Structure of 6-Oxo Camphor Hydrolase H122A Mutant Bound to Its Natural Product, (2S,4S)-α-Campholinic Acid
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m403514200
ISSN1083-351X
AutoresPhilip M. Leonard, Gideon Grogan,
Tópico(s)Biofuel production and bioconversion
ResumoThe crotonase homolog, 6-oxo camphor hydrolase (OCH), catalyzes the desymmetrization of bicyclic β-diketones to optically active keto acids via an enzymatic retro-Claisen reaction, resulting in the cleavage of a carbon-carbon bond. We have previously reported the structure of OCH (Whittingham, J. L., Turkenburg, J. P., Verma, C. S., Walsh, M. A., and Grogan, G. (2003) J. Biol. Chem. 278, 1744–1750), which suggested the involvement of five residues, His-45, His-122, His-145, Asp-154, and Glu-244, in catalysis. Here we report mutation studies on OCH that reveal that H145A and D154N mutants of OCH have greatly reduced values of kcat/Km derived from a very large increase in Km for the native substrate, 6-oxo camphor. In addition, H122A has a greatly reduced value of kcat, and its Km is five times that of the wild-type. The location of the active site is confirmed by the 1.9-Å structure of the H122A mutant of OCH complexed with the minor diastereoisomer of (2S,4S)-α-campholinic acid, the natural product of the enzyme. This shows the pendant acetate of the product hydrogen bonded to a His-145/Asp-154 dyad and the endocyclic carbonyl of the cyclopentane ring hydrogen bonded to Trp-40. The results are suggestive of a base-catalyzed mechanism of C-C bond cleavage and provide clues to the origin of prochiral selectivity by the enzyme and to the recruitment of the crotonase fold for alternate modes of transition state stabilization to those described for other crotonase superfamily members. The crotonase homolog, 6-oxo camphor hydrolase (OCH), catalyzes the desymmetrization of bicyclic β-diketones to optically active keto acids via an enzymatic retro-Claisen reaction, resulting in the cleavage of a carbon-carbon bond. We have previously reported the structure of OCH (Whittingham, J. L., Turkenburg, J. P., Verma, C. S., Walsh, M. A., and Grogan, G. (2003) J. Biol. Chem. 278, 1744–1750), which suggested the involvement of five residues, His-45, His-122, His-145, Asp-154, and Glu-244, in catalysis. Here we report mutation studies on OCH that reveal that H145A and D154N mutants of OCH have greatly reduced values of kcat/Km derived from a very large increase in Km for the native substrate, 6-oxo camphor. In addition, H122A has a greatly reduced value of kcat, and its Km is five times that of the wild-type. The location of the active site is confirmed by the 1.9-Å structure of the H122A mutant of OCH complexed with the minor diastereoisomer of (2S,4S)-α-campholinic acid, the natural product of the enzyme. This shows the pendant acetate of the product hydrogen bonded to a His-145/Asp-154 dyad and the endocyclic carbonyl of the cyclopentane ring hydrogen bonded to Trp-40. The results are suggestive of a base-catalyzed mechanism of C-C bond cleavage and provide clues to the origin of prochiral selectivity by the enzyme and to the recruitment of the crotonase fold for alternate modes of transition state stabilization to those described for other crotonase superfamily members. Enzymatic desymmetrization processes are of great interest to the synthetic organic chemist as they provide quantitative routes to optically active synthetic intermediates from prochiral starting materials (1Schoffers E. Golebiowski A. Johnson C. Tetrahedron. 1996; 52: 3769-3826Google Scholar). We have described the application of the enzyme 6-oxo camphor hydrolase (OCH) 1The abbreviations used are: OCH, 6-oxo camphor hydrolase; ECH, enoyl-CoA hydratase. 1The abbreviations used are: OCH, 6-oxo camphor hydrolase; ECH, enoyl-CoA hydratase. to the desymmetrization of bicyclic β-diketones for the preparation of optically active keto acids (2Grogan G. Graf J. Jones A. Parsons S. Turner N.J. Flitsch S.L. Angew. Chem. Int. Ed. Engl. 2001; 40: 1111-1114Google Scholar), which may be of use in the preparation of chiral synthons of a range of bioactive compounds. This unusual enzymatic process is the biological equivalent of a retro-Claisen or retro-Dieckmann reaction. The carbon-carbon bond of a β-dicarbonyl species is cleaved to yield a carboxylic acid and a methylene group. The natural substrate for the enzyme is bornane-2, 6-dione, or 6-oxo camphor 1 (Fig. 1), a natural catabolite of the monoterpene camphor when processed by the natural host strain, Rhodococcus sp. NCIMB 9784 (3Chapman P.J. Meerman G. Gunsalus I.C. Srinivasan R. Rinehart K.L. J. Am. Chem. Soc. 1966; 88: 618-619Google Scholar). We have previously demonstrated (2Grogan G. Graf J. Jones A. Parsons S. Turner N.J. Flitsch S.L. Angew. Chem. Int. Ed. Engl. 2001; 40: 1111-1114Google Scholar) that the enzyme yields a 6:1 diastereomeric ratio of the major product, (2R,4S)-campholinic acid 2 and the diastereoisomer 3 of the (2S,4S) configuration. The cleavage of β-dicarbonyl species in nature is mechanistically diverse (4Grogan G. J. Mol. Catal. B Enzym. 2002; 19: 73-82Google Scholar), incorporating, among others, enzymes such as polyvinyl ketone hydrolase (5Shimao M. Tamogami T. Kishida S. Harayama S. Microbiology. 2000; 146: 649-657Google Scholar) from Pseudomonas sp., a serine triad-type hydrolase, and acetylacetone-cleaving enzyme Dke1 (6Straganz G. Brecker L. Weber H.J. Steiner W. Ribbons D.W. Biochem. Biophys. Res. Commun. 2002; 297: 232-236Google Scholar), a dioxygenase from Acinetobacter johnsonii. On cloning and sequencing the gene encoding OCH, camK, from Rhodococcus sp. 9784 (7Grogan G. Roberts G.A. Bougioukou D. Turner N.J. Flitsch S.L. J. Biol. Chem. 2001; 276: 12565-12572Google Scholar), we were surprised to find that its closest amino acid sequence homologs in the SwissProt data base were enzymes of the crotonase superfamily. Crotonases are a mechanistically diverse group of enzymes that catalyze a variety of chemical reactions and for which a number of x-ray crystal structures have been reported, including enzymes catalyzing asymmetric double bond hydration (8Engel C.K. Mathieu M. Zeelen J.P. Hiltunen J.K. Wierenga R.K. EMBO J. 1996; 15: 5135-5145Google Scholar), decarboxylation (9Benning M.M. Haller T. Gerlt J.A. Holden H.M. Biochemistry. 2000; 39: 4630-4639Google Scholar), dehalogenation (10Benning M.M. Taylor K.L. Liu R-Q. Yang G. Xiang H. Wesenberg G. Dunaway-Mariano D. Holden H.M. Biochemistry. 1996; 35: 8103-8109Google Scholar), the isomerization of double bonds in fatty acids (11Modis Y. Filppula S.A. Novikov D.K. Norledge B. Hiltunen J.K. Wierenga R.K. Structure. 1998; 6: 957-970Google Scholar), and ring closure to form an aromatic ring (12Truglio J.J. Theis K. Feng Y. Gajda R. Machutta C. Tonge P.J. Kisker C. J. Biol. Chem. 2003; 278: 42352-42360Google Scholar). Each crotonase superfamily member shares common characteristics despite apparent differences in reaction chemistry. The substrate in each homolog described hitherto is an acyl coenzyme A thioester. Each reaction is thought to proceed via an intermediate enolate that appears to be stabilized by a conserved oxyanion hole in the enzyme tertiary structure, formed by the peptidic backbone N-Hs of the central residue of a conserved GG(A)G halfway through the sequence motif and the fifth residue of a more N-terminal hexad (13Holden H.M. Benning M.M. Haller T. Gerlt J.A. Acc. Chem. Res. 2001; 34: 145-157Google Scholar). The crotonase superfamily has thus served as a useful paradigm for illustrating the mechanisms of divergent evolution of enzyme action through the recruitment of the crotonase (enoyl-CoA hydratase) fold for alternate reaction chemistry. It was clear from the sequencing results that OCH represented a further diverged member of the crotonase superfamily; not only was the natural substrate not an acyl coenzyme A thioester but the distinctive GG(A)G motif for oxyanion stabilization was not present, being replaced instead by an NHP sequence (7Grogan G. Roberts G.A. Bougioukou D. Turner N.J. Flitsch S.L. J. Biol. Chem. 2001; 276: 12565-12572Google Scholar).The divergence of OCH from the parent crotonase, enoyl-CoA hydratase (ECH) (8Engel C.K. Mathieu M. Zeelen J.P. Hiltunen J.K. Wierenga R.K. EMBO J. 1996; 15: 5135-5145Google Scholar), was further emphasized by comparison of the enzyme structures (14Whittingham J.L. Turkenburg J.P. Verma C.S. Walsh M.A. Grogan G. J. Biol. Chem. 2003; 278: 1744-1750Google Scholar). While retaining the overall quaternary arrangement of enoyl-CoA hydratase, the tertiary fold of the poorly sequence-conserved C-terminal helix of OCH appeared to assume a different motif, curling around the β-α-β-superhelix of the N-terminal portion in contrast to forming a second discrete domain as observed in ECH and some other crotonase superfamily structures such as 4-chlorobenzoyl dehalogenase (10Benning M.M. Taylor K.L. Liu R-Q. Yang G. Xiang H. Wesenberg G. Dunaway-Mariano D. Holden H.M. Biochemistry. 1996; 35: 8103-8109Google Scholar). This stark difference in tertiary structure was also observed for methylmalonyl-CoA decarboxylase (9Benning M.M. Haller T. Gerlt J.A. Holden H.M. Biochemistry. 2000; 39: 4630-4639Google Scholar) from Escherichia coli and yeast Δ3-Δ2-enoyl-CoA isomerase of the crotonase superfamily (15Mursula A.M. van Aalten D.M.F. Kalervo Hiltunen J. Wierenga R.K. J. Mol. Biol. 2001; 309: 845-853Google Scholar). At that stage, despite repeated co-crystallization experiments with the natural substrate, we were not able to acquire a structure of OCH with either its natural substrate or product bound. However, a large cavity in the enzyme, completed by the recruitment of the C-terminal helical domain, was identified as a possible active site based on the protrusion of a number of acid-base amino acid residues into its center. Using the known prochiral selectivity of OCH, we were able to model both the substrate 6-oxo camphor and major diastereomeric product (2R,4S)-α-campholinic acid into the putative active site and to intimate a putative mechanistic role for the acid-base residues His-45, His-122, His-145, Asp-154, and Glu-244.In this study, we report the kinetic analysis of mutants H45A, H122A, H145A, D154N, and E244Q of OCH and describe rational use of the low kcat plus low Km mutant H122A for the acquisition of a 1.9-Å crystal structure of OCH H122A bound to the minor diastereoisomer of the reaction product, (2S,4S)-α-campholinic acid. The kinetic data, in conjunction with the mutant crystal structure, support a possible mechanistic role for a His-145/Asp-154 dyad in a general base-catalyzed mechanism and also illuminate the important roles of Trp-40 and Phe-82 in substrate recognition and determination of prochiral selectivity. The cumulative data are also suggestive of an atypical mode of transition state binding for a crotonase homolog, which would by definition place OCH and more closely related gene products in an enzyme family distinct from other crotonases hitherto described.EXPERIMENTAL PROCEDURESChemicals—All chemicals were obtained from Sigma unless otherwise specified. 6-oxo camphor was obtained as detailed in Ref. 7Grogan G. Roberts G.A. Bougioukou D. Turner N.J. Flitsch S.L. J. Biol. Chem. 2001; 276: 12565-12572Google Scholar. Plasmid pET-26b(+) was obtained from Novagen Ltd. Primers for mutagenesis of camK were obtained from MWG Biotech.Bacterial Strains, Plasmids, and Culture Conditions—Wild-type and mutant OCH genes were expressed in E. coli strain BL21(DE3). The plasmid pGG3 had been obtained previously by ligating the camK gene into pET-26b(+) plasmid (14Whittingham J.L. Turkenburg J.P. Verma C.S. Walsh M.A. Grogan G. J. Biol. Chem. 2003; 278: 1744-1750Google Scholar). E. coli was cultured in Luria Bertani broth with 30 μg ml–1 kanamycin at 37 °C. Cultures were routinely grown to an optical density of A600 = 0.6, induced for expression of wild-type or mutant OCH by the addition of 1 mm isopropyl-β-d-thiogalactopyranoside, and then grown for a further 3 h at 37 °C.Site-directed Mutagenesis of OCH—The H45A, H122A, H145A, D154N, and E244Q mutants were constructed using the QuikChange® mutagenesis kit (Stratagene) with the pGG3 plasmid as a template. The primers used to create the mutants were 5′-GTGTGGACCTCAACCGCAGCCGACGAGCTGGCCTACTG-3′ and 5′-CAGTAGGCCAGCTCGTCGGCTGCGGTTGAGGTCCACAC-3′ for H45A, 5′-CAACGGACCGGTGACCAACGCCCCGGAGATCCCCGTCATG-3′ and 5′-CATGACGGGGATCTCCGGGGCGTTGGTCACCGGTCCGTTG-3′ for H122A, 5′-CACCTTCCAGGACGGACCGGCCTTCCCTTCCGGCATCGTG-3′ and 5′-CACGATGCCGGAAGGGAAGGCCGGTCCGTCCTGGAAGGTG-3′ for H145A, 5′-CCGGCATCGTGCCCGGGAACGGCGCCCACGTGGTG-3′ and 5′-CACCACGTGGGCGCCGTTCCCGGGCACGATGCCGG-3′ for D154N, and 5′-GTCTCGGCCTCGCGCACCAAGCGCTCGCCGCCATC-3′ and 5′-GATGGCGGCGAGCGCTTGGTGCGCGAGGCCGAGAC-3′ for E244Q. The following parameters were used during thermal cycling: 1 cycle of 99 °C for 30 s, followed by 20 cycles of 99 °C for 30 s, 55 °C for 60 s, and 68 °C for 840 s. Mutant plasmids were purified using standard methods and sequenced to confirm the presence of the substituted bases.Purification and Assay of OCH Mutants—All five mutant genes were expressed and purified using an identical protocol as detailed in Ref. 7Grogan G. Roberts G.A. Bougioukou D. Turner N.J. Flitsch S.L. J. Biol. Chem. 2001; 276: 12565-12572Google Scholar. Levels of soluble OCH mutant proteins were obtained with comparable yield and solubility to the wild-type. The activity of the OCH mutants was evaluated as described previously (7Grogan G. Roberts G.A. Bougioukou D. Turner N.J. Flitsch S.L. J. Biol. Chem. 2001; 276: 12565-12572Google Scholar) by measuring the decrease in absorption at 294 nm over time, indicating the rate of cleavage of the 6-oxo camphor substrate by enzyme. It was possible to construct Michaelis-Menten plots for each mutant by measuring the initial rate of catalysis over a range of substrate concentrations. Lineweaver-Burk plots were used to obtain the Km and kcat for the natural substrate for each mutant enzyme.Crystallization of the H122A Mutant—Crystals of the H122A mutant of OCH were grown by the vapor diffusion hanging drop technique. A 10 mg/ml protein solution containing 50 mm Tris-HCl, pH 7.1, 1 mm dithiothreitol, and 20 μm phenylmethylsulfonyl fluoride was mixed in a 50:50 ratio with reservoir to form the hanging drops. The reservoir solution consisted of 0.1 m 2-(N-morpholino)ethanesulfonic acid, pH 5.6, 0.2 m calcium acetate, and 26% (v/v) polyethylene glycol monomethyl ether, 2,000 Da. Prior to data collection, crystals were transferred to a saturated solution of 6-oxo camphor in reservoir and soaked for 30 min. The H122A mutant crystallized in space group P21, with cell dimensions a = 83.27 Å, b = 132.00 Å, c = 135.43 Å, β = 94.115°.Data Collection and Data Processing—A data set extending to 1.9-Å resolution was collected on a single crystal of the H122A mutant enzyme that had been flash frozen at 120 K using the soak solution, which acted as a cryoprotectant because of the presence of polyethylene glycol monomethyl ether, 2,000 Da. The data were collected on beamline ID14-EH3 at the European Synchrotron Radiation Facility, Grenoble, France, using a MAR165 CCD detector. The data were processed, scaled, and merged using the HKL suite (16Otwinowski Z. Minor V. Carter C.W. Sweet R.M. Macromolecular Crystallography, Part A. 276. Academic Press, New York, London1997: 307-326Google Scholar). Data collection and processing statistics are given in Table I.Table IH122A data collection, refinement, and final model statisticsData collection statisticsBeamlineID14-EH3Wavelength (Å)Space groupP21Resolution (Å)20-1.9 (1.96-1.9)Unique reflections271694 (19032)Completeness (%)100 (100)Rsym (%)6.0 (28.2)Multiplicity3.8 (3.7)I/σI22.2 (4.9)Refinement statisticsRcryst (%)16.4Rfree (%)19.6Root mean square bond lengths (Å)0.017Root mean square bond angles (degree)1.662Ramachandran95.3Average B main chain (Å2)19.4Average B side chain (Å2)22.5Average B waters (Å2)26.5Average B ligand (Å2)23.8Model composition asymmetric unitResidues2984Water molecules1929Ligand molecules12 Open table in a new tab Structure Solution and Refinement—The structure was initially phased by molecular replacement using the CCP4 (17Number Collaborative Computational Project Acta Crystogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Google Scholar) program AMORE (18Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Google Scholar), starting with a model from the 2-Å resolution structure of OCH (Protein Data Bank entry 1o8u). The OCH hexamer was used as a search model with all water atoms removed. The resulting H122A model contained 12 monomers in the asymmetric unit corresponding to 2 hexamers. Following molecular replacement, initial refinement was performed using the CCP4 (17Number Collaborative Computational Project Acta Crystogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Google Scholar) program REFMAC5 (19Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Google Scholar). Rigid body refinement was performed for 10 cycles. The model was then subjected to positional refinement and initial maximum likelihood weighted 2 Fo – Fc and Fo – Fc maps were calculated. The model was adjusted to fit the electron density maps using the XTALVIEW molecular graphics program (20McRee D.E. J. Mol. Graph. 1992; 10: 44-46Google Scholar). Electron density was present in both maps corresponding to the bound product of catalysis, (2S,4S)-α-campholinic acid. The CCP4 (17Number Collaborative Computational Project Acta Crystogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Google Scholar) program SKETCHER was used to make the product molecule model; a library file was created for use in REFMAC5 (19Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Google Scholar) using the CCP4 (17Number Collaborative Computational Project Acta Crystogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Google Scholar) program LIBCHECK. This was built into each of the 12 sites in the asymmetric unit. Further positional refinement was performed with non-crystallographic symmetry restraints applied between all 12 monomers, and the model was adjusted again. The water structure was built into the model using the CCP4 (17Number Collaborative Computational Project Acta Crystogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Google Scholar) program ARP_WATERS in ARP/wARP version 5.0 (21Lamzin V.S. Wilson K.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 129-147Google Scholar), followed by more rounds of positional refinement. During the refinement process, 5% of the data were randomly selected and excluded from refinement for Rfree cross-validation. The refinement statistics of the final model are given in Table I.Accession Number—The coordinates for the structure of the H122A mutant of OCH have been deposited with the Protein Data Bank under the designation 1szo.RESULTSKinetic Analysis of OCH Mutants—The 2-Å structure of OCH and subsequent modeling studies (14Whittingham J.L. Turkenburg J.P. Verma C.S. Walsh M.A. Grogan G. J. Biol. Chem. 2003; 278: 1744-1750Google Scholar) were suggestive of mechanistic roles for His-45, His-122, His-145, Asp-154, and Glu-244. On an essentially intuitive basis, therefore, site-directed mutants H45A, H122A, H145A, D154N, and E244Q of OCH were chosen for study and prepared using the Stratagene QuikChange® kit. Kinetic analysis of the mutants was carried out using the established UV spectrophotometric assay for OCH based on the disappearance of the native substrate, 6-oxo camphor, at 294 nm. The results are shown in Table II. Each mutant shows a reduced kcat, but this effect is most noticeable for the H145A and H122A mutants. The H45A, H122A, and E244Q mutants display a Km for the native substrate comparable with that of the wild-type enzyme, whereas the H145A and D154N mutants display an approximate 100-fold increase. In terms of catalytic efficiency, the kinetic constants for H145A contribute to the most reduced second order rate constant (kcat/Km) for any of the mutants, a factor ∼106 lower than the wild-type. The second order rate constants for reactions catalyzed by E244Q and H45A suggest that these residues have a less significant overall effect on catalysis than the other three mutated residues.Table IIKinetic parameters for wild-type (recombinant) and mutant 6-oxo camphor hydrolasesEnzymekcatKmkcat/Kms-1mm-1 s-1Wild-type OCH642.860.041.61 × 107H45A1.140.019.50 × 104H122A0.360.201.80 × 103H145A0.283.338.40 × 101D154N1.424.013.55 × 102E244Q20.400.082.55 × 105 Open table in a new tab Overall Structure of the H122A Mutant-Product Complex— The crystal structure of the H122A mutant complexed with (2S,4S)-α-campholinic acid was determined to 1.9-Å resolution. The structure (Fig. 2) revealed that the location of the active site had been correctly identified in our previous publication (14Whittingham J.L. Turkenburg J.P. Verma C.S. Walsh M.A. Grogan G. J. Biol. Chem. 2003; 278: 1744-1750Google Scholar), bound on one side by helices α-2, α-3, and α-9, the last of these being the C-terminal helix. The quality of the final model was analyzed using the CCP4 (17Number Collaborative Computational Project Acta Crystogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Google Scholar) program PROCHECK (22Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Google Scholar). The Ramachandran plot shows that 95.3% of residues occupy the most favored regions, with a further 4.2% occupying additional allowed regions. Only residue His-145 adopts dihedral angles significantly outside the allowed regions of the Ramachandran plot. His-145 is located within a type I β-turn immediately preceding a 310 helix, between strand 8 and helix α-5, and the electron density of this residue is unambiguous.Fig. 2Stereoview close-up of the active site of the H122A mutant of OCH with bound (2S,4S)-α-campholinic acid and selected side chain residues shown in ball-and-stick representation, with electron density from the final maximum likelihood weighted 2 Fo – Fc map to 1.9 Å and calculated contoured at 1 σ also shown. Side chain residues are labeled. The active site is located between helices α-2, α-3, and α-9, shown as ribbons in silver. This figure and Figs. 3 and 4 were generated with the programs MOLSCRIPT (28Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Google Scholar), BOBSCRIPT (29Esnouf R.M. J. Mol. Graph. 1997; 15: 132-134Google Scholar), and RASTER3D (30Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Google Scholar).View Large Image Figure ViewerDownload (PPT)The H122A mutant forms a hexamer, which can be described as a dimer of trimers, identical to that of the wild-type enzyme (14Whittingham J.L. Turkenburg J.P. Verma C.S. Walsh M.A. Grogan G. J. Biol. Chem. 2003; 278: 1744-1750Google Scholar). The α-carbons of the mutant hexamer superimpose on those of the wild-type hexamer with a root mean square displacement of 0.38 Å and a maximum displacement of 2.50 Å, calculated using the CCP4 program LSQKAB (23Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Google Scholar). The differences between the two structures can be attributed to the presence of the bound product molecule in the active site (Fig. 3). There are three regions of change with respect to the wild-type structure upon binding of the product molecule, defined by Glu-76 → Asn-83, Pro-118 → Glu-124, and Gln-141 → Ile-150. The first region contains a 310 helix and is positioned just before helix α-3. The region contains two phenylalanines, the first of which, Phe-79, appears to move to close off the entrance to the active site as suggested previously (14Whittingham J.L. Turkenburg J.P. Verma C.S. Walsh M.A. Grogan G. J. Biol. Chem. 2003; 278: 1744-1750Google Scholar). The second phenylalanine, Phe-82, facilitates this movement by forming a stacking interaction between the plane of its aromatic ring and the pro-(R) face of the cyclopentane ring of the product, suggesting a mechanism whereby the entrance to the active site is closed during catalysis by direct interaction with the substrate. The second region is positioned after helix α-3 and strand 6 and contains a type IV β-turn. The mutated residue Ala-122 is contained within this region, and the movement may reflect its role in catalysis, but it may also be an artifact of the mutation. The final region contains a type I β-turn followed by a 310 helix. This region contains His-145, which hydrogen bonds at a distance of 2.77 Å to one of the carboxylate oxygens of the pendant acetate at C-4 of the bound product.Fig. 3Stereoview of superimposition of native OCH wild-type structure and H122A mutant in the active site region, showing movement of three labeled secondary structural motifs on ligand binding. The wild-type structure, including side chains, is shown in silver. All side chains are shown in all-bonds representation with selected side chains labeled.View Large Image Figure ViewerDownload (PPT)The density of the bound ligand in each of the 12 subunits in the model is unambiguous, as illustrated in Fig. 2. The ligand observed is not the major product of carbon-carbon bond cleavage observed with the wild-type enzyme, which exhibits the cis-stereochemistry of the 2-methyl and 4-acetate groups as confirmed by x-ray crystallography of the enzymatic product generated in vitro (2Grogan G. Graf J. Jones A. Parsons S. Turner N.J. Flitsch S.L. Angew. Chem. Int. Ed. Engl. 2001; 40: 1111-1114Google Scholar), but rather the (2S,4S)-diastereomer of the trans conformation. The structure provides useful information on important active site interactions between the product molecule and the active site. In addition, the product is bound in a closed conformation, with the structure giving the impression of the substrate molecule just after carbon-carbon bond cleavage, and retains the three-dimensional character of the substrate.In addition to those already described, further interactions are apparent between active site residue side chains and the product (Fig. 4). The endocyclic carbonyl of the product is hydrogen bonded at a distance of 2.56 Å to the N-H group of Trp-40. The carboxylate oxygen of the pendant acetate at C4 that hydrogen bonds to His-145 also hydrogen bonds to Glu-244 at a distance of 2.50 Å. The other carboxylate oxygen of the acetate hydrogen bonds with Asp-154 at a distance of 2.67 Å.It is possible that His-145 and Asp-154 form a catalytic dyad. There may also be an indirect interaction between this carboxylate oxygen and His-45 via an active site water that is hydrogen bonded to both the carboxylate oxygen at a distance of 2.76 Å and His-45 at a distance of 2.72 Å. Another active site water hydrogen bonds the first water at a distance of 2.72 Å and also the backbone carbonyls of Asp-154 and Gly-97 at distances of 2.66 and 2.90 Å, respectively. Finally, the geminal dimethyl group of the product is situated in a hydrophobic cleft formed by Leu-84, Ile-150, and Phe-82, making hydrophobic contacts at distances of 4.04, 4.12, and 3.59 Å, respectively. All three residues are in regions of change one and three with respect to the wild-type structure described above and appear to move closer upon ligand binding.Fig. 4Stereoview of active site of OCH H122A mutant showing hydrogen bonding interactions of product (2S,4S)-α-campholinic acid, with active site residue side chains and water molecules shown in ball-and-stick representation. Side chains and hydrogen bond distances (Å) are labeled.View Large Image Figure ViewerDownload (PPT)Superimposition of the ligand-bound mutant and wild-type structure also reveal water molecules in the native structure in place of the endocyclic carbonyl of the product and the oxygen atoms of the pendant C4 acetate. This is perhaps suggestive of water activation by either Asp-154 or His-145 in a general base-catalyzed mechanism as discussed below.DISCUSSIONA variety of mechanisms have evolved in nature for the cleavage of β-diketone molecules between the two carbonyl groups. Pentane 2,4-dione (acetylacetone) is cleaved by a serine hydrolase-type enzyme from Pseudomonas to yield acetate and acetone (5Shimao M. Tamogami T. Kishida S. Harayama S. Microbiology. 2000; 146: 649-657Google Scholar) and can also be cleaved oxidatively by the enzyme Dke1 from Acinetobacter johnsonii to yield methylglyoxal and acetate (6Straganz G. Brecker L. Weber H.J. Steiner W. Ribbons D.W. Biochem. Biophys. Res. Commun. 2002; 297: 232-236Google Scholar). Mechanistic investigations of these enzymes are complicated by the existence of the substrate in both diketo and enolate forms in solution. In contrast, all the substrates shown to be cleaved by 6-oxo camphor hydrolase thus far are non-enolizable, either as a result of quaternary substitution at the carbon center between the carbonyl groups or because of restrictions imposed by Bredt's rule in the bicyclic systems. From the results described above, it appears that in the case of these non-enolizable ketones, further enzyme chemistry using amino acids recruited from the crotonase fold has been adopted for β-diketone cleavage, and indeed a previously unreported mechanism of carbon-carbon bond cleavage appears to operate.Although OCH is unique among the crotonase superfamily in that it does not act on an acyl-CoA thioester, the retro-Dieckmann reaction catalyzed is reminiscent of the carbon-carbon bond cleavage catalyzed by the crotonase homolog BadI from Rhodopseudomonas palustris (24Pelletier D.A. Harwood C.S. J. Bacteriol. 1998; 180: 2330-2336Google Scholar). BadI catalyzes the cleavage of 2-ketocyclohexanecarboxyl-CoA (Fig. 5); a recent abstract (25Eberhard E.D. G
Referência(s)