From the
Oxford Centre for Molecular Sciences and The Dyson Perrins Laboratory and the ¶Physical and Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, United Kingdom and the **School of Biological Sciences, Nanyang Technological University, 1 Nanyang Walk, Singapore 637616, Republic of Singapore
Received for publication, December 20, 2002 , and in revised form, February 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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Bycroft, Salmond, and coworkers (7) 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 (8). 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 (4). Eight of the genes, carAH, are organized as an operon controlled by CarR, an LuxR type transcriptional activator (9, 10). Five of the genes, carAE, 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 CarAC (11, 12).
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) (2, 13). This can then be cyclized in an ATP-driven process catalyzed by CarA, to give a (3S,5S)-carbapenam (14). This reaction is closely related to -lactam formation during biosynthesis of the
-lactamase inhibitor clavulanic acid (15, 16, 17) but is very different from
-lactam formation during penicillin biosynthesis (18). In this proposed pathway and consistent with the reports of both Bycroft et al. (14) and Li et al. (12), the role of CarC is to catalyze both epimerization at C-5 and desaturation across the C-2/C-3 bond of the carbapenam.
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There has been some uncertainty over the absolute stereo-chemistry of the carbapenams involved in carbapenem biosynthesis. Originally, two -lactams assigned as (3R,5R)- and (3S,5R)-carbapenams were isolated (19). The former was subsequently reassigned as a (3S,5S)-carbapenam following synthesis of a derivative of the enantiomeric (3R,5R)-carbapenam (14). Tanaka et al. (20) have queried this reassignment, but recent synthetic work has substantiated the assignment of the natural carbapenam as (3S,5S) (21).
CarC is related to clavaminic acid synthase (CAS)1 (approximately 23% sequence identity) (8), which catalyzes three reactions, comprising hydroxylation, ring closure, and desaturation processes, during clavulanic acid biosynthesis (22). 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.
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EXPERIMENTAL PROCEDURES |
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BioassayAssay 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/MSLC/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 x 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 x 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 AnalysisMolecular 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).
CrystallizationCarC 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 DeterminationDiffraction 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 (23) (Table I). Fifteen selenium positions were located and refined with SOLVE (24). Phases were calculated from these positions with SHARP (25). Density modification and non-crystallographic symmetry averaging were carried out using DM of the CCP4 suite (23). 5% of the reflections were randomly selected to provide an Rfree test set.
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Model Building and RefinementUsing the program O (26) all residues were built except for the N-terminal methionine, the C-terminal isoleucine, and two flexible loops linking 3/
4 and
7/
8 (the exact residues missing varied between monomers). Structure refinement was performed using REFMAC5 (27), 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 (28) 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 ModelingHydrogens were added to the complex using the HBUILD routine of CHARMM (version 27) (29). 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 (30). 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 (31) and on mechanistic grounds but cannot be ruled out. Each of these models contained 4119 atoms. A combined quantum mechanical/molecular mechanical (32, 33) (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-N2, Asp-103-O
1, His-251-N
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 (34) 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/631G* electrostatic potential in vacuum (35), using Gaussian98 (36). 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 (37). The QM region was treated with the semi-empirical quantum mechanical method AM1 (38) implemented within CHARMM (33). 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 mol1 Å1. 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.
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RESULTS AND DISCUSSION |
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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 -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. (12) and Bycroft et al. (14, 21). The level of substrate conversion effected by CarA with the trans-CMP was low compared with that of
-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 (11). The organization of
-lactam biosynthesis proteins into a metabolon has also been suggested for clavulanic acid (40).
Crystallization and OligomerizationCrystals 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).
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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 5to
6 on the A subunit interact with the loop linking the
5 and
6 strands on the B-subunit.
With respect to interactions between the ABC and DEF trimers, A interacts most closely with E (the symmetry-equivalent of B), whereas B interacts with D (the symmetry-equivalent of A). C, however, interacts with F, its own symmetry-equivalent. This difference results from a crystallographic 2-fold symmetry axis that runs between A/E and B/D pairs but between C and F monomers (Fig. 1b). In the case of the A/E interactions, residues from the N terminus, 2 and
8, from the A subunit interact with their counterparts on the E subunit to form the hexamer.
Overall Structure of the MonomerThe structures of the A, B, and C monomers are similar but not identical (the r.m.s. deviations of the C atoms for the AB, BC, and CA pairs were 0.27, 0.26, and 0.17 Å, respectively). In the following discussions, the descriptions refer to the B monomer. The CarC main chain contains 14
-strands, 8 of which (
1
4,
6, and
11
14) combine to form the distorted double-stranded
-helix (DSBH or jellyroll) motif characteristic of the 2OG oxygenase superfamily that includes CAS (41), taurine dioxygenase (TauD) (42), and proline 3-hydroxylase (43) (Fig. 2). A DSBH is found in a wide range of metal binding (including the Cu(II)-utilizing quercetin 2,3-dioxygenase (44)) and non-metal-binding proteins.
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The presence of an extended insert (residues 135225 (4
6 and
7
10)) between the fourth and fifth strands of the DBSH places CarC within a distinct sub-group of 2OG oxygenases that includes CAS (45) but not deacetoxycephalosporin C synthase (DAOCS). The structural similarity between CarC and CAS (23% sequence identity) reinforces proposals that clavam and carbapenem biosynthesis have a close evolutionary relationship (8).
Active Site and 2-Oxoglutarate BindingThe active site is predominantly made up of residues from strands 13,
14, and
6 of the DSBH (His-251, Arg-253, Arg-263, Arg-267, Gln-269, and Thr-130). The remainder of the active site is constructed from two loops: those linking
3/
4 (His-101, Asp-103, Gly-104, Gly-105, Leu-106, and Ala-107) and those linking
9/
10 (Tyr-191, Phe-194, and Trp-202) with the latter on
10. The entrance to the active site is formed by the extended loop linking
3/
4 (residues 85111) and, probably, by the loops linking
3/
4 and
7/
8, which are disordered.
The 5-carboxylate of 2OG is bound by the side chains of Arg-263 (14, 3.2 and 2.9 Å), Thr-130 (
6, 2.7 Å), and Arg-253 (
13, 3.0 Å). Arg-263 and Thr-130 are conserved in CAS and TauD (42), although the presence of Arg-253 is distinct to CarC. The 2-keto- and 1-carboxylate groups of 2OG bind in a bi-dentate manner to the single iron, which is ligated by side chains from His-101, Asp-103, and His-251, all either part of or close to the DSBH (Fig. 3). These residues form a conserved 2-His-1-carboxylate triad of residues (46). CarC differs from CAS, but not TauD, in that its carboxylate ligand comes from an Asp rather than a Glu residue, highlighting the special nature of CAS in this regard. In the CarC·Fe·2OG·L-NAP structure, a water molecule is ligated to the ferrous iron opposite His-101 thus giving a six-coordinate arrangement. In the CarC·Fe·2OG structure, the 2OG is ligated such that its 2-keto-group is opposite Asp-103, consistent with results for other 2OG oxygenases. The relative arrangement of Fe(II) ligands in the structures of anthocyanidin synthase and DAOCS positions the 2OG 1-carboxylate opposite His-251 (using the CarC numbering system); however, in TauD and CAS the 2OG 1-carboxylate is opposite His-101 (using the CarC numbering system). With CarC the 2OG 1-carboxylate appears to be in an intermediate position but is closer to being opposite to His-251 (angle for N
His-251·Fe·1-carboxylate of 2OG, 150°; angle for N
His-101·Fe·1-carboxylate of 2OG, 111°; for the L-NAP structure).
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When a CAS·Fe·2OG·substrate complex was exposed to NO, acting as a dioxygen analogue, a rearrangement occurred placing the 2OG 1-carboxylate opposite His-251 (using the CarC numbering system), suggesting that in all cases dioxygen binding may occur trans to His-101 (31). However, as for related oxygenases, the substrates or substrate analogues (i.e. L-NAP for CarC) may not be correctly positioned to effect substrate oxidation if the ferryl species is formed opposite to His-101. It has been proposed that, following decarboxylation of 2OG, the ferryl species rearranges to be opposite His-251 and adjacent to the substrate (31). Alternatively, it cannot be ruled out that the 1-carboxylate can rearrange such that dioxygen can then bind opposite to His-251.
Substrate BindingIn addition to the Fe(II)·2OG complex and associated residues, the active site cavity comprises a largely hydrophobic region including Leu-98, Leu-106, Phe-194, Tyr-191, and Trp-202. The latter three are arranged such that the indole ring of Trp-202 is sandwiched by the aromatic rings of Phe-194 and Tyr-191 in an approximately orthogonal manner. These residues form a hydrophobic wall on one side of the active site cavity (Figs. 2 and 5). On another face of the active site the backbone amide N-Hs of Gly-104, Gly-105, and Leu-106 and the side chains of Ser-109 and Tyr-191 form a notable sequence of H-bond donors. The phenolic OH of Tyr-191 points toward the proposed substrate binding area.
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A 2OG turnover assay was used to screen a limited range of substrate analogues for binding to CarC. L-NAP and D-NAP were found to inhibit 2OG turnover ( 60 and 25% of uncoupled turnover respectively). L-NAP was then used in cocrystallization experiments. The occupancy level of L-NAP differs between the three monomers A, B and C, from partial occupancy in B and C to very limited occupancy in A.
The CarC·Fe(II)·2OG·L-NAP structure is very similar to that of the CarC·Fe(II)·2OG complex. A water molecule is probably ligated to the ferrous iron opposite to His-101, possibly reflecting the observation that L-NAP inhibits (uncoupled) 2OG turnover. A strategy for potential inhibition of 2OG oxygenases is thus to hinder oxygen binding via stabilization of octahedral iron coordination chemistry. Due to the similar size of the acetyl and carboxylate groups of the L-NAP and its partial occupancy, it was not possible to unequivocally assign the orientation of L-NAP within the active site. Thus two possible orientations of L-NAP (I and II) related by a rotation of 180° are equally likely. In both orientations, two of the methylenes of L-NAP buttress against the side chain of Trp-202 in a hydrophobic interaction. In orientation I, the acetyl group is directed toward Arg-267 and Gln-269 and the backbone amide N-Hs of Gly-104. The hydrogens on the opposite face of the proline ring to the carboxylate are directed toward the iron. In orientation II, the carboxylate is directed toward the side chains of Arg-267 and Gln-269 and the backbone amide N-Hs of Gly-104. The hydrogens on the same face as the carboxylate are directed toward the iron. The proposed interaction of the carboxylate with Arg-267 in orientation II, is supported by the precedent of CAS, where the equivalent arginine (Arg-297, CAS) forms interactions with both the 1-carboxylate of 2OG and the substrate carboxylate, suggesting that this residue plays a key role in catalysis (Fig. 4).
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Because the natural CarC substrate was not available in sufficient quantities for crystallization work, we used QM/MM modeling studies to investigate possible modes of carbapenam binding. Initial minimization of the model based on the CarC·Fe(II)·2OG·L-NAP crystal structure, in which the L-NAP is bound in orientation II resulted in a similar overall structure (r.m.s. deviation for non-hydrogen atoms, without waters: 1.5 Å), except for small changes in the loop between residues Gly-104 and Ser-109. Minimizations were then performed with the (3S,5S)- and the (3S,5R)-carbapenam models using both orientations I and II of L-NAP as initial "templates," i.e. by overlaying the pyrrolidine-carboxylate rings. Different binding possibilities are observed in the models, which suggest the Gly-104 to Ser-109 loop may be involved in binding a substrate carboxylate or lactam carbonyl. However, in each case, the results indicated similar energies for both the (3S,5S)- and the (3S,5R)-carbapenams (Fig. 5).
Mechanistic DiscussionStudies with other 2OG oxygenases have indicated that conformational changes occur upon substrate binding (47) such that the substrate binds to an "open" conformation but is isolated at the active site when it is oxidized. Thus the current crystallographic and modeled CarC structures may not precisely represent the active site conformation in which substrate binding and/or oxidation occurs. Nonetheless, they identify key residues involved in catalysis and suggest possible mechanisms for the CarC reaction.
Given the precedents with CAS and TauD, and the observation that the carboxylate of L-NAP may bind to the active site arginine (Arg-267), it is possible that the carboxylate of the (3S,5S)-carbapenam substrate binds to Arg-267 and Gln-269 during its oxidation, in a similar manner to that observed in orientation II of L-NAP and the associated (3S,5S)-carbapenam model; modeling suggests direct abstraction of a hydrogen at C-5 of the (3S,5S)-carbapenam may occur (Fig. 5). However in this case it would seem that re-orientation of the substrate would be required if a ferryl intermediate was to both abstract a hydrogen and re-hydrogenate "directly" at the C-5 position. The modeling studies indicate that a (3S,5R)-carbapenam, with or without a C-5 radical, could be accommodated in the active site, but there is no clear driving force for such a conformational change.
Instead, orientation I of L-NAP and associated models may represent the productive conformation. In this case epimerization may occur via abstraction of the C-3 or C-2 hydrogens (Scheme 2, a and b) or less likely, the C-1 hydrogen, followed by opening and ring closing of the bicyclic -lactam system. Desaturation can then occur as in CAS. This proposal is attractive, because re-orientation of the substrate in the active site is not required for a ferryl intermediate to effect both epimerization and desaturation. In this proposal re-hydrogenation of a radical intermediate can lead to the (3S,5R)-carbapenam in a shunt pathway.
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Other possibilities can be envisaged. Epimerization via abstraction at C-6 seems unlikely, because it would invoke a high energy primary radical and desaturation would either require significant re-orientation or unprecedented rearrangements/H-shifts. Given the precedent of ribonucleotide reductase (48) and others, the possibility that epimerization occurs via a process involving a protein based radical should be considered. However, analysis of the active site does not reveal clear candidates: Tyr-191 and Trp-202 were considered possibilities for such a role but appear incorrectly positioned. Thus, we favor a mechanism solely mediated via hydrogen transfers to the Fe(IV)=O and Fe(III)OH intermediates, which are believed to occur with reactions catalyzed by related oxygenases involving rearrangements and desaturations, such as DAOCS (47) and CAS (41, 49).
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FOOTNOTES |
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* This work was supported by the European Union, the Biotechnology and Biological Sciences Research Council, the Engineering and Physical Sciences Research Council, the Wellcome Trust, and Amura (for case awards to M. C. S. and L. X. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
|| To whom correspondence may be addressed. Tel.: 44-1-865-275-625; Fax: 44-1-865-275-674; E-mail: christopher.schofield{at}chem.ox.ac.uk (C. J. S.) or RCWilmouth{at}ntu.edu.sg (R. C. W.).
1 The 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 -helix; TauD, taurine dioxygenase; DAOCS, deacetoxycephalosporin C synthase; i-PrOH, 2-propanol; trans-CMP, ((5S-carboxymethyl)-S-proline).
2 M. C. Sleeman, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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