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The crystal structure of isopenicillin N synthase with a dipeptide substrate analogue
Archives of Biochemistry and Biophysics 530 (2013) 48–53
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Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
The crystal structure of isopenicillin N synthase with a dipeptide substrate analogue Adam Daruzzaman a, Ian J. Clifton a, Robert M. Adlington a, Jack E. Baldwin a,⇑, Peter J. Rutledge b,⇑ a b
Chemistry Research Laboratory, University of Oxford, Mansfield Rd, Oxford OX1 3TA, UK School of Chemistry F11, The University of Sydney, NSW 2006, Australia
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Article history: Received 19 September 2012 and in revised form 7 December 2012 Available online 19 December 2012 Keywords: Antibiotics Biosynthesis Enzyme mechanism Metalloenzymes Non-heme iron oxidase Penicillin
a b s t r a c t Isopenicillin N synthase (IPNS) converts its linear tripeptide substrate d-L-a-aminoadipoyl-L-cysteinyl-Dvaline (ACV) to bicyclic isopenicillin N (IPN), the key step in penicillin biosynthesis. Solution-phase incubation experiments have shown that IPNS will accept and oxidise a diverse array of substrate analogues, including tripeptides that incorporate L-homocysteine as their second residue, and tripeptides with truncated side-chains at the third amino acid such as d-L-a-aminoadipoyl-L-cysteinyl-D-a-aminobutyrate (ACAb), d-L-a-aminoadipoyl-L-cysteinyl-D-alanine (ACA) and d-L-a-aminoadipoyl-L-cysteinyl-glycine (ACG). However IPNS does not react with dipeptide substrates. To probe this selectivity we have crystallised the enzyme with the dipeptide d-L-a-aminoadipoyl-L-homocysteine (AhC) and solved a crystal structure for the IPNS:Fe(II):AhC complex to 1.40 Å resolution. This structure reveals an unexpected mode of peptide binding at the IPNS active site, in which the homocysteinyl thiolate does not bind to iron. Instead the primary mode of binding sees the homocysteinyl carboxylate coordinated to the metal, while its side-chain is oriented into the region of the active site normally occupied by the benzyl group of protein residue Phe211. Ó 2012 Elsevier Inc. All rights reserved.
Introduction Isopenicillin N synthase (IPNS) is a member of the non-heme iron enzyme family and catalyses the oxidative cyclisation of d-La-aminoadipoyl-L-cysteinyl-D-valine (ACV, 1) to isopenicillin N (IPN, 2) (Scheme 1) [1]. IPNS has been studied extensively using spectroscopic [2–4], mechanistic [1,5], theoretical [6,7] and crystallographic experiments [8–10]. It is generally agreed that the IPNS-catalysed conversion of ACV 1 to IPN 2 involves the high-valent iron-oxo intermediate 3 [10,11]. Solution phase experiments have shown that IPNS will accept a wide range of tripeptide substrate analogues and catalyse a diverse array of oxidative chemistry [5]. Truncated tripeptides such as d-La-aminoadipoyl-L-cysteinyl-D-a-aminobutyrate (ACAb, 4), d-L-aaminoadipoyl-L-cysteinyl-D-alanine (ACA, 5) and d-L-a-aminoadipoyl-L-cysteinyl-glycine (ACG, 6) are oxidised by IPNS
Abbreviations used: AC, d-L-a-aminoadipoyl-L-cysteinyl-; ACA, d-L-a-aminoadipoyl-L-cysteinyl-D-alanine; ACAb, d-L-a-aminoadipoyl-L-cysteinyl-D-a-aminobutyrate; ACG, d-L-a-aminoadipoyl-L-cysteinyl-glycine; ACV, d-L-a-aminoadipoyl-Lcysteinyl-D-valine; AhC, d-L-a-aminoadipoyl-L-homocysteine; AhC-, d-L-a-aminoadipoyl-L-homocysteinyl; AhCmC, d-L-a-aminoadipoyl-L-homocysteinyl-D-S-methylcysteine; AhCV, d-L-a-aminoadipoyl-L-homocysteinyl-D-valine; IPN, isopenicillin N; IPNS, isopenicillin N synthase. ⇑ Corresponding authors. Fax: +61 2 9351 3329 (P.J. Rutledge), fax: +44 1865 285002 (J.E. Baldwin). E-mail addresses: [email protected] (J.E. Baldwin), peter.rutledge@ sydney.edu.au (P.J. Rutledge). 0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2012.12.012
(Fig. 1). ACAb 4 is converted to three bicyclic products (two epimeric C2-methyl penams and a cepham) [12]; ACA 5 is turned over but gives undetermined product(s) that show no antibiotic activity nor any evidence of b-lactam character [13]; and ACG 6 gives rise to a hydrated aldehyde product via two-electron oxidation of the tripeptide (in contrast to the four-electron oxidation required for IPN formation from ACV 1) [14]. However IPNS does not turn over more severely truncated analogues that are missing the third amino acid altogether – i.e. dipeptides [5]. ACV analogues in which the second residue (L-cysteine) is replaced by L-homocysteine are oxidised by IPNS [15–17]. d-L-aAminoadipoyl-L-homocysteinyl-D-valine (AhCV, 7) yields monocyclic c-lactam products 8 upon reaction with IPNS (Scheme 2), thought to proceed via initial formation of the monocyclic c-lactam 9 (homologue of the ACV-derived b-lactam 3) [15]. However the iron(IV)-oxo moiety in 9 does not extract the b-hydrogen from the AhCV valine – for steric and/or stereoelectronic reasons – and is instead quenched by other means. This leaves the enzyme-bound c-lactam to collapse to an acyl iminium ion, subsequently hydrolysed to the c-lactam alcohol products 8. AhC-D-allylglycine [15], AhC-D-cysteine [16] and selectively deuterated [15] and fluorinated [17] AhCV analogues have all been studied as mechanistic probes, and all undergo oxidation at the hCys residue when incubated with IPNS. Protein crystallography has greatly enhanced our understanding of IPNS catalysis [8–10]. Crystal structures of IPNS with its natural substrate ACV [9], its product IPN [10,18], and various
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Scheme 1. IPNS converts the linear tripeptide ACV 1 to bicyclic IPN 2, a reaction that is generally agreed to proceed via the monocyclic intermediate 3 [1,10,11].
Scheme 2. Reaction of AhCV 7 with IPNS generates the hydroxy c-lactams 8, a reaction thought to proceed via the monocyclic intermediate 9 [15].
substrate analogues [13,19–21] have revealed different modes of substrate binding, and elucidated the interplay between active site geometry and reactivity. Pseudo-time-resolved studies using highpressure oxygenation to trigger reaction inside IPNS crystals have brought further insight, allowing the enzyme mechanism to be studied within the crystalline protein [10,22–26]. Crystal structures have previously been reported for complexes of IPNS with the truncated tripeptide analogues ACAb 4, ACA 5 and ACG 6, with and without the dioxygen analogue nitric oxide (NO) also bound [13,19]. These structures revealed new binding modes and metal-centred rearrangements at the IPNS active site, and showed that reducing the size and hydrophobicity of the valinyl side-chain of ACV 1 allows additional water molecules into the active site, even with NO also bound [13,19]. There is thus a very delicate interplay between steric interactions and hydrophilic/ hydrophobic effects around the catalytic metal centre, and IPNS catalysis is strongly influenced by differences both in hydrophobicity and steric bulk. To probe this interplay of sterics and hydrophobicity further, we report herein the crystal structure of IPNS complexed to a dipeptide substrate analogue. The question as to why IPNS does not execute a half reaction when presented with an appropriate dipeptide substrate analogue has long remained unresolved. It seems plausible that binding of the thiolate of a dipeptide analogue to the active site iron would facilitate oxygen binding and trigger partial turnover, but this has not been observed. During recent work to synthesise new AhCV analogues [27], the dipeptide d-L-a-aminoadipoyl-L-homocysteine (AhC, 10) was prepared. We have crystallised this analogue with IPNS to investigate what happens to substrate binding when the tripeptide is truncated to the extent that the third residue is missing altogether.
for 30 min, cooled to room temperature and concentrated in vacuo. The crude reside was azeotroped with toluene (2 10 mL) to remove residual TFA and dissolved in water (20 mL). The aqueous solution was washed with ethyl acetate (2 10 mL). The combined organic phases were extracted with water (2 10 mL) and the pooled aqueous phases back-extracted with ethyl acetate (10 mL). The combined aqueous extracts were lyophilised to give a crusty yellow solid, which was purified by reversed phase HPLC to give 11 as a fluffy white solid (17 mg, 65%); Rt 7 min 30 s; 1H (500 MHz, CDCl3): 1.54–1.73 (2H, m, NHCHCH2CH2CH2), 1.74–1.90 (2H, m, NHCHCH2CH2CH2), 2.09–2.11 (1H, m, 1 of CHCH2CH2S), 2.30 (2H, t, J 7.0, NHCHCH2CH2CH2), 2.52–2.60 (1H, m, 1 of CHCH2CH2S), 3.25–3.33 (1H, m, 1 of CHCH2CH2S), 3.35–3.32 (1H, m, 1 of CHCH2CH2S), 3.67 (1H, t, J 6.0, NHCHCH2CH2CH2), 4.62 (1H, X of ABX, JXA 13.0, JXB 7.0, CHCH2CH2S); 13C (500 MHz, CDCl3): 19.8 (NHCHCH2CH2CH2), 26.5 (CHCH2CH2S), 28.7 (NHCHCH2CH2CH2), 28.9 (CHCH2CH2S), 33.9 (NHCHCH2CH2CH2), 58.3 (CHCH2CH2S), 173.4 (CO2H), 175.1 (amide NHC=O) 209.6 (thioester SC = O); HRMS Calculated for C10H17N2O4S [MH]+: 261.0909; found 261.0909. NMR assignments are based on COSY, HSQC and HMBC. HMBC indicates coupling between C1 (carbonyl carbon) and the protons of C4 of the thiolactone moiety. Thus the cyclic structure 11 is assigned rather than the ring opened form 10 (Scheme 3).
Crystallography and structure determination Crystals of the IPNS:Fe(II):AhC complex were grown under anaerobic conditions by combining IPNS, iron(II) sulfate and thiolactone 11 as previously reported for tripeptide substrates [28,29]. Crystals for X-ray diffraction were selected using a light microscope, removed from the anaerobic environment and exchanged into a cryoprotectant buffer (1:1 mixture of well buffer
Materials and Methods Synthesis of (S)-2-amino-6-oxo-6-(((S)-2-oxotetrahydrothiophen-3yl)amino)hexanoic acid 11 N-tert-Butyloxycarbonyl-a-para-methoxybenzyl-d-(L-a-aminoadipoyl)-S-para-methoxybenzyl-L-homocysteinyl-S-methyl-D-cysteine benzhydryl ester 12 (90 mg, 0.10 mmol) was dissolved in trifluoroacetic acid (TFA, 4 mL) and anisole (0.50 mL, 4.6 mmol). The reaction mixture was heated at 80–90 °C (reflux) under argon
Scheme 3. Structure of the dipeptide d-L-a-aminoadipoyl-L-homocysteine (AhC, 10) in equilibrium with the cyclic thiolactone form 11.
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Scheme 4. Thiolactone 11 is isolated as the major product from the acid-mediated deprotection of tripeptide derivative 12, along with the deprotected tripeptide 13; conditions: TFA, anisole, reflux, RPHPLC, giving 11 (65%) and 13 (12%).
Fig. 1. Truncated tripeptides that have previously been crystallised with IPNS: d-L-a-aminoadipoyl-L-cysteinyl-D-a-aminobutyrate (ACAb, 4), d-L-a-aminoadipoyl-L-cysteinylD-alanine (ACA, 5) and d-L-a-aminoadipoyl-L-cysteinyl-glycine (ACG, 6) [13,19].
and saturated lithium sulfate in 40% v/v glycerol), then cryo-cooled in liquid nitrogen. Data were collected at the European Synchrotron Radiation Source (ESRF), Grenoble, France; the temperature was maintained at 100 K throughout data collection using an Oxford Cryosystems Cryostream. Data were processed using MOSFLM [30] and programs from the CCP4 suite [31], then refined using REFMAC5 [32] and Coot for model building [33]. Initial phases were generated using co-ordinates for the protein from the previously published IPNS:Fe(II):ACV structure [9], and manual rebuilding of protein side-chains was performed as necessary. Crystallographic coordinates and structure factors have been deposited in the Worldwide Protein Data Bank, under accession number 4bb3.
Results & discussion Synthesis of d-L-a-aminoadipoyl-L-homocysteine AhC, 10 The dipeptide AhC 10 exists in equilibrium with the cyclic form, thiolactone 11 (Scheme 3). The cyclic thioester 11 was isolated from the acid-mediated deprotection of the protected tripeptide derivative N-tert-butyloxycarbonyl-a-p-methoxybenzyl-d-(L-aaminoadipoyl)-S-p-methoxybenzyl-L-homocysteinyl-S-methyl-Dcysteine benzhydryl ester 12 (Scheme 4), alongside the tripeptide d-L-a-aminoadipoyl-L-homocysteinyl-D-S-methylcysteine (AhCmC, 13). Thiolactone 11 presumably arises from intramolecular cleavage of the hCys–mCys amide bond by the side-chain thiol under the acidic conditions of the deprotection reaction.
Fig. 2. Stereo view of the active site of the IPNS:Fe(II):AhC complex, showing electron density as: 2mFoDfc density [42] at 1.5r (green) and mFoDfc density at ±3r (cyan (+3r) and magenta (3r)) where r is the RMS value of each map over an asymmetric unit. Figure generated using ccp4 mg [43].
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The crystal structure of the IPNS:Fe(II):AhC complex
Table 1 X-ray data collection and crystallographic statistics. IPNS:Fe(II):AhC
a
X-ray source Wavelength (Å) PDB acquisition code Resolution (Å) Space group Unit cell dimensions (a Å, b Å, c Å)
ESRF beamline ID14 EH2 0.93 4bb3 1.40 P212121 46.51, 71.20, 100.88
Resolution shell (Å) Total number of reflections Number of unique reflections Completeness (%) Average I/r (I) Rmerge (%)a Rmeas (%)b
50.44–1.4 293474 66412 99.2 14.7 6.5 7.2
Rcryst (%)c Rfree (%)d RMS deviatione Average B factors (Å2)f Number of water molecules
17.27 18.83 0.022 (2.1) 14.0, 16.6, 19.9, 26.6 299
1.47–1.4 32787 9119 95.1 3.3 41.3 48.4
Rmerge = RjRp h|Iffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi h,j – hIhi|/RjRhhIhi 100. Rmeas = Rhkl N=ðN 1Þi |IðhklÞ Ix ðhklÞ|/RhklRI Ii (hkl) 100 [40,41]. P P c Rcryst = ||Fobs|- |Fcalc|| |Fobs| 100. d Rfree = based on 5% of the total reflections. e RMS deviation from ideality for bonds (followed by the value for angles). f Average B factors in order: main chain; side-chain; substrate and iron; solvent (water and 1 sulfate). b
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Anticipating that the thiolactone 11 would give rise to the linear dipeptide AhC 10 under the conditions used for crystallisation with IPNS (1.8 M lithium sulfate/ 100 mM TrisHCl buffer, pH 8.5), the cyclic compound 11 was subjected to these conditions following the previously reported procedure [29]. Diffraction quality crystals grew as with other substrate analogues, and the X-ray crystal structure reveals the IPNS:Fe(II):AhC complex at 1.40 Å resolution (Fig. 2, Table 1). The overall structure of the protein mirrors very closely complexes in which IPNS is coordinated to tripeptide substrates [9,10,21,34]. The active site metal ion is held by the characteristic triad of amino acid side-chains from the protein: His214, Asp216 and His270, the ‘2-His-1-carboxylate’ facial triad diagnostic of this enzyme family [35]. The dipeptide substrate analogue is bound at the active site, but not as anticipated. Its aminoadipoyl side-chain is oriented as seen in the IPNS:Fe(II):ACV complex, adopting a similar staggered conformation and tethered by the familiar salt bridge to Arg87. However the side-chain thiolate of the homocysteinyl residue does not ligate to the metal, but instead extends away from the iron centre into the region of space normally occupied by the side-chain of Phe211. The carboxylate of the homocysteinyl residue ligates to iron, which has octahedral coordination geometry. The remaining two binding sites at the metal are occu-
Fig. 3. Comparison of substrate binding to iron in the complexes of IPNS with a. ACV 1 (PDB id 1bk0); b. ACA 5 (1wo5); c. ACG 6 (1wo3); and d. AhC 10 (4bb3). Note the very different position of the (homo)cysteinyl sulfur atom (yellow) in d. Figure generated using ccp4 mg [43]. (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this paper.)
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pied by water molecules, opposite His214 (as seen in the IPNS:Fe(II):ACV structure), and trans to Asp216 (the site at which the co-substrate dioxygen would normally bind). Additionally, another well-defined water molecule occupies the region normally filled by the third-residue (‘valinyl’) carboxylate. No such ligand is present in this area of the apo-IPNS:Mn(II) complex, which is instead occupied by the C-terminal residue of the protein (Gln330) [8]. The side-chain of Phe211 is displaced by the homocysteine side-chain and takes up the orientation that is observed in the substrate-free apo-enzyme complex [8] and the complex of IPNS with the penam product 2-a-hydroxymethyl isopenicillin N [23]. The side-chain of Leu324 is disordered beyond Cd; it seems likely that this disorder arises from an interaction with the substrate, and the homocysteinyl thiolate sits in the right region of the active site to disturb Leu324. Other residues of the C-terminal tail (Leu324– Thr331) are not disturbed: this section of the protein is clearly defined and folded over the active site pocket in the ‘substratebound’ conformation. Mobility in the C-terminus has been linked to product departure from the IPNS active site [23], for which there is no evidence in this complex. It is apparent from the maps shown in Fig. 2 that there is some disorder in the electron density arising from AhC 10. The primary binding mode described above is present in 80% occupancy. Attempts to include partial occupancy by a second substrate orientation in the refinement model gave data consistent with low occupancy of second dipeptide binding mode (data not shown), however this could not be modelled more precisely. Conclusions Previous crystal structures with the truncated substrate analogues ACAb 4 [19], ACA 5 and ACG 6 [13] and d-(L-a-aminoadipoyl)-L-cysteinyl-D-vinylglycine (ACvG 14) [23] have shown that reducing the steric bulk of the third amino acid alters the coordination geometry at iron in the IPNS active site (Fig. 3). Similarly, substrate analogues that are L-configured at their third residue (where the native substrate ACV is D-configured) allow additional water molecules into this region of the IPNS active site, hydrogen bonded to the carboxylate of the L-configured third residue [34,36,37]. However it has never been previously observed that a substrate incorporating a free thiol does not coordinate to iron through this sulfur atom, as seen here with AhC 10 (Fig. 2, 3d). While dipeptide substrate analogues are not turned over to oxidised products, this study demonstrates that they can in fact bind to IPNS. Anchoring of the a-aminoadipoyl tail to Arg87 is key to this interaction, but thiolate ligation to iron is not required. Previous studies have demonstrated a strong affinity between thiolate or sulfide ligands and the metal centre at the IPNS active site [10,25,38,39]. On this basis, it was anticipated that if dipeptides such as AhC 10 or d-(L-a-aminoadipoyl)-L-cysteine were to bind in the enzyme active site, they would coordinate to iron via the (homo)cysteinyl thiolate. However the structure of the IPNS:Fe(II):AhC complex reveals that iron–sulfur ligation is not required, and this truncated substrate binds to iron through the carboxylate of its homocysteine residue. As a result, the a- and b-carbon atoms and thiolate of the homocysteine residue extend ‘behind’ the ligated substrate and rest in the cleft normally occupied by Phe211. The side-chain of that residue is displaced, and the sidechain of nearby Leu324 disturbed. The additional water ligands occupying the otherwise vacant space close to iron would increase the hydrophilicity of the active site region and impede access to the oxygen binding site. Moreover, it has previously been proposed that ligation of the ACV thiolate to iron lowers the Fe(II)/Fe(III) redox potential,
allowing dioxygen to bind and initiating the reaction cycle [9]. It follows that in the absence of thiolate ligation (as in the IPNS:Fe(II):AhC complex) the Fe(II)/Fe(III) redox potential is not lowered, dioxygen binding is not facilitated, and reaction is not triggered. We propose that this combination of increased hydrophilicity and altered redox potential at iron prevents IPNS from promoting any partial turnover or other oxidative reaction with the dipeptide 10, and by analogy the cysteine-containing homologue d-(L-a-aminoadipoyl)-L-cysteine and other related dipeptides. Acknowledgments We thank Professor Dr Nicolai Burzlaff, Dr Jon Elkins, Professor Chris Schofield, and the scientists at the ESRF in Grenoble for help and discussions. Financial support was provided by the MRC, BBSRC and EPSRC. References [1] J.E. Baldwin, C.J. Schofield, in: M.I. Page (Ed.), The Chemistry of b-Lactams, Blackie, Glasgow, 1992, pp. 1–78. [2] V.J. Chen, A.M. Orville, M.R. Harpel, C.A. Frolik, K.K. Surerus, E. Munck, J.D. Lipscomb, J. Biol. Chem. 264 (1989) 21677–21681. [3] R.A. Scott, S. Wang, M.K. Eidsness, A. Kriauciunas, C.A. Frolik, V.J. Chen, Biochemistry 31 (1992) 4596–4601. [4] C.R. Randall, L. Shu, Y.-M. Chiou, K.S. Hagen, M. Ito, N. Kitajima, R.J. Lachicotte, Y. Zang, L. Que, Inorg. Chem. 34 (1995) 1036–1039. [5] J.E. Baldwin, M. Bradley, Chem. Rev. 90 (1990) 1079–1088. [6] M. Wirstam, P.E.M. Siegbahn, J. Am. Chem. Soc. 122 (2000) 8539–8547. [7] M. Lundberg, P.E.M. Siegbahn, K. Morokuma, Biochemistry 47 (2007) 1031– 1042. [8] P.L. Roach, I.J. Clifton, V. Fulop, K. Harlos, G.J. Barton, J. Hajdu, I. Andersson, C.J. Schofield, J.E. Baldwin, Nature 375 (1995) 700–704. [9] P.L. Roach, I.J. Clifton, C.M.H. Hensgens, N. Shibata, C.J. Schofield, J. Hadju, J.E. Baldwin, Nature 387 (1997) 827–830. [10] N.I. Burzlaff, P.J. Rutledge, I.J. Clifton, C.M.H. Hensgens, M. Pickford, R.M. Adlington, P.L. Roach, J.E. Baldwin, Nature 401 (1999) 721–724. [11] J.E. Baldwin, in: A.G. Brown, S.M. Roberts (Eds.), Special Publication No. 52, The Royal Society of Chemistry, London, 1985, pp. 62–85. [12] G.A. Bahadur, J.E. Baldwin, J.J. Usher, E.P. Abraham, G.S. Jayatilake, R.L. White, J. Am. Chem. Soc. 103 (1981) 7650–7651. [13] A.J. Long, I.J. Clifton, P.L. Roach, J.E. Baldwin, P.J. Rutledge, C.J. Schofield, Biochemistry 44 (2005) 6619–6628. [14] J.E. Baldwin, M. Bradley, R.M. Adlington, W.J. Norris, N.J. Turner, Tetrahedron 47 (1991) 457–480. [15] J.E. Baldwin, W.J. Norris, R.T. Freeman, M. Bradley, R.M. Adlington, S. Long-Fox, C.J. Schofield, J. Chem. Soc. (1988) 1128–1130. [16] J.E. Baldwin, J.M. Blackburn, M. Sako, C.J. Schofield, J. Chem. Soc. Chem. Commun. (1989) 970–972. [17] J.E. Baldwin, G.P. Lynch, C.J. Schofield, J. Chem. Soc. Chem. Commun. (1991) 736–738. [18] A.C. Stewart, I.J. Clifton, R.M. Adlington, J.E. Baldwin, P.J. Rutledge, ChemBioChem 8 (2007) 2003–2007. [19] A.J. Long, I.J. Clifton, P.L. Roach, J.E. Baldwin, C.J. Schofield, P.J. Rutledge, Biochem. J. 372 (2003) 687–693. [20] A.R. Grummitt, P.J. Rutledge, I.J. Clifton, J.E. Baldwin, Biochem. J. 382 (2004) 659–666. [21] A.R. Howard-Jones, J.M. Elkins, I.J. Clifton, P.L. Roach, R.M. Adlington, J.E. Baldwin, P.J. Rutledge, Biochemistry 46 (2007) 4755–4762. [22] J.M. Ogle, I.J. Clifton, P.J. Rutledge, J.M. Elkins, N.I. Burzlaff, R.M. Adlington, P.L. Roach, J.E. Baldwin, Chem. Biol. 8 (2001) 1231–1237. [23] J.M. Elkins, P.J. Rutledge, N.I. Burzlaff, I.J. Clifton, R.M. Adlington, P.L. Roach, J.E. Baldwin, Org. Biomol. Chem. 1 (2003) 1455–1460. [24] A. Daruzzaman, I.J. Clifton, R.M. Adlington, J.E. Baldwin, P.J. Rutledge, ChemBioChem 7 (2006) 351–358. [25] W. Ge, I.J. Clifton, J.E. Stok, R.M. Adlington, J.E. Baldwin, P.J. Rutledge, J. Am. Chem. Soc. 130 (2008) 10096–10102. [26] W. Ge, I.J. Clifton, A.R. Howard-Jones, J.E. Stok, R.M. Adlington, J.E. Baldwin, P.J. Rutledge, ChemBioChem 10 (2009) 2025–2031. [27] A. Daruzzaman, I.J. Clifton, R.M. Adlington, J.E. Baldwin, P.J. Rutledge, ChemBioChem 14 (2013) in press, http://dx.doi.org/10.1002/cbic.201200728. [28] P.L. Roach, I.J. Clifton, C.M.H. Hensgens, N. Shibata, A.J. Long, R.W. Strange, S.S. Hasnain, C.J. Schofield, J.E. Baldwin, J. Hajdu, Eur. J. Biochem. 242 (1996) 736– 740. [29] P.J. Rutledge, N.I. Burzlaff, J.M. Elkins, M. Pickford, J.E. Baldwin, P.L. Roach, Analyt. Biochem. 308 (2002) 265–268. [30] A.G.W. Leslie, Acta Crystallogr. D 55 (1999) 1696–1702. [31] Collaborative Crystallography Project Number 4, Acta Crystallogr. D 50 (1994) 760–763.
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Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
The crystal structure of isopenicillin N synthase with a dipeptide substrate analogue Adam Daruzzaman a, Ian J. Clifton a, Robert M. Adlington a, Jack E. Baldwin a,⇑, Peter J. Rutledge b,⇑ a b
Chemistry Research Laboratory, University of Oxford, Mansfield Rd, Oxford OX1 3TA, UK School of Chemistry F11, The University of Sydney, NSW 2006, Australia
a r t i c l e
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Article history: Received 19 September 2012 and in revised form 7 December 2012 Available online 19 December 2012 Keywords: Antibiotics Biosynthesis Enzyme mechanism Metalloenzymes Non-heme iron oxidase Penicillin
a b s t r a c t Isopenicillin N synthase (IPNS) converts its linear tripeptide substrate d-L-a-aminoadipoyl-L-cysteinyl-Dvaline (ACV) to bicyclic isopenicillin N (IPN), the key step in penicillin biosynthesis. Solution-phase incubation experiments have shown that IPNS will accept and oxidise a diverse array of substrate analogues, including tripeptides that incorporate L-homocysteine as their second residue, and tripeptides with truncated side-chains at the third amino acid such as d-L-a-aminoadipoyl-L-cysteinyl-D-a-aminobutyrate (ACAb), d-L-a-aminoadipoyl-L-cysteinyl-D-alanine (ACA) and d-L-a-aminoadipoyl-L-cysteinyl-glycine (ACG). However IPNS does not react with dipeptide substrates. To probe this selectivity we have crystallised the enzyme with the dipeptide d-L-a-aminoadipoyl-L-homocysteine (AhC) and solved a crystal structure for the IPNS:Fe(II):AhC complex to 1.40 Å resolution. This structure reveals an unexpected mode of peptide binding at the IPNS active site, in which the homocysteinyl thiolate does not bind to iron. Instead the primary mode of binding sees the homocysteinyl carboxylate coordinated to the metal, while its side-chain is oriented into the region of the active site normally occupied by the benzyl group of protein residue Phe211. Ó 2012 Elsevier Inc. All rights reserved.
Introduction Isopenicillin N synthase (IPNS) is a member of the non-heme iron enzyme family and catalyses the oxidative cyclisation of d-La-aminoadipoyl-L-cysteinyl-D-valine (ACV, 1) to isopenicillin N (IPN, 2) (Scheme 1) [1]. IPNS has been studied extensively using spectroscopic [2–4], mechanistic [1,5], theoretical [6,7] and crystallographic experiments [8–10]. It is generally agreed that the IPNS-catalysed conversion of ACV 1 to IPN 2 involves the high-valent iron-oxo intermediate 3 [10,11]. Solution phase experiments have shown that IPNS will accept a wide range of tripeptide substrate analogues and catalyse a diverse array of oxidative chemistry [5]. Truncated tripeptides such as d-La-aminoadipoyl-L-cysteinyl-D-a-aminobutyrate (ACAb, 4), d-L-aaminoadipoyl-L-cysteinyl-D-alanine (ACA, 5) and d-L-a-aminoadipoyl-L-cysteinyl-glycine (ACG, 6) are oxidised by IPNS
Abbreviations used: AC, d-L-a-aminoadipoyl-L-cysteinyl-; ACA, d-L-a-aminoadipoyl-L-cysteinyl-D-alanine; ACAb, d-L-a-aminoadipoyl-L-cysteinyl-D-a-aminobutyrate; ACG, d-L-a-aminoadipoyl-L-cysteinyl-glycine; ACV, d-L-a-aminoadipoyl-Lcysteinyl-D-valine; AhC, d-L-a-aminoadipoyl-L-homocysteine; AhC-, d-L-a-aminoadipoyl-L-homocysteinyl; AhCmC, d-L-a-aminoadipoyl-L-homocysteinyl-D-S-methylcysteine; AhCV, d-L-a-aminoadipoyl-L-homocysteinyl-D-valine; IPN, isopenicillin N; IPNS, isopenicillin N synthase. ⇑ Corresponding authors. Fax: +61 2 9351 3329 (P.J. Rutledge), fax: +44 1865 285002 (J.E. Baldwin). E-mail addresses: [email protected] (J.E. Baldwin), peter.rutledge@ sydney.edu.au (P.J. Rutledge). 0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2012.12.012
(Fig. 1). ACAb 4 is converted to three bicyclic products (two epimeric C2-methyl penams and a cepham) [12]; ACA 5 is turned over but gives undetermined product(s) that show no antibiotic activity nor any evidence of b-lactam character [13]; and ACG 6 gives rise to a hydrated aldehyde product via two-electron oxidation of the tripeptide (in contrast to the four-electron oxidation required for IPN formation from ACV 1) [14]. However IPNS does not turn over more severely truncated analogues that are missing the third amino acid altogether – i.e. dipeptides [5]. ACV analogues in which the second residue (L-cysteine) is replaced by L-homocysteine are oxidised by IPNS [15–17]. d-L-aAminoadipoyl-L-homocysteinyl-D-valine (AhCV, 7) yields monocyclic c-lactam products 8 upon reaction with IPNS (Scheme 2), thought to proceed via initial formation of the monocyclic c-lactam 9 (homologue of the ACV-derived b-lactam 3) [15]. However the iron(IV)-oxo moiety in 9 does not extract the b-hydrogen from the AhCV valine – for steric and/or stereoelectronic reasons – and is instead quenched by other means. This leaves the enzyme-bound c-lactam to collapse to an acyl iminium ion, subsequently hydrolysed to the c-lactam alcohol products 8. AhC-D-allylglycine [15], AhC-D-cysteine [16] and selectively deuterated [15] and fluorinated [17] AhCV analogues have all been studied as mechanistic probes, and all undergo oxidation at the hCys residue when incubated with IPNS. Protein crystallography has greatly enhanced our understanding of IPNS catalysis [8–10]. Crystal structures of IPNS with its natural substrate ACV [9], its product IPN [10,18], and various
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Scheme 1. IPNS converts the linear tripeptide ACV 1 to bicyclic IPN 2, a reaction that is generally agreed to proceed via the monocyclic intermediate 3 [1,10,11].
Scheme 2. Reaction of AhCV 7 with IPNS generates the hydroxy c-lactams 8, a reaction thought to proceed via the monocyclic intermediate 9 [15].
substrate analogues [13,19–21] have revealed different modes of substrate binding, and elucidated the interplay between active site geometry and reactivity. Pseudo-time-resolved studies using highpressure oxygenation to trigger reaction inside IPNS crystals have brought further insight, allowing the enzyme mechanism to be studied within the crystalline protein [10,22–26]. Crystal structures have previously been reported for complexes of IPNS with the truncated tripeptide analogues ACAb 4, ACA 5 and ACG 6, with and without the dioxygen analogue nitric oxide (NO) also bound [13,19]. These structures revealed new binding modes and metal-centred rearrangements at the IPNS active site, and showed that reducing the size and hydrophobicity of the valinyl side-chain of ACV 1 allows additional water molecules into the active site, even with NO also bound [13,19]. There is thus a very delicate interplay between steric interactions and hydrophilic/ hydrophobic effects around the catalytic metal centre, and IPNS catalysis is strongly influenced by differences both in hydrophobicity and steric bulk. To probe this interplay of sterics and hydrophobicity further, we report herein the crystal structure of IPNS complexed to a dipeptide substrate analogue. The question as to why IPNS does not execute a half reaction when presented with an appropriate dipeptide substrate analogue has long remained unresolved. It seems plausible that binding of the thiolate of a dipeptide analogue to the active site iron would facilitate oxygen binding and trigger partial turnover, but this has not been observed. During recent work to synthesise new AhCV analogues [27], the dipeptide d-L-a-aminoadipoyl-L-homocysteine (AhC, 10) was prepared. We have crystallised this analogue with IPNS to investigate what happens to substrate binding when the tripeptide is truncated to the extent that the third residue is missing altogether.
for 30 min, cooled to room temperature and concentrated in vacuo. The crude reside was azeotroped with toluene (2 10 mL) to remove residual TFA and dissolved in water (20 mL). The aqueous solution was washed with ethyl acetate (2 10 mL). The combined organic phases were extracted with water (2 10 mL) and the pooled aqueous phases back-extracted with ethyl acetate (10 mL). The combined aqueous extracts were lyophilised to give a crusty yellow solid, which was purified by reversed phase HPLC to give 11 as a fluffy white solid (17 mg, 65%); Rt 7 min 30 s; 1H (500 MHz, CDCl3): 1.54–1.73 (2H, m, NHCHCH2CH2CH2), 1.74–1.90 (2H, m, NHCHCH2CH2CH2), 2.09–2.11 (1H, m, 1 of CHCH2CH2S), 2.30 (2H, t, J 7.0, NHCHCH2CH2CH2), 2.52–2.60 (1H, m, 1 of CHCH2CH2S), 3.25–3.33 (1H, m, 1 of CHCH2CH2S), 3.35–3.32 (1H, m, 1 of CHCH2CH2S), 3.67 (1H, t, J 6.0, NHCHCH2CH2CH2), 4.62 (1H, X of ABX, JXA 13.0, JXB 7.0, CHCH2CH2S); 13C (500 MHz, CDCl3): 19.8 (NHCHCH2CH2CH2), 26.5 (CHCH2CH2S), 28.7 (NHCHCH2CH2CH2), 28.9 (CHCH2CH2S), 33.9 (NHCHCH2CH2CH2), 58.3 (CHCH2CH2S), 173.4 (CO2H), 175.1 (amide NHC=O) 209.6 (thioester SC = O); HRMS Calculated for C10H17N2O4S [MH]+: 261.0909; found 261.0909. NMR assignments are based on COSY, HSQC and HMBC. HMBC indicates coupling between C1 (carbonyl carbon) and the protons of C4 of the thiolactone moiety. Thus the cyclic structure 11 is assigned rather than the ring opened form 10 (Scheme 3).
Crystallography and structure determination Crystals of the IPNS:Fe(II):AhC complex were grown under anaerobic conditions by combining IPNS, iron(II) sulfate and thiolactone 11 as previously reported for tripeptide substrates [28,29]. Crystals for X-ray diffraction were selected using a light microscope, removed from the anaerobic environment and exchanged into a cryoprotectant buffer (1:1 mixture of well buffer
Materials and Methods Synthesis of (S)-2-amino-6-oxo-6-(((S)-2-oxotetrahydrothiophen-3yl)amino)hexanoic acid 11 N-tert-Butyloxycarbonyl-a-para-methoxybenzyl-d-(L-a-aminoadipoyl)-S-para-methoxybenzyl-L-homocysteinyl-S-methyl-D-cysteine benzhydryl ester 12 (90 mg, 0.10 mmol) was dissolved in trifluoroacetic acid (TFA, 4 mL) and anisole (0.50 mL, 4.6 mmol). The reaction mixture was heated at 80–90 °C (reflux) under argon
Scheme 3. Structure of the dipeptide d-L-a-aminoadipoyl-L-homocysteine (AhC, 10) in equilibrium with the cyclic thiolactone form 11.
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Scheme 4. Thiolactone 11 is isolated as the major product from the acid-mediated deprotection of tripeptide derivative 12, along with the deprotected tripeptide 13; conditions: TFA, anisole, reflux, RPHPLC, giving 11 (65%) and 13 (12%).
Fig. 1. Truncated tripeptides that have previously been crystallised with IPNS: d-L-a-aminoadipoyl-L-cysteinyl-D-a-aminobutyrate (ACAb, 4), d-L-a-aminoadipoyl-L-cysteinylD-alanine (ACA, 5) and d-L-a-aminoadipoyl-L-cysteinyl-glycine (ACG, 6) [13,19].
and saturated lithium sulfate in 40% v/v glycerol), then cryo-cooled in liquid nitrogen. Data were collected at the European Synchrotron Radiation Source (ESRF), Grenoble, France; the temperature was maintained at 100 K throughout data collection using an Oxford Cryosystems Cryostream. Data were processed using MOSFLM [30] and programs from the CCP4 suite [31], then refined using REFMAC5 [32] and Coot for model building [33]. Initial phases were generated using co-ordinates for the protein from the previously published IPNS:Fe(II):ACV structure [9], and manual rebuilding of protein side-chains was performed as necessary. Crystallographic coordinates and structure factors have been deposited in the Worldwide Protein Data Bank, under accession number 4bb3.
Results & discussion Synthesis of d-L-a-aminoadipoyl-L-homocysteine AhC, 10 The dipeptide AhC 10 exists in equilibrium with the cyclic form, thiolactone 11 (Scheme 3). The cyclic thioester 11 was isolated from the acid-mediated deprotection of the protected tripeptide derivative N-tert-butyloxycarbonyl-a-p-methoxybenzyl-d-(L-aaminoadipoyl)-S-p-methoxybenzyl-L-homocysteinyl-S-methyl-Dcysteine benzhydryl ester 12 (Scheme 4), alongside the tripeptide d-L-a-aminoadipoyl-L-homocysteinyl-D-S-methylcysteine (AhCmC, 13). Thiolactone 11 presumably arises from intramolecular cleavage of the hCys–mCys amide bond by the side-chain thiol under the acidic conditions of the deprotection reaction.
Fig. 2. Stereo view of the active site of the IPNS:Fe(II):AhC complex, showing electron density as: 2mFoDfc density [42] at 1.5r (green) and mFoDfc density at ±3r (cyan (+3r) and magenta (3r)) where r is the RMS value of each map over an asymmetric unit. Figure generated using ccp4 mg [43].
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The crystal structure of the IPNS:Fe(II):AhC complex
Table 1 X-ray data collection and crystallographic statistics. IPNS:Fe(II):AhC
a
X-ray source Wavelength (Å) PDB acquisition code Resolution (Å) Space group Unit cell dimensions (a Å, b Å, c Å)
ESRF beamline ID14 EH2 0.93 4bb3 1.40 P212121 46.51, 71.20, 100.88
Resolution shell (Å) Total number of reflections Number of unique reflections Completeness (%) Average I/r (I) Rmerge (%)a Rmeas (%)b
50.44–1.4 293474 66412 99.2 14.7 6.5 7.2
Rcryst (%)c Rfree (%)d RMS deviatione Average B factors (Å2)f Number of water molecules
17.27 18.83 0.022 (2.1) 14.0, 16.6, 19.9, 26.6 299
1.47–1.4 32787 9119 95.1 3.3 41.3 48.4
Rmerge = RjRp h|Iffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi h,j – hIhi|/RjRhhIhi 100. Rmeas = Rhkl N=ðN 1Þi |IðhklÞ Ix ðhklÞ|/RhklRI Ii (hkl) 100 [40,41]. P P c Rcryst = ||Fobs|- |Fcalc|| |Fobs| 100. d Rfree = based on 5% of the total reflections. e RMS deviation from ideality for bonds (followed by the value for angles). f Average B factors in order: main chain; side-chain; substrate and iron; solvent (water and 1 sulfate). b
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Anticipating that the thiolactone 11 would give rise to the linear dipeptide AhC 10 under the conditions used for crystallisation with IPNS (1.8 M lithium sulfate/ 100 mM TrisHCl buffer, pH 8.5), the cyclic compound 11 was subjected to these conditions following the previously reported procedure [29]. Diffraction quality crystals grew as with other substrate analogues, and the X-ray crystal structure reveals the IPNS:Fe(II):AhC complex at 1.40 Å resolution (Fig. 2, Table 1). The overall structure of the protein mirrors very closely complexes in which IPNS is coordinated to tripeptide substrates [9,10,21,34]. The active site metal ion is held by the characteristic triad of amino acid side-chains from the protein: His214, Asp216 and His270, the ‘2-His-1-carboxylate’ facial triad diagnostic of this enzyme family [35]. The dipeptide substrate analogue is bound at the active site, but not as anticipated. Its aminoadipoyl side-chain is oriented as seen in the IPNS:Fe(II):ACV complex, adopting a similar staggered conformation and tethered by the familiar salt bridge to Arg87. However the side-chain thiolate of the homocysteinyl residue does not ligate to the metal, but instead extends away from the iron centre into the region of space normally occupied by the side-chain of Phe211. The carboxylate of the homocysteinyl residue ligates to iron, which has octahedral coordination geometry. The remaining two binding sites at the metal are occu-
Fig. 3. Comparison of substrate binding to iron in the complexes of IPNS with a. ACV 1 (PDB id 1bk0); b. ACA 5 (1wo5); c. ACG 6 (1wo3); and d. AhC 10 (4bb3). Note the very different position of the (homo)cysteinyl sulfur atom (yellow) in d. Figure generated using ccp4 mg [43]. (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this paper.)
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pied by water molecules, opposite His214 (as seen in the IPNS:Fe(II):ACV structure), and trans to Asp216 (the site at which the co-substrate dioxygen would normally bind). Additionally, another well-defined water molecule occupies the region normally filled by the third-residue (‘valinyl’) carboxylate. No such ligand is present in this area of the apo-IPNS:Mn(II) complex, which is instead occupied by the C-terminal residue of the protein (Gln330) [8]. The side-chain of Phe211 is displaced by the homocysteine side-chain and takes up the orientation that is observed in the substrate-free apo-enzyme complex [8] and the complex of IPNS with the penam product 2-a-hydroxymethyl isopenicillin N [23]. The side-chain of Leu324 is disordered beyond Cd; it seems likely that this disorder arises from an interaction with the substrate, and the homocysteinyl thiolate sits in the right region of the active site to disturb Leu324. Other residues of the C-terminal tail (Leu324– Thr331) are not disturbed: this section of the protein is clearly defined and folded over the active site pocket in the ‘substratebound’ conformation. Mobility in the C-terminus has been linked to product departure from the IPNS active site [23], for which there is no evidence in this complex. It is apparent from the maps shown in Fig. 2 that there is some disorder in the electron density arising from AhC 10. The primary binding mode described above is present in 80% occupancy. Attempts to include partial occupancy by a second substrate orientation in the refinement model gave data consistent with low occupancy of second dipeptide binding mode (data not shown), however this could not be modelled more precisely. Conclusions Previous crystal structures with the truncated substrate analogues ACAb 4 [19], ACA 5 and ACG 6 [13] and d-(L-a-aminoadipoyl)-L-cysteinyl-D-vinylglycine (ACvG 14) [23] have shown that reducing the steric bulk of the third amino acid alters the coordination geometry at iron in the IPNS active site (Fig. 3). Similarly, substrate analogues that are L-configured at their third residue (where the native substrate ACV is D-configured) allow additional water molecules into this region of the IPNS active site, hydrogen bonded to the carboxylate of the L-configured third residue [34,36,37]. However it has never been previously observed that a substrate incorporating a free thiol does not coordinate to iron through this sulfur atom, as seen here with AhC 10 (Fig. 2, 3d). While dipeptide substrate analogues are not turned over to oxidised products, this study demonstrates that they can in fact bind to IPNS. Anchoring of the a-aminoadipoyl tail to Arg87 is key to this interaction, but thiolate ligation to iron is not required. Previous studies have demonstrated a strong affinity between thiolate or sulfide ligands and the metal centre at the IPNS active site [10,25,38,39]. On this basis, it was anticipated that if dipeptides such as AhC 10 or d-(L-a-aminoadipoyl)-L-cysteine were to bind in the enzyme active site, they would coordinate to iron via the (homo)cysteinyl thiolate. However the structure of the IPNS:Fe(II):AhC complex reveals that iron–sulfur ligation is not required, and this truncated substrate binds to iron through the carboxylate of its homocysteine residue. As a result, the a- and b-carbon atoms and thiolate of the homocysteine residue extend ‘behind’ the ligated substrate and rest in the cleft normally occupied by Phe211. The side-chain of that residue is displaced, and the sidechain of nearby Leu324 disturbed. The additional water ligands occupying the otherwise vacant space close to iron would increase the hydrophilicity of the active site region and impede access to the oxygen binding site. Moreover, it has previously been proposed that ligation of the ACV thiolate to iron lowers the Fe(II)/Fe(III) redox potential,
allowing dioxygen to bind and initiating the reaction cycle [9]. It follows that in the absence of thiolate ligation (as in the IPNS:Fe(II):AhC complex) the Fe(II)/Fe(III) redox potential is not lowered, dioxygen binding is not facilitated, and reaction is not triggered. We propose that this combination of increased hydrophilicity and altered redox potential at iron prevents IPNS from promoting any partial turnover or other oxidative reaction with the dipeptide 10, and by analogy the cysteine-containing homologue d-(L-a-aminoadipoyl)-L-cysteine and other related dipeptides. Acknowledgments We thank Professor Dr Nicolai Burzlaff, Dr Jon Elkins, Professor Chris Schofield, and the scientists at the ESRF in Grenoble for help and discussions. Financial support was provided by the MRC, BBSRC and EPSRC. References [1] J.E. Baldwin, C.J. Schofield, in: M.I. Page (Ed.), The Chemistry of b-Lactams, Blackie, Glasgow, 1992, pp. 1–78. [2] V.J. Chen, A.M. Orville, M.R. Harpel, C.A. Frolik, K.K. Surerus, E. Munck, J.D. Lipscomb, J. Biol. Chem. 264 (1989) 21677–21681. [3] R.A. Scott, S. Wang, M.K. Eidsness, A. Kriauciunas, C.A. Frolik, V.J. Chen, Biochemistry 31 (1992) 4596–4601. [4] C.R. Randall, L. Shu, Y.-M. Chiou, K.S. Hagen, M. Ito, N. Kitajima, R.J. Lachicotte, Y. Zang, L. Que, Inorg. Chem. 34 (1995) 1036–1039. [5] J.E. Baldwin, M. Bradley, Chem. Rev. 90 (1990) 1079–1088. [6] M. Wirstam, P.E.M. Siegbahn, J. Am. Chem. Soc. 122 (2000) 8539–8547. [7] M. Lundberg, P.E.M. Siegbahn, K. Morokuma, Biochemistry 47 (2007) 1031– 1042. [8] P.L. Roach, I.J. Clifton, V. Fulop, K. Harlos, G.J. Barton, J. Hajdu, I. Andersson, C.J. Schofield, J.E. Baldwin, Nature 375 (1995) 700–704. [9] P.L. Roach, I.J. Clifton, C.M.H. Hensgens, N. Shibata, C.J. Schofield, J. Hadju, J.E. Baldwin, Nature 387 (1997) 827–830. [10] N.I. Burzlaff, P.J. Rutledge, I.J. Clifton, C.M.H. Hensgens, M. Pickford, R.M. Adlington, P.L. Roach, J.E. Baldwin, Nature 401 (1999) 721–724. [11] J.E. Baldwin, in: A.G. Brown, S.M. Roberts (Eds.), Special Publication No. 52, The Royal Society of Chemistry, London, 1985, pp. 62–85. [12] G.A. Bahadur, J.E. Baldwin, J.J. Usher, E.P. Abraham, G.S. Jayatilake, R.L. White, J. Am. Chem. Soc. 103 (1981) 7650–7651. [13] A.J. Long, I.J. Clifton, P.L. Roach, J.E. Baldwin, P.J. Rutledge, C.J. Schofield, Biochemistry 44 (2005) 6619–6628. [14] J.E. Baldwin, M. Bradley, R.M. Adlington, W.J. Norris, N.J. Turner, Tetrahedron 47 (1991) 457–480. [15] J.E. Baldwin, W.J. Norris, R.T. Freeman, M. Bradley, R.M. Adlington, S. Long-Fox, C.J. Schofield, J. Chem. Soc. (1988) 1128–1130. [16] J.E. Baldwin, J.M. Blackburn, M. Sako, C.J. Schofield, J. Chem. Soc. Chem. Commun. (1989) 970–972. [17] J.E. Baldwin, G.P. Lynch, C.J. Schofield, J. Chem. Soc. Chem. Commun. (1991) 736–738. [18] A.C. Stewart, I.J. Clifton, R.M. Adlington, J.E. Baldwin, P.J. Rutledge, ChemBioChem 8 (2007) 2003–2007. [19] A.J. Long, I.J. Clifton, P.L. Roach, J.E. Baldwin, C.J. Schofield, P.J. Rutledge, Biochem. J. 372 (2003) 687–693. [20] A.R. Grummitt, P.J. Rutledge, I.J. Clifton, J.E. Baldwin, Biochem. J. 382 (2004) 659–666. [21] A.R. Howard-Jones, J.M. Elkins, I.J. Clifton, P.L. Roach, R.M. Adlington, J.E. Baldwin, P.J. Rutledge, Biochemistry 46 (2007) 4755–4762. [22] J.M. Ogle, I.J. Clifton, P.J. Rutledge, J.M. Elkins, N.I. Burzlaff, R.M. Adlington, P.L. Roach, J.E. Baldwin, Chem. Biol. 8 (2001) 1231–1237. [23] J.M. Elkins, P.J. Rutledge, N.I. Burzlaff, I.J. Clifton, R.M. Adlington, P.L. Roach, J.E. Baldwin, Org. Biomol. Chem. 1 (2003) 1455–1460. [24] A. Daruzzaman, I.J. Clifton, R.M. Adlington, J.E. Baldwin, P.J. Rutledge, ChemBioChem 7 (2006) 351–358. [25] W. Ge, I.J. Clifton, J.E. Stok, R.M. Adlington, J.E. Baldwin, P.J. Rutledge, J. Am. Chem. Soc. 130 (2008) 10096–10102. [26] W. Ge, I.J. Clifton, A.R. Howard-Jones, J.E. Stok, R.M. Adlington, J.E. Baldwin, P.J. Rutledge, ChemBioChem 10 (2009) 2025–2031. [27] A. Daruzzaman, I.J. Clifton, R.M. Adlington, J.E. Baldwin, P.J. Rutledge, ChemBioChem 14 (2013) in press, http://dx.doi.org/10.1002/cbic.201200728. [28] P.L. Roach, I.J. Clifton, C.M.H. Hensgens, N. Shibata, A.J. Long, R.W. Strange, S.S. Hasnain, C.J. Schofield, J.E. Baldwin, J. Hajdu, Eur. J. Biochem. 242 (1996) 736– 740. [29] P.J. Rutledge, N.I. Burzlaff, J.M. Elkins, M. Pickford, J.E. Baldwin, P.L. Roach, Analyt. Biochem. 308 (2002) 265–268. [30] A.G.W. Leslie, Acta Crystallogr. D 55 (1999) 1696–1702. [31] Collaborative Crystallography Project Number 4, Acta Crystallogr. D 50 (1994) 760–763.
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