 °C, the protein solution (25 ml) was applied to a 2.5 × 10-cm Fast Flow Q Sepharose column and eluted with an increasing gradient of potassium chloride (0–0.5 m). Active fractions were pooled, precipitated, and dialyzed as above. The resulting protein solution (6 ml) was taken to a concentration of 1.7m ammonium sulfate and divided into two aliquots, which were separately loaded onto a prepacked H/R 5/5 phenyl Superose column (1 ml) and eluted with a decreasing gradient of salt. Active fractions were identified, and their purity was assessed by SDS-polyacrylamide gel electrophoresis prior to being pooled. The enzyme was stored at −80 °C for at least six weeks with no discernible loss of activity. The isoelectric point of the purified protein was determined by isoelectric focusing using an Amersham Pharmacia Biotech Phastsystem. The native molecular weight was determined by calibrated gel filtration chromatography on a Superose 12 gel filtration column against a range of commercially available standards (Amersham Pharmacia Biotech). The void volume of the column was determined using blue dextran 2000. N-terminal sequencing was performed according to the method of Hayeset al. (11Hayes J.D. Kerr L.A. Cronshaw A.D. Biochem. J. 1989; 264: 437-445Crossref PubMed Scopus (82) Google Scholar), and 18 residues were unambiguously assigned as Met-Lys-Gln-Leu-Ala-Thr-Pro-Phe-Gln- Glu-Tyr-Ser-Gln-Lys-Tyr-Glu-Asn-Ile. Liquid chromatography-mass spectrometry of pure 6-oxocamphor hydrolase was performed on a Waters/Alliance 2690 HPLC system fitted with a Phenomenex Jupiter C18 300-Å, 250 mm × 2 mm × 5 μm column. The flow rate was 200 μl min−1, and a gradient of 0–95% water/acetonitrile was employed. The liquid chromatography apparatus was fitted to a Waters 286 UV detector and a Micromass Platform II single quadrupole mass spectrometer utilizing an electrospray ionization source controlled via the VG Mass-Lynx software (VG Biotechnology Ltd., Altrincham, Cheshire, United Kingdom). The source temperature was 140 °C. Capillary voltage was 3.3 kV, and the cone voltage was ramped from 40–90 V over a range of 500–2000m/z. The instrument was calibrated over thisMr range with horse heart myoglobin (Sigma). Genomic DNA was prepared by a modified version of the method described by Kulakovaet al. (12Kulakova A.N. Stafford T.M. Larkin M.J. Kulakov L.A. Plasmid. 1995; 33: 208-217Crossref PubMed Scopus (35) Google Scholar). Approximately 0.5 g of wet cell paste was washed twice in 5 ml of 10 mm EDTA, pH 8.0, and then resuspended in 5 ml of 75 mm NaCl, 25 mm EDTA, pH 8.0, 20 mm Tris/HCl, pH 8.0, containing 5 mg/ml lysozyme. The mixture was incubated for 2 h at 37 °C before adding 50 μl of proteinase K solution (20 mg/ml) and 300 μl of 10% (w/v) SDS. This was incubated for a further 2 h at 55 °C with occasional inversion. The solution was extracted once with phenol (equilibrated with Tris/HCl, pH 8.0) and then twice with chloroform. DNA was precipitated with isopropanol and spooled onto a glass rod. After rinsing with 70% (v/v) ethanol, the DNA was air-dried and dissolved in 0.5 ml of TE buffer (10 mm Tris HCl, pH8, 1 mm EDTA pH8). A partially degenerate oligonucleotide (36-mer) designed against the N-terminal sequence (residues 7–18) of the 6-oxocamphor hydrolase activity was synthesized as a hybridization probe: 5′-CCSTTCCAGGAGTACWSSCAGAAGTACGAGAACATC-3′ (where S represents G or C, and W represents A or T). The oligonucleotide mix was radiolabeled by a kinase reaction using T4 polynucleotide kinase and [γ-32P]ATP under standard conditions (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Total DNA, digested to completion with different restriction endonucleases and blotted onto a Hybond-N membrane (Amersham Pharmacia Biotech) was hybridized to the radiolabeled oligonucleotide mix at 55 °C for 48 h. The membrane was then washed twice at room temperature with 300 mm NaCl, 30 mm sodium citrate containing 0.1% (w/v) SDS for 15 min and twice at 55 °C with 30 mmNaCl, 3 mm sodium citrate containing 0.1% (w/v) SDS for 15 min. Following autoradiography, a single cross-hybridizing band was detected in each lane. EcoRI, SacI, andSmaI digests of genomic DNA were separated on preparative gels; the regions spanning ∼4.5, 3.6, and 2.0 kbp, 1The abbreviations used are:kbpkilobase pairbpbase pairORFopen reading frameECHenoyl-CoA hydratase4CBD4-chlorobenzoyl-CoA dehalogenase respectively, were excised; and the DNA was extracted and shotgun-cloned into pUC18 vector. Following transformation into E. coli XL1 Blue, positive clones were isolated by a colony lift procedure (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) using hybridization and washing conditions identical to those used for the Southern blot. Clones were verified using a dot blot procedure (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) prior to sequencing. kilobase pair base pair open reading frame enoyl-CoA hydratase 4-chlorobenzoyl-CoA dehalogenase Double-stranded DNA sequencing (14Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52597) Google Scholar) of plasmid DNA prepared from positive clones was carried out with an automated DNA sequencer (ABI PRISM 377, PerkinElmer Life Sciences). All sequencing was carried out on both strands. Computer-assisted sequence analysis was performed using the DNAStrider and MacVector software packages. Data base homology searches (SwissProt release 39 protein data base) were carried out using the NCBI BLAST server. The nucleotide sequence data reported in this paper has been deposited at EMBL and GenBankTM with the accession number AF323755. TableI shows that the purification of 6-oxocamphor hydrolase from crude cell extract proceeds with a yield of 5% and a factor of 35.7. SDS-polyacrylamide gel electrophoresis of the purified protein (Fig. 2) suggested a denatured molecular mass of ∼35,000 Da, but electrospray mass spectrometry confirmed a smaller subunit mass of 28,488 Da. Analysis of the native protein by gel filtration chromatography revealed an apparent native molecular mass of 83,000 Da (average of two determinations). This suggests the protein exists in solution as a trimer (assuming the molecule is not highly elongated, which would give it a much larger than expected Stokes radius). An isoelectric point of 8.5 was recorded for the enzyme.Table IPurification of 6-oxocamphor hydrolase from Rhodococcus sp. NCIMB 9784Purification stageTotal proteinActivityTotal activitySpecific activityYieldmgμmol min−1ml−1unitsunits mg−1%Cell extract809.625.38121.610.010040–80% Ammonium sulphate-cut337.078.07802.023.296Phenyl sepharose75.7139.43486.346.043Fast Flow Q6.9191.71150.4166.714Phenyl superose1.198.3393.2357.55 Open table in a new tab 6-Oxocamphor hydrolase was observed to have the same pI as penta-2,4-dione hydrolase and a comparable native molecular mass, the latter being a monomer of 75,000 Da (14Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52597) Google Scholar). The pH optimum of the enzyme was ascertained to be 7.0, and the enzyme displayed 25% higher activity in 50 mm phosphate buffer than 50 mmTris/HCl at the same pH. The specific activity of 6-oxocamphor hydrolase was determined to be 357.5 units mg−1, the Km to be 0.05 mm, and the Kcat to be 167 s−1 for 6-oxocamphor. The high specific activity of the enzyme is reflected in an inability to detect any 6-oxocamphor in fermentation extractions of Rhodococcus sp. NCIMB 9784, which yield a high proportion of 6-endo-hydroxycamphor. It is evident that the transformation of the hydroxycamphor to the diketone represents a bottleneck in the metabolism of camphor by this organism. 1 mmCu2+ was found to inhibit 6-oxocamphor hydrolase activity (72% relative activity), whereas 1 mm Zn2+(104%) had no significant effect. In common with penta-2,4-dione hydrolase (15Kawagoshi Y. Fujita M. World J. Microbiol. Biotechnol. 1998; 14: 95-100Crossref Scopus (30) Google Scholar), 6-oxocamphor hydrolase was greatly inhibited by Hg2+ ions (1 mm gave only 2% relative activity). High salt concentrations were shown to strongly inhibit penta-2,4-dione hydrolase, with no observable activity at 1m NaCl (15Kawagoshi Y. Fujita M. World J. Microbiol. Biotechnol. 1998; 14: 95-100Crossref Scopus (30) Google Scholar). No such inhibition was observed with 6-oxocamphor hydrolase. Gel filtration studies indicated no significant alteration in the apparent molecular size of 6-oxocamphor hydrolase at this concentration of NaCl. The effect of EDTA and various inhibitors of both thiol nucleophile-dependent hydrolases and the serine hydrolase inhibitor phenylmethylsulfonyl fluoride were tested. 6-oxocamphor hydrolase was inhibited to some degree by thiol active reagents, such as 1 mm N-ethylmaleimide (68% relative activity), but most notably 1 mm hydroxymercuribenzoate (14%); 1 mm EDTA had a slight activating effect (119%). Phenylmethylsulfonyl fluoride (1 mm) had almost no effect on activity. The gene encoding the 6-oxocamphor hydrolase was cloned by hybridization with a mixture of oligonucleotides designed against the N-terminal sequence of the purified protein activity. Three overlapping fragments of DNA (2.0 kbp SmaI, 3.6 kbpSacI, and 4.3 kbp EcoRI) were isolated by this procedure (Fig. 3) and cloned into pUC18 (clones S2.0, Sa3.6, and E4.3, respectively). Sequence analysis of the clones revealed several potential open reading frames (ORFs) (Fig.4). All displayed the typical codon usage pattern found in Rhodococcus sp., with a strong bias toward GC-rich codons.Figure 4Map of the 6240-bpSacI–EcoRI DNA region ofRhodococcus sp. NCIMB 9784. Open reading frames are denoted by arrows showing the direction of transcription. Only the restriction sites mentioned in the text are shown. The three overlapping clones (Sa3.6, S2.0, and E4.3) isolated by hybridization to an oligonucleotide mix designed against the N-terminal amino acid sequence of 6-oxocamphor hydrolase are shown by thethick bars at the bottom. The site of hybridization is denoted by asterisks.View Large Image Figure ViewerDownload (PPT) The deduced polypeptide translation of one such ORF (camK) matches the N-terminal sequence obtained from the isolated 6-oxocamphor hydrolase activity. camK encodes a protein of 257 amino acids, and the predicted ATG start codon is positioned 5 bp 3′ of a purine-rich region that may act as a ribosome binding site (Fig.5). Furthermore, the calculated mass of the polypeptide (28,482 Da) is very close to the experimentally determined mass of the purified protein by electrospray mass spectrometry (28,488 Da). Comparison of the translated sequence with the SwissProt data base revealed significant homology to the crotonase superfamily of enzymes from several sources. The best alignments were obtained with crotonase (enoyl-CoA hydratase) from Clostridium acetobutylicum (16Boynton Z.L. Bennett G.N. Rudolph F.B. J. Bacteriol. 1996; 178: 3015-3024Crossref PubMed Google Scholar) and E. coli (17Haller T. Buckel T. Retey J. Gerlt J.A. Biochemistry. 2000; 39: 4622-4629Crossref PubMed Scopus (124) Google Scholar), revealing 45 and 42% homology, respectively (data not shown). Significantly, close homology was also observed with 2-ketocyclohexanecarboxyl coenzyme A hydrolase from Rhodopseudomonas palustris (18Pelletier D.A. Harwood C.S. J. Bacteriol. 1998; 180: 2330-2336Crossref PubMed Google Scholar) and 4-chlorobenzoyl-CoA dehalogenase from a Pseudomonas sp. (19Benning M.M. Taylor K.L. Liu R.-Q. Yang G. Xiang H. Wesenberg G. Dunaway-Mariano D. Holden H.M. Biochemistry. 1996; 35: 8103-8109Crossref PubMed Scopus (154) Google Scholar). A sequence comparison of the translated sequence against representative members of the crotonase superfamily is given in Fig.6.Figure 5DNA sequence of the 6240-bpSacI–EcoRI region encoding the 6-oxocamphor hydrolase (camK) activity ofRhodococcus sp. NCIMB 9784. Each deduced ORF is labeled, and the direction of transcription is indicated. N-terminal amino acid sequence determined from the purified 6-oxocamphor hydrolase activity is underlined. The stop codons are denoted byasterisks, and the proposed Shine-Dalgarno sequences areunderlined. This sequence has been submitted to GenBankTM (accession number AF323755).View Large Image Figure ViewerDownload (PPT)Figure 6Amino acid sequence alignment of 6-oxocamphor hydrolase (6-OCH) with other members of the crotonase superfamily of enzymes. ECH , enoyl-CoA hydratase of rat mitochondria (27Müller-Newen G. Janssen U. Stoffel W. Eur. J. Biochem. 1995; 228: 68-73Crossref PubMed Scopus (77) Google Scholar); 2KCH, 2-ketocyclohexanecarboxyl-CoA hydrolase from Rhodopseudomonas palustris (18Pelletier D.A. Harwood C.S. J. Bacteriol. 1998; 180: 2330-2336Crossref PubMed Google Scholar);4CBD , 4-chlorobenzoyl-CoA dehalogenase from aPseudomonas sp. (19Benning M.M. Taylor K.L. Liu R.-Q. Yang G. Xiang H. Wesenberg G. Dunaway-Mariano D. Holden H.M. Biochemistry. 1996; 35: 8103-8109Crossref PubMed Scopus (154) Google Scholar). Regions of similarity are highlighted in the boxes.View Large Image Figure ViewerDownload (PPT) The 3′ end of the gene encoding 6-oxocamphor hydrolase (camK) has an overlap of 1 nucleotide, encompassing the TGA stop codon and a predicted GTG start codon of another open reading frame (ORF1). ORF1 appears to be translationally coupled tocamK and encodes a protein of 167 amino acids. A BLAST search of the SwissProt data base indicates homology to maoCgene from Klebsiella aerogenes (20Sugino H. Sasaki M. Azakami H. Yamashita M. Murooka Y. J. Bacteriol. 1992; 174: 2485-2492Crossref PubMed Google Scholar), which belongs to the aldehyde dehydrogenase family and to the short-chain dehydrogenase/reductase family of enzymes (e.g.17β-estradiol dehydrogenase from rat). Downstream of ORF1 is an ORF2 encoding a polypeptide of 408 amino acids that displays similarity to nonspecific lipid transfer proteins from various species (e.g. 49% homology to chicken protein). The function of this open reading frame in relation to camphor metabolism is not known. Upstream of camK, an ORF encoding a polypeptide of 206 amino acids (ORF3) was identified on the opposite strand. This divergently transcribed open reading frame has a potential ribosome binding site located 5 bp 5′ of the proposed ATG start codon. Sequence analysis of the translated product revealed significant homology to a number of transcriptional repressor proteins of the TetR/AcrR family from various microbial sources (e.g. 47% homology to the acrab operon repressor from E. coli (24Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner F.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. G.oeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (6004) Google Scholar)). A potential DNA-binding motif based on sequence homologies was identified (38SVRDLGEALGIQPGSVYAHI) that may form a helix-turn-helix motif. Further upstream of the proposed transcriptional regulator is ORF4, transcribed in the same direction as camK. Two alternative potential ATG start codons in the same reading frame were identified at positions 492 and 498 in the nucleotide sequence (Fig. 5). Because both ATG codons are positioned just downstream (5 and 6 bp, respectively) of a potential ribosome binding site, it is not clear which one represents the start of the ORF. For clarity, we have assigned the more 5′ ATG as being the start codon. The ORF encodes a polypeptide of 396 amino acids. Sequence analysis of the translated product revealed convincing homology to a number of ferredoxin reductase proteins in the data base. Conservation of sequence was particularly obvious in the regions involved in adenine nucleotide binding (data not shown). The best homology was found with the ferredoxin (rhodocoxin) reductase (ThcB) involved in the biodegradation of thiocarbamate fromRhodococcus sp. NI86/21 (21Nagy I. Schoofs G. Compernolle F. Proost P. Vanderleyden J. De Mot R. J. Bacteriol. 1995; 177: 676-687Crossref PubMed Google Scholar) (46% homology) and the putidaredoxin reductase involved in the hydroxylation of camphor byP. putida (22Koga H. Rauchfuss B. Gunsalus I.C. Biochem. Biophys. Res. Commun. 1985; 130: 412-417Crossref PubMed Scopus (37) Google Scholar) (46% homology). We have recently carried out initial studies on the substrate specificity of 6-oxocamphor hydrolase (23Grogan G. Graf J. Jones A. Parsons S. Turner N.J. Flitsch S.L. Angew. Chem. Int. Ed. Engl., in press. 2000; Google Scholar) (refer to Fig. 7). Acyclic diketones such as pentane-2,5-dione (a substrate for the β-diketone hydrolase from Pseudomonas vesicularis var. povalolyticus(15Kawagoshi Y. Fujita M. World J. Microbiol. Biotechnol. 1998; 14: 95-100Crossref Scopus (30) Google Scholar)) and 3,3-dimethylpentane-2,5-dione were not substrates. 2,2-Disubstituted cyclohexa-1,3-diones were the only monocyclic diketones transformed, although the transformation of two of these, 2-methyl-2-propylcyclohexa-1,3-dione 5 and 2-methyl-2-butylcyclohexa-1,3-dione 6, resulted in racemic keto acid products (which were converted to their methyl esters7 and 8, respectively, for analysis). Transformations of the bicyclic diketone substrates bicyclo[2.2.1]heptane 2,6-dione 9 and bicyclo[2.2.2]octane-2,6-dione 10 were, however, shown to yield (S)-keto acid products of 84%, (converted to methyl ester 11) and 95% enantiomeric excess (methyl ester12), respectively (Fig. 7). It is noteworthy that all substrates accepted by the enzyme are nonenolizable either due to quaternary substitution between the carbonyl groups (e.g. 3, 5, 6) or due to ring-strain-related restrictions imposed by Bredt's rule (3, 9, 10). The recent completion of the E. coli genome (24Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner F.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. G.oeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (6004) Google Scholar) has revealed seven genes encoding paralogues of enoyl-CoA hydratase, three with unknown function (17Haller T. Buckel T. Retey J. Gerlt J.A. Biochemistry. 2000; 39: 4622-4629Crossref PubMed Scopus (124) Google Scholar). This suggests that there remain activities of the crotonase superfamily yet to be described. In this paper, we present evidence that 6-oxocamphor hydrolase, which catalyzes the asymmetric hydrolysis of β-diketones, represents a new addition to the spectrum of activities catalyzed by the crotonase superfamily. The activity of crotonase, or enoyl-CoA hydratase (ECH), has been the subject of intensive study over many years owing to its central role in the β-oxidation pathway. The essential activity of ECH in this regard has been the stereospecific reversible hydration of enoyl-CoA molecules of varying fatty acid length to yield β-hydroxy thioesters (25Wakil S.J. Biochim. Biophys. Acta. 1957; 19: 497-504Crossref Scopus (35) Google Scholar, 26Willadesen P. Eggerer H. Eur. J. Biochem. 1975; 54: 247-252Crossref PubMed Scopus (98) Goog