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• W1997588995 abstract "The proposed biosynthetic pathway to the carbapenem antibiotics proceeds via epimerization/desaturation of a carbapenam in an unusual process catalyzed by an iron- and 2-oxoglutarate-dependent oxygenase, CarC. Crystal structures of CarC complexed with Fe(II) and 2-oxoglutarate reveal it to be hexameric (space group C2221), consistent with solution studies. CarC monomers contain a double-stranded β-helix core that supports ligands binding a single Fe(II) to which 2-oxoglutarate complexes in a bi-dentate manner. A structure was obtained with l-N-acetylproline acting as a substrate analogue. Quantum mechanical/molecular mechanical modeling studies with stereoisomers of carbapenams and carbapenems were used to investigate substrate binding. The combined work will stimulate further mechanistic studies and aid in the engineering of carbapenem biosynthesis. The proposed biosynthetic pathway to the carbapenem antibiotics proceeds via epimerization/desaturation of a carbapenam in an unusual process catalyzed by an iron- and 2-oxoglutarate-dependent oxygenase, CarC. Crystal structures of CarC complexed with Fe(II) and 2-oxoglutarate reveal it to be hexameric (space group C2221), consistent with solution studies. CarC monomers contain a double-stranded β-helix core that supports ligands binding a single Fe(II) to which 2-oxoglutarate complexes in a bi-dentate manner. A structure was obtained with l-N-acetylproline acting as a substrate analogue. Quantum mechanical/molecular mechanical modeling studies with stereoisomers of carbapenams and carbapenems were used to investigate substrate binding. The combined work will stimulate further mechanistic studies and aid in the engineering of carbapenem biosynthesis. Carbapenems possess a broad spectrum of antibacterial activity and are relatively stable to serine β-lactamases that are a major cause of resistance to penicillins and cephalosporins (1Livermore D.M. Woodford N. Curr. Opin. Microbiol. 2000; 3: 489-495Crossref PubMed Scopus (310) Google Scholar, 2McGowan S.J. Bycroft B.W. Salmond G.P.C. Trends Microbiol. 1998; 6: 203-208Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Carbapenems, such as thienamycin, were first isolated from bacterial extracts, including those from Serratia marcescens, Erwinia carotovora, and Streptomyces cattleya (3Kahan J.S. Kahan F.M. Goegelman R. Currie S.A. Jackson M. Stapley E.O. Miller T.W. Miller A.K. Hendlin D. Mochales S. Hernandez S. Woodruff H.B. Birnbaum J. J. Antibiot. 1979; 32: 1-12Crossref PubMed Scopus (500) Google Scholar, 4Parker W.L. Rathnum M.L. Wells Jr., J.S. Trejo W.H. Principe P.A. Sykes R.B. J. Antibiot. 1982; 35: 653-660Crossref PubMed Scopus (113) Google Scholar). Early attempts to improve the fermentation titers to commercially useful levels were unsuccessful (5Williamson J.M. CRC Crit. Rev. Biotech. 1986; 4: 111-131Crossref Scopus (16) Google Scholar), and carbapenem use is, in part, limited by production costs. Synthetic methodology for carbapenem production has been developed (6Nicolaou K.C. Sorensen E.J. Classics in Total Synthesis: Targets, Strategies, Methods. Wiley, New York1996: 249-263Google Scholar) but is less efficient than the direct fermentation or “semi-synthetic” procedures used for the production of penicillins and cephalosporins. Studies on carbapenem biosynthesis are of interest because they may enable engineering of the pathway to produce either medicinally useful antibiotics directly or intermediates for their production. Bycroft, Salmond, and coworkers (7Thomson N.R. Crow M.A. McGowan S.J. Cox A. Salmond G.P.C. Mol. Microbiol. 2000; 36: 539-556Crossref PubMed Scopus (253) Google Scholar) have discovered that the low fermentation titers of carbapenems are due to the operation of a “quorum sensing” machinery in their regulation, opening the way to increased fermentation yields. The sequences of the nine genes (in E. carotovora) responsible for the biosynthesis of (5R)-carbapenem, the simplest known natural carbapenem have been reported (8McGowan S.J. Sebaihia M. Porter L.E. Stewart G.S.A.B. Williams P. Bycroft B.W. Salmond G.P.C. Mol. Microbiol. 1996; 22: 415-426Crossref PubMed Scopus (102) Google Scholar). Although this compound is not a medicinally useful carbapenem due to its lack of a C-6 side chain, it nonetheless shares an identical nucleus with carbapenems that are (4Parker W.L. Rathnum M.L. Wells Jr., J.S. Trejo W.H. Principe P.A. Sykes R.B. J. Antibiot. 1982; 35: 653-660Crossref PubMed Scopus (113) Google Scholar). Eight of the genes, carA–H, are organized as an operon controlled by CarR, an LuxR type transcriptional activator (9McGowan S. Sebaihia M. Jones S. Yu B. Bainton N. Chan P.F. Bycroft B. Stewart G.S.A.B. Williams P. Salmond G.P.C. Microbiology. 1995; 141: 541-550Crossref PubMed Scopus (144) Google Scholar, 10Cox A.R.J. Thomson N.R. Bycroft B. Stewart G.S.A.B. Williams P. Salmond G.P.C. Microbiology. 1998; 144: 201-209Crossref PubMed Scopus (42) Google Scholar). Five of the genes, carA–E, are believed to be directly involved in the production of the (5R)-carbapenem nucleus, and it has been shown that it can be produced in Escherichia coli, albeit at lower levels, using only CarA–C (11McGowan S.J. Sebaihia M. O'Leary S. Hardie K.R. Williams P. Stewart G.S.A.B. Bycroft B.W. Salmond G.P.C. Mol. Microbiol. 1997; 26: 545-556Crossref PubMed Scopus (70) Google Scholar, 12Li R. Stapon A. Blanchfield J.T. Townsend C.A. J. Am. Chem. Soc. 2000; 122: 9296-9297Crossref Scopus (63) Google Scholar). It has been proposed that glutamate semi-aldehyde, formed by the action of CarD and CarE, condenses with acetyl-CoA in a CarB-catalyzed reaction to give a monocyclic pyrolidine intermediate (5S-carboxymethyl)-S-proline (trans-CMP) (Scheme 1) (2McGowan S.J. Bycroft B.W. Salmond G.P.C. Trends Microbiol. 1998; 6: 203-208Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 13McGowan S.J. Holden M.T.G. Bycroft B.W. Salmond G.P.C. Antonie Van Leeuwenhoek. 1999; 75: 135-141Crossref PubMed Scopus (28) Google Scholar). This can then be cyclized in an ATP-driven process catalyzed by CarA, to give a (3S,5S)-carbapenam (14Bycroft B.W. Chhabra S.R. J. Chem. Soc. Chem. Commun. 1989; : 423-425Crossref Google Scholar). This reaction is closely related to β-lactam formation during biosynthesis of the β-lactamase inhibitor clavulanic acid (15McNaughton H.J. Thirkettle J.E. Zhang Z. Schofield C.J. Jensen S.E. Barton B. Greaves P. Chem. Commun. 1998; : 2325-2326Crossref Scopus (41) Google Scholar, 16Miller M.T. Bachmann B.O. Townsend C.A. Rosenzweig A.C. Nat. Struct. Biol. 2001; 8: 684-689Crossref PubMed Scopus (58) Google Scholar, 17Bachmann B.O. Li R. Townsend C.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9082-9086Crossref PubMed Scopus (98) Google Scholar) but is very different from β-lactam formation during penicillin biosynthesis (18Burzlaff N.I. Rutledge P.J. Clifton I.J. Hensgens C.M. Pickford M. Adlington R.M. Roach P.L. Baldwin J.E. Nature. 1999; 401: 721-724Crossref PubMed Scopus (168) Google Scholar). In this proposed pathway and consistent with the reports of both Bycroft et al. (14Bycroft B.W. Chhabra S.R. J. Chem. Soc. Chem. Commun. 1989; : 423-425Crossref Google Scholar) and Li et al. (12Li R. Stapon A. Blanchfield J.T. Townsend C.A. J. Am. Chem. Soc. 2000; 122: 9296-9297Crossref Scopus (63) Google Scholar), the role of CarC is to catalyze both epimerization at C-5 and desaturation across the C-2/C-3 bond of the carbapenam. There has been some uncertainty over the absolute stereo-chemistry of the carbapenams involved in carbapenem biosynthesis. Originally, two β-lactams assigned as (3R,5R)- and (3S,5R)-carbapenams were isolated (19Bycroft B.W. Maslen C. Box S.J. Brown A.G. Tyler J.W. J. Chem. Soc. Chem. Commun. 1987; : 1623-1625Crossref Google Scholar). The former was subsequently reassigned as a (3S,5S)-carbapenam following synthesis of a derivative of the enantiomeric (3R,5R)-carbapenam (14Bycroft B.W. Chhabra S.R. J. Chem. Soc. Chem. Commun. 1989; : 423-425Crossref Google Scholar). Tanaka et al. (20Tanaka H. Sakagami H. Ogasawara K. Tetrahedron Lett. 2002; 43: 93-96Crossref Scopus (21) Google Scholar) have queried this reassignment, but recent synthetic work has substantiated the assignment of the natural carbapenam as (3S,5S) (21Bycroft B. Chhabra S.R. Kellam B. Smith P. Tetrahedron Lett. 2003; 44: 973-976Crossref Scopus (19) Google Scholar). CarC is related to clavaminic acid synthase (CAS) 1The abbreviations used are: CAS, clavaminic acid synthase; 2OG, 2-oxoglutarate; LC/MS, liquid chromatography/mass spectrometry; PEG, polyethylene glycol; SeMet, selenomethionine; l-NAP, l-N-acetylproline; QM/MM, quantum mechanical/molecular mechanical; r.m.s., root mean square; DSBH, double-stranded β-helix; TauD, taurine dioxygenase; DAOCS, deacetoxycephalosporin C synthase; i-PrOH, 2-propanol; trans-CMP, ((5S-carboxymethyl)-S-proline). (approximately 23% sequence identity) (8McGowan S.J. Sebaihia M. Porter L.E. Stewart G.S.A.B. Williams P. Bycroft B.W. Salmond G.P.C. Mol. Microbiol. 1996; 22: 415-426Crossref PubMed Scopus (102) Google Scholar), which catalyzes three reactions, comprising hydroxylation, ring closure, and desaturation processes, during clavulanic acid biosynthesis (22Lloyd M.D. Merritt K.D. Lee V. Sewell T.J. Wha-Son B. Baldwin J.E. Schofield C.J. Elson S.W. Baggaley K.H. Nicholson N.H. Tetrahedron. 1999; 55: 10201-10220Crossref Scopus (47) Google Scholar). Although the desaturation process catalyzed by CarC follows the precedent set by the CAS desaturation reaction, its assigned epimerization reaction is unique. To understand the mechanism of the highly unusual CarC reactions, structural information is required. Here we report the in vitro enzyme-mediated production of an active carbapenem antibiotic and describe crystal structures of CarC that define its active site structure thus enabling mechanistic and engineering studies aimed at altering its selectivity. CarC Cloning, Expression, and Purification—A PCR-amplified DNA product corresponding to the E. carotovora carC gene (8McGowan S.J. Sebaihia M. Porter L.E. Stewart G.S.A.B. Williams P. Bycroft B.W. Salmond G.P.C. Mol. Microbiol. 1996; 22: 415-426Crossref PubMed Scopus (102) Google Scholar) was engineered as an NdeI-BamHI fragment into the pET24a expression vector (Novagen) and transformed into E. coli BL21(DE3) supercompetent cells. Cells were grown in shake flasks at 37 °C using 2TY medium containing 50 μg/ml kanamycin. At A600 0.8, cells were induced with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside and growth allowed to continue for 4 h, CarC expression was ∼20% of the total soluble protein. Cultured cells were resuspended in 50 mm Tris-HCl, pH 8.0, 1 mm dithiothreitol, 2 mm EDTA, 100 mm NaCl, 0.16 mg/ml lysozyme, with 8 μl phenylmethylsulfonyl fluoride per gram of cell pellet (50 mm phenylmethylsulfonyl fluoride stock made up in 100% isopropanol) and 0.05% polyethyleneimine. Suspended cells were sonicated (HeatSystems), and the lysate was centrifuged (35,000 × g) for 30 min. The resultant supernatant was filtered (0.22-μm, Millipore), then loaded onto a DEAE-Sepharose FF column (30 ml) (Amersham Biosciences) at 4 °C. Elution employed a linear gradient of 150–500 mm NaCl in 50 mm Tris-HCl, pH 8.0, and 1 mm EDTA. Fractions containing CarC were pooled, concentrated, and applied to an S75-Superdex column equilibrated in 150 mm Tris-HCl, pH 8.0, and 1 mm EDTA. Fractions containing CarC, judged to be ∼90% pure (by SDS-PAGE), were pooled and concentrated. CarC was exchanged into 10 mm Tris-HCl, pH 8.0, using a PD-10 gel-filtration column (Amersham Biosciences) and concentrated to 40 mg/ml prior to crystallization (molecular masses measured by negative ion electrospray MS: 31,480 ± 5 Da; calculated mass of CarC without N-terminal Met: 31,479.8 Da). Bioassay—Assay mixtures were transferred into holed (11-mm diameter) bioassay plates (E. coli X580) and incubated at 37 °C overnight. A typical assay mixture consisted of Tris-HCl, pH 9 (10 mm), 2-oxoglutarate (2OG) (10 mm), MgCl2 (2 mm), ATP (3 mm), CMP (3 mm), CarA (2.5 mg/ml), CarC (1.6 mg/ml), and FeSO4 (1 mm). The assay was incubated at 37 °C for 30 min. When trans-CMP was used as a substrate an antibiosis zone equivalent to ∼24 nmol/ml cephamycin C was observed. LC/MS—LC/MS was performed using a Waters high-performance liquid chromatography system connected to a Micro-Mass ZMD mass spectrometer in the negative ion mode. Assay mixtures as for the bioassays were mixed with MeOH (equal volume), chilled on ice for 10 min, and then centrifuged (17,800 × g) for 5 min before analysis. Controls were carried out under identical conditions but with deactivated enzymes. The assay mixture (20 μl) was injected onto a Synergi Polar-RP (250 mm × 4.6 mm) column (Phenomenex) equilibrated in water at 1 ml/min. After 15 min a gradient to 90% MeOH was run over 5 min. These conditions were maintained for 5 min before returning to 100% water over 5 min and re-equilibration for 10 min. Quantitative Gel Filtration and Native PAGE Analysis—Molecular weight analysis by gel filtration used an Amersham Biosciences calibration kit and a 24-ml Superdex 200 HR 10/30 column equilibrated with 150 mm Tris-HCl, pH 8.0, at a flow rate of 0.5 ml/min. Native-PAGE employed 15% Tris-glycine polyacrylamide gels lacking SDS and run at 50 V for 10 h. The following standards were used (10 mg/ml): ovalbumin (43 kDa), bovine serum albumin (67-kDa monomer, 134-kDa dimer, and 268-kDa tetramer), and catalase (232 kDa). Crystallization—CarC was crystallized using the hanging-drop vapor diffusion method. Droplets (2 μl) containing CarC (24 mg/ml), 10 mm 2OG, and 5 mm FeSO4 were mixed with 2 μl of well buffer and equilibrated against 500 μl of the same well buffer. Anaerobic crystallization (<0.5 ppm of O2) under argon was carried out in a Belle Technology glove box. After ∼1 week a mixture of thin needles and microcrystals was obtained from two solutions: (i) 30% i-PrOH, 0.1 m Tris-HCl, pH 8.5, 0.2 m NH4Ac and (ii) 8% (w/v) PEG 8000, 0.1 m Tris-HCl, pH 8.5. Crystals suitable for x-ray diffraction were obtained from 4% (w/v) PEG 8000, 400 mm NH4Ac, 10% (w/v) i-PrOH, 100 mm Tris-HCl (pH 8.5), 5 mm FeSO4, and 10 mm dipotassium 2OG. Selenomethionine (SeMet) substituted CarC was produced using a metabolic inhibition protocol and LeMaster media supplemented with 50 μg/ml L-SeMet. Electrospray ionization-MS analysis determined a mean incorporation of 5 selenium atoms per CarC monomer. SeMet CarC was crystallized from 4% (w/v) PEG 8000, 400 mm NH4Ac, 10% (w/v) i-PrOH, 100 mm Tris-HCl (pH 8.5), 5 mm FeSO4, and 10 mm dipotassium 2OG. l-N-Acetylproline (l-NAP) was cocrystallized with CarC using a protein solution containing CarC (12 mg/ml), l-NAP (10 mm), FeSO4 (2.5 mm), 2OG (5 mm), i-PrOH (5%), PEG 8000 (2%), NH4Ac (200 mm), and Tris-HCl, pH 8.5 (50 mm). For low temperature data collection, crystals were soaked in well buffer supplemented with 20% (w/v) ethylene glycol and flash-frozen in liquid N2. Data Collection and Phase Determination—Diffraction data for SeMet and l-NAP cocrystallized CarC were collected at 100 K on beamline 14.2 of the Synchrotron Radiation Source, Daresbury, UK with an ADSC Quantum 4 detector. The data were processed using MOSFLM and SCALA of the CCP4 suite (23Collaborative Computational Project Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) (Table I). Fifteen selenium positions were located and refined with SOLVE (24Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). Phases were calculated from these positions with SHARP (25De La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar). Density modification and non-crystallographic symmetry averaging were carried out using DM of the CCP4 suite (23Collaborative Computational Project Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). 5% of the reflections were randomly selected to provide an Rfree test set.Table IData collection, phasing, and refinement statisticsSelenomethionine·Fe(II)·2OGL-NAP·Fe(II)·2OGPeakEdgeRemoteSpace groupC2221C2221Dimensiona (Å)79.980.4b (Å)163.9164.1c (Å)146.5146.3Molecules per asymmetric unit33Temperature (K)100100Wavelength (λ)0.97850.97901.00000.9780Resolution (Å)aValues in parentheses are for the outermost resolution shell.2.40-30 (2.40-2.46)2.30-25 (2.30-2.36)Observations173615170325178678157442Unique reflections37852379903799941822Completeness (%)aValues in parentheses are for the outermost resolution shell.99.7 (99.4)99.8 (99.2)99.9 (100)97 (98.6)Rmerge (%)aValues in parentheses are for the outermost resolution shell.6.3 (29.1)6.6 (32.2)5.9 (24.6)6.4 (23.5)<I/σ>aValues in parentheses are for the outermost resolution shell.9.9 (2.5)7.4 (2.4)7.6 (3.1)7.5 (3.2)Rcryst0.2230.230Rfree0.2680.275r.m.s.d.br.m.s.d., root mean square deviation from ideality.Bond length (Å)0.0180.014Bond angle (°)1.8871.725Disordered residuesA67-78/161-17268-78/161-172B68-72/161-17069-72/161-171C69-79/162-17267-79/161-172Average B-factorcProtein, solvent, and substrate, respectively.23, 3422, 33, 79PDB ID code1NX41NX8a Values in parentheses are for the outermost resolution shell.b r.m.s.d., root mean square deviation from ideality.c Protein, solvent, and substrate, respectively. Open table in a new tab Model Building and Refinement—Using the program O (26Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) all residues were built except for the N-terminal methionine, the C-terminal isoleucine, and two flexible loops linking α3/β4 and β7/β8 (the exact residues missing varied between monomers). Structure refinement was performed using REFMAC5 (27Murshudov G.N. Vagin A.A. Lebedev A. Wilson K.S. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 247-255Crossref PubMed Scopus (1010) Google Scholar), with inclusion of the iron atoms, 2OG molecules, and 232 water molecules. There were no outliers in the Ramachandran plot (92.5%, 7.3%, 0.2% in the core, allowed, and generously allowed regions, respectively). Molecular replacement using AMoRe (28Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar) was used to phase the data set collected with l-NAP cocrystals, and REFMAC5 was employed for the structure refinement. There was one Ramachandran outlier whose conformation appeared to be supported by the electron density (Leu-108 in subunit B) (92.8%, 7.0%, 0.0%, and 0.2% in the core, allowed, generously allowed, and disallowed regions, respectively). The electron density maps indicated that l-NAP was present in subunits B and C but at a much lower level, if at all, in A. Note that, although the submitted PDB file (1NX8) indicates one orientation (orientation I, see “Results and Discussion”), the orientation of l-NAP within the active site could not be unequivocally inferred from the electron density maps and a second orientation (II) is possible. Molecular Modeling—Hydrogens were added to the complex using the HBUILD routine of CHARMM (version 27) (29Brooks B.R. Bruccoleri R.E. Olafson B.D. States D.J. Swaminathan S. Karplus M. J. Comput. Chem. 1983; 4: 187-217Crossref Scopus (14019) Google Scholar). The final model included the protein atoms, the l-NAP ligand, 2OG, Fe(II), and 39 crystallographic waters, 4119 atoms in total. Models of the structure complexed with (3S,5S)- and (3S,5R)-carbapenams were prepared using QUANTA (30Inc Accelrys QUANTA. Burlington, MA1997Google Scholar). Modeling studies were carried out with both carbapenams, using orientations I and II of l-NAP as initial “templates.” In each case, a full occupancy oxygen atom was added (at a distance of 2.2 Å from Fe(II)) in the ligation position opposite to His-251 (the proposed catalytic cycle involves an Fe(IV)=O intermediate). Without this oxygen, the minimization resulted in the substrate carboxylate ligating to Fe(II). The involvement of such an iron-substrate carboxylate complex in catalysis seems unlikely both with respect to precedent (31Zhang Z. Ren J. Harlos K. McKinnon C.H. Clifton I.J. Schofield C.J. FEBS Lett. 2002; 517: 7-12Crossref PubMed Scopus (140) Google Scholar) and on mechanistic grounds but cannot be ruled out. Each of these models contained 4119 atoms. A combined quantum mechanical/molecular mechanical (32Warshel A. Levitt M. J. Mol. Biol. 1976; 103: 227-249Crossref PubMed Scopus (3695) Google Scholar, 33Field M.J. Bash P.A. Karplus M. J. Comput. Chem. 1990; 11: 700-733Crossref Scopus (2185) Google Scholar) (QM/MM) potential was used to perform minimizations of the model systems, while keeping Fe(II) and the atoms that are bound to it (His-101-N[epsis ]2, Asp-103-Oδ1, His-251-N[epsis ]2, 2-keto, and 1-carboxylate oxygens of 2OG, and the additional oxygen atom in the two carbapenam systems) fixed in their original positions (Table II). In each case the ligand was described by a QM potential and the rest of the system by a coupled MM potential. The CHARMM standard all-atom parameters (34MacKerell Jr., A.D. Bashford D. Bellott M. Dunbrack Jr., R.L. Evanseck J.D. Field M.J. Fischer S. Gao J. Guo H. Ha S. Joseph-McCarthy D. Kuchnir L. Kuczera K. Lau F.T.K. Mattos C. Michnick S. Ngo T. Nguyen D.T. Prodhom B. Reiher I.W. E. Roux B. Schlenkrich M. Smith J.C. Stote R. Straub J. Watanabe M. Wiorkiewicz-Kuczera J. Yin D. Karplus M. J. Phys. Chem. B. 1998; 102: 3586-3616Crossref PubMed Scopus (11819) Google Scholar) were used in the MM region, except for the oxygen in the empty ligation position on Fe(II), which was given a charge of –1, and for the 2OG, whose charges were calculated by fitting them to the B3LYP/6–31G* electrostatic potential in vacuum (35Besler B.H. Merz K.M. Kollman P.A. J. Comput. Chem. 1990; 11: 431-439Crossref Scopus (2954) Google Scholar), using Gaussian98 (36Frisch M.J. Trucks G.W. Schlegel H.B. Scuseria G.E. Robb M.A. Cheeseman J.R. Zakrzewski V.G. Montgomery J.A. Stratmann R.E. Burant J.C. Dapprich S. Millam J.M. Daniels A.D. Kudin K.N. Strain M.C. Farkas O. Tomasi J. Barone V. Cossi M. Cammi R. Mennucci B. Pomelli C. Adamo C. Clifford S. Ochterski J. Petersson G.A. Ayala P.Y. Cui Q. Morokuma K. Malick D.K. Rabuck A.D. Raghavachari K. Foresman J.B. Cioslowski J. Ortiz J.V. Stefanov B.B. Liu G. Liashenko A. Piskorz P. Komaromi I. Gomperts R. Martin R.L. Fox D.J. Keith T. Al-Laham M.A. Peng C.Y. Nanayakkara A. Gonzalez C. Challacombe M. Gill P.M.W. Johnson B.G. Chen W. Wong M.W. Andres J.L. Head-Gordon M. Replogle E.S. Pople J.A. Gaussian98. Gaussian Inc., Pittsburgh, PA1998Google Scholar). For non-bonded interactions, the electrostatics terms were truncated with a force switch function between 10 and 14 Å and the van der Waals terms with a shift function with a cutoff distance of 14 Å (37Steinbach P.J. Brooks B.R. J. Comput. Chem. 1994; 15: 667-683Crossref Scopus (915) Google Scholar). The QM region was treated with the semi-empirical quantum mechanical method AM1 (38Dewar M.J.S. Zoebisch E.G. Healy E.F. Stewart J.J.P. J. Am. Chem. Soc. 1985; 107: 3902-3909Crossref Scopus (15086) Google Scholar) implemented within CHARMM (33Field M.J. Bash P.A. Karplus M. J. Comput. Chem. 1990; 11: 700-733Crossref Scopus (2185) Google Scholar). The QM/MM minimizations included the steepest descent method followed by the Adopted Basis Newton-Raphson method implemented in CHARMM, until the average r.m.s. gradient was less than 0.01 kcal mol–1 Å–1. Energies and geometric parameters were obtained from the minimizations to compare the stability of the different models. The procedure was repeated for the (5R)- and (5S)-carbapenems (without the oxygen atom in the empty ligation position opposite to His-251) and for the two carbapenems except with 2OG replaced with succinate. In the latter case the results were almost identical to those obtained with 2OG.Table IIInteratomic distances and relative energy data for QM/MM calculations of the stability of carbapenems modeled into the active site of CarC CHARMM/AM1ModelRelative energyDistanceaAll distances are between hydrogens and heavy atoms. The values are in Angstroms.Fe C2-HFe C2-H′Fe C3-HFe C5-HFe C6-HFe C6-H′Arg-267-HH12 8-OArg-267-HH22 8-OGly-104-NH 8-O′Gly-105-NH 8-O′Leu-106-NH 8-O′Ala-107-NH 8-O′Tyr-191-OH 7-OGly-104-NH 7-OGly-105-NH 7-OLeu-106-NH 7-OLeu-106-NH 7-O(kcal/mol)1. (3S,5S)bModels built based on L-NAP orientation II.-70286.78.17.74.25.46.92.01.93.33.32.12.11.94.12.53.05.02. (3S,5R)bModels built based on L-NAP orientation II.-70326.68.08.37.34.45.72.11.91.92.43.44.62.03.82.52.04.63. (3S,5S)cModels built based on L-NAP orientation I.-70225.25.05.88.710.09.02.01.85.46.55.95.84.83.42.12.42.14. (3S,5R)cModels built based on L-NAP orientation I.-70235.24.95.28.58.69.82.01.85.16.35.65.46.25.04.33.82.15. (5R)10.56.95.16.59.18.84.31.82.11.83.62.22.82.34.36. (5S)10.78.66.36.39.29.14.42.02.01.83.92.02.42.64.3a All distances are between hydrogens and heavy atoms. The values are in Angstroms.b Models built based on L-NAP orientation II.c Models built based on L-NAP orientation I. Open table in a new tab The CarC Reaction—The proposed carbapenam-3-carboxylic acid intermediate was prepared from the appropriate β-amino acid precursor using CarA. 2M. C. Sleeman, unpublished results. The carC and carA genes were cloned from the E. carotovora genomic DNA and expressed in E. coli, and the corresponding proteins were purified by standard techniques. The (5S-carboxymethyl)-S-proline (trans-CMP) putative substrate for CarA was prepared via minor modification of reported methodology (39Kanno O. Shimoji Y. Ohya S. Kawamoto I. J. Antibiot. 2000; 53: 404-414Crossref PubMed Scopus (6) Google Scholar). Assays with CarA alone, and combined CarA/CarC assays, containing the appropriate cofactors and potential CarA substrates, were conducted. With the trans-CMP substrate, no antibiotic activity was observed in assays with CarA alone. With trans-CMP in the presence of both CarA and CarC, a clear zone of activity was observed. Liquid chromatography-MS of CarA assay mixtures using trans-CMP led to the observation of a new peak with a mass corresponding to a carbapenam (negative ion electrospray: 154 Da [M-H]–) absent in controls. With trans-CMP in the combined CarA/CarC reactions, peaks were observed for masses corresponding to both a carbapenam and a carbapenem (negative ion electrospray: 152 Da [M-H]–), again absent in controls. Assuming that CarA does not catalyze epimerization, the results imply that CarA can mediate β-lactam ring formation from trans-CMP to give (3S,5S)-carbapenam (Scheme 1, path a). They also suggest that the (3S,5S)-carbapenam can be converted by CarC to the (5R)-carbapenem. Because it cannot be entirely ruled out that the (3S,5S)-CMP used in this study was contaminated with a low level of its (3R,5R) enantiomer, on the basis of our data alone the possibility that the natural substrate for CarC is a (3R,5R) carbapenam cannot be discounted (Scheme 1, path b); however, this would be in conflict with the results and conclusions of both Li et al. (12Li R. Stapon A. Blanchfield J.T. Townsend C.A. J. Am. Chem. Soc. 2000; 122: 9296-9297Crossref Scopus (63) Google Scholar) and Bycroft et al. (14Bycroft B.W. Chhabra S.R. J. Chem. Soc. Chem. Commun. 1989; : 423-425Crossref Google Scholar, 21Bycroft B. Chhabra S.R. Kellam B. Smith P. Tetrahedron Lett. 2003; 44: 973-976Crossref Scopus (19) Google Scholar). The level of substrate conversion effected by CarA with the trans-CMP was low compared with that of β-lactam synthetase (from the clavulanic acid biosynthesis pathway) with its natural substrate, possibly indicating an alternative in vivo substrate (see Scheme 1) or that a multiprotein complex is required to effect full activity (11McGowan S.J. Sebaihia M. O'Leary S. Hardie K.R. Williams P. Stewart G.S.A.B. Bycroft B.W. Salmond G.P.C. Mol. Microbiol. 1997; 26: 545-556Crossref PubMed Scopus (70) Google Scholar). The organization of β-lactam biosynthesis proteins into a metabolon has also been suggested for clavulanic acid (40Kershaw N.J. McNaughton H.J. Hewitson K.S. Hernández H. Griffin J. Hughes C. Greaves P. Barton B. Robinson C.V. Schofield C.J. Eur. J. Biochem. 2002; 269: 2052-2059Crossref PubMed Scopus (30) Google Scholar). Crystallization and Oligomerization—Crystals of CarC complexed with Fe(II) and 2OG were obtained under anaerobic conditions. Crystals were also obtained anaerobically for CarC together with Fe(II), 2OG, and a substrate analogue (N-acetyl-L-proline) (see below). The structure was solved by molecular replacement using the model of SeMet-substituted CarC complexed with Fe(II). Analysis of crystallographic symmetry revealed that CarC crystallizes as a hexamer comprised of two trimers (ABC and DEF in which A = D, B = E, and C = F) (C2221) (Fig. 1). Each asymmetric unit contains three monomers in a trimeric arrangement, with a hexamer being generated by a 2-fold crystallographic symmetry axis. Gel filtration and native gel electrophoresis studies also indicated that the predominant form of CarC in solution is also hexameric with low levels of monomeric and trimeric forms also being observed (molecular mass by analytical gel filtration: ∼200 kDa). Within each trimer the monomers are arranged such that the active sites are well separated and directed toward the exterior of the hexamer, which possesses a large central channel. Hydrogen bonds and electrostatic interactions form links between the monomers and link the ABC and DEF trimers to form the hexamer; residues from α5to α6 on th" @default.
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• W1997588995 title "Crystal Structure of Carbapenem Synthase (CarC)" @default.
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