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FEBS Letters 587 (2013) 2705–2709

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The crystal structure of an isopenicillin N synthase complex with an ethereal analogue reveals in the oxygen ⇑ ⇑ Ian J. Clifton a, Wei Ge a, Robert M. Adlington a, Jack E. Baldwin a, , Peter J. Rutledge b,

a Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK b School of Chemistry, The University of Sydney, NSW 2006, Australia

article info abstract

Article history: Isopenicillin N synthase (IPNS) is a non-heme oxidase central to the of b-lactam Received 10 May 2013 . IPNS converts the tripeptide d-(L-a-aminoadipoyl)-L-cysteinyl-D- (ACV) to isopeni- Revised 4 July 2013 cillin N while reducing molecular oxygen to water. The substrate analogue d-(L-a-aminoadipoyl)-L- Accepted 4 July 2013 cysteinyl-O-methyl-D-threonine (ACmT) is not turned over by IPNS. Epimeric d-(L-a-aminoadipoyl)- Available online 13 July 2013 L-cysteinyl-O-methyl-D-allo-threonine (ACmaT) is converted to a bioactive penam product. ACmT and ACmaT differ from each other only in the stereochemistry at the b-carbon atom of their third Edited by Peter Brzezinski residue. These substrates both contain a methyl ether in place of the isopropyl group of ACV. We report an X-ray crystal structure for the anaerobic IPNS:Fe(II):ACmT complex. This structure reveals Keywords: an additional water bound to the , held by -bonding to the ether Non-heme iron oxidase oxygen atom of the substrate analogue. Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Biosynthesis Protein crystallography Metalloenzyme

1. Introduction d-(L-a-aminoadipoyl)-L-cysteinyl-O-methyl-D-threonine (ACmT, 3) contains a methyl ether in place of the valinyl isopropyl group of Isopenicillin N synthase (IPNS) is a member of the non-heme ACV. ACmT 3 is not turned over by IPNS but the epimeric tripeptide iron oxidase family of [1–3]. IPNS uses an iron(II) d-(L-a-aminoadipoyl)-L-cysteinyl-O-methyl-D-allo-threonine (AC- and molecular oxygen to catalyse cyclisation of its tripeptide sub- maT 4) does react, and is converted by IPNS to 2-a-methoxy-2-b- strate d-(L-a-aminoadipoyl)-L-cysteinyl-D-valine (ACV, 1) to bicy- methylpenam 5 (Scheme 2a) [16,17]. Penam 5 has antibiotic activ- clic isopenicillin N (IPN, 2), the central step in penicillin ity comparable to isopenicillin N 2 [16]. biosynthesis (Scheme 1) [4]. IPNS catalysis has been extensively This result stands in contrast to incubation experiments with studied using spectroscopy [5–8], crystallography [9–11], compu- the isosteric tripeptides that contain D-isoleucine (ACI, 6) and D- tational modeling [12,13], and turnover experiments with sub- allo-isoleucine (ACaI, 7)(Scheme 2b). ACI 6 and ACaI 7 are both strate analogues [4,14]. It is generally agreed that the IPNS cyclized to penicillin products by IPNS, with retention of configu- reaction cycle proceeds via initial b-lactam formation and a high- ration in the C–S bond-forming step [18,19]. However neither of valent iron-oxo intermediate [11,15]. the corresponding -containing compounds AC-D-thioisoleu- A diverse array of tripeptide analogues have been incubated cine (ACtI, 8) and AC-D-thio-allo-isoleucine (ACtaI, 9) is turned with IPNS in the parallel effort to access new antibiotic structures over by the [14,20]. It was concluded in 1989 that the and characterize the enzyme mechanism [4,14]. The ACV analogue difference between the two series (3 and 4 on the one hand, 6 and 7 on the other) ‘‘must derive from some property of the methoxy function,’’ and proposed that ‘‘a hydrogen bond between Abbreviations: AC-, d-L-a-aminoadipoyl-L-cysteinyl-; ACaI, AC-D-allo-isoleucine; the methoxy group and an active site group restricts rotation ACI, AC-D-isoleucine; ACmaT, AC-O-methyl-D-allo-threonine; ACmT, AC-O-methyl- D-threonine; ACtaI, AC-D-thio-allo-isoleucine; ACtI, AC-D-thioisoleucine; ACV, AC-D- around C(2)–C(3) in the intermediate ... so the [intermediate] de- valine; IPN, isopenicillin N; IPNS, isopenicillin N synthase rived from the allo-threonine peptide can be cyclised, but that de- ⇑ Corresponding authors. Fax: +44 1865 285002 (J.E. Baldwin), +61 2 9351 3329 rived from the threonine [peptide] is restrained in a conformation (P.J. Rutledge). in which the b-hydrogen atom cannot be attacked by the active E-mail addresses: [email protected] (J.E. Baldwin), peter.rutledge@- sydney.edu.au (P.J. Rutledge). species’’ [21].

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.07.016 2706 I.J. Clifton et al. / FEBS Letters 587 (2013) 2705–2709

Scheme 1. The IPNS-catalysed reaction: conversion of the linear tripeptide ACV 1 to bicyclic IPN 2, biosynthetic progenitor of all penicillin and antibiotics (L- AA = d-(L-a-aminoadipoyl)).

Scheme 2. (a) The reaction of IPNS with the epimeric substrate analogues ACmT 3 and ACmaT 4: ACmT 3 is not turned over by the enzyme, while ACmaT 4 gives a bioactive penam 5. (b) Structures of the isosteric tripeptides ACI 6,ACaI 7, ACtI, 8 and ACtaI, 9 (L-AA = d-(L-a-aminoadipoyl)).

X-ray crystal structures of apo-IPNS [9] and the enzyme:sub- Benoiton (Scheme 3) [34]. Although the percentage conversion in strate complex [10] paint a detailed structural picture of the en- this etherification reaction is low (24–32% in our hands) unreacted zyme active site. Time-resolved studies using high-pressure starting material (45–49%) is readily recovered and recycled; the oxygenation to initiate reaction within crystalline IPNS have en- product 11 and residual starting material were isolated together abled step-by-step elucidation of the reaction mechanism on a after work-up and could be separated by partitioning the resultant structural level [11,22–25], while structures of the enzyme with oil between small volumes of DCM and water. N-Boc-O-methyl-D- ACV analogues reveal a range of binding modes and the interplay threonine 11 was isolated from the DCM phase and further purified between steric and electronic effects at the IPNS active site [26– by crystallisation from chloroform. 32]. Seeking. a structural explanation for the failure of ACmT 3 to The Boc group was removed using toluenesulfonic acid [35] and react with IPNS, we have crystallised the protein with this sub- the resulting carboxylic acid converted to the benzhydryl ester 12 strate analogue and solved the crystal structure of the IPNS:- using diphenyldiazomethane [36] (Scheme 3). Protected amino Fe(II):ACmT complex. acid 12 was coupled to previously reported dipeptide 13 [23] using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) and 1- 2. Materials and methods hydroxybenzotriazole hydrate (HOBt) [37] under standard condi- tions to give protected tripeptide 14. Global deprotection with tri- 2.1. Synthesis of d-(L-a-aminoadipoyl)-L-cysteinyl-O-methyl-D- fluoroacetic acid [38] gave the tripeptide 3 which was collected as threonine 3 the trifluoroacetate salt by precipitation from ether. Purification by reversed-phase HPLC (octadecylsilane 250 10 mm; k = 254 nm, 5 D-Threonine 10 (Aldrich) was N-Boc-protected [33] then con- AUFS; 4 mL/min, 10 mM NH4HCO3 in water/methanol as eluant: verted to N-Boc-O-methyl-D-threonine 11 using sodium iso-prop- running time 0–5 min, 0% methanol; 5–12 min, gradient of 0– oxide and methyl iodide in THF as described by Chen and 25% methanol; 13–18 min, 2.5% methanol; 18–20 min 0% metha-

t i Scheme 3. Synthesis of target tripeptide 3; (i) NaOH, H2O/ BuOH, (Boc)2O, rt, 23 h, 100%; (ii) NaO Pr, CH3I, THF, 0 °C, 48 h, 32%; (iii) TsOH, toluene, 40 °C; (iv) Ph2CN2,CH3CN/

Et2O, rt, 55% (over 2 steps); (v) EDCI, HOBt, Et3N, DCM, rt, 20 h, 100%; (vi) TFA, anisole, reflux, 30 min, 100%; RP-HPLC. I.J. Clifton et al. / FEBS Letters 587 (2013) 2705–2709 2707

Table 1 Data collection and statistics.

IPNS:Fe(II):ACmT X-ray source Rigaku rotating anode Wavelength (Å) 1.5418 PDB acquisition code 3zoi Resolution (Å) 1.81

Space group P212121 Unit Cell Dimensions (a Å, b Å, c Å) 46.48, 70.79, 100.51 Resolution shell (Å) 21.01–1.81 1.91–1.81 Total number of reflections 92909 10764 Number of unique reflections 27150 3414 Completeness (%) 98.2 92.1 Average I/r(I) 14.3 2.9 a Rmerge (%) 6.4 33.5 b Rmeas (%) 7.6 40.6 c Rcryst (%) 16.2 d Rfree (%) 20.2 RMS deviatione 0.020 (1.8) Average B factors (Å2)f 18.6 21.0 16.3 24.6 Number of water 233 P P P P a Rmerge ¼P j phffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffijIh;j—hIhij=P j hhIhi100. P P b Rmeas ¼ P hkl N=ðN 1Þ PijIiðhklÞIðhklÞj= hkl lIiðhklÞ100 [54,55]. c Rcryst ¼ jjFobsjjFcalcjj= jFobsj100. 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).

Fig. 1. Stereo view of the active site of the anaerobic IPNS:Fe(II):ACmT complex, showing (i) a 2mFoDfc electron density map (cyan) contoured at 1.5r around the substrate

ACmT; and (ii) a mFoDFc omit map (pink) contoured at 4r around key water molecules (calculated from a model in which all were removed, random noise (0.2 Å max) added to remaining coordinates, then re-refined). Figure generated using ccp4 mg [53]. nol v/v; the tripeptide was treated with tris(2-carboxyethyl)phos- (1:1 mixture of well buffer and saturated lithium sulfate in 40% phine hydrochloride for 20 min prior to injection onto the HPLC v/v glycerol), then flash-frozen in liquid nitrogen [40,41]. column to reduce any disulfides [39]) gave tripeptide 3 Data were collected at 100 K using Cu-Ka radiation from a Rig- as a fine white powder; Rt 11 min 40 s; dH (500 MHz, D2O): 1.05 aku rotating anode generator and a MAR Research 300 mm image + (3H, d, J 6.5, CHCH3), 1.52–1.70 (2H, m, H3NCHCH2CH2CH2), plate detector at The Department of Molecular Biophysics, Oxford + + 1.73–1.88 (2H, m, H3NCHCH2CH2CH2), 2.33 (2H, t, J 7.5, H3- University. The temperature was maintained at 100 K using an Ox- NCHCH2CH2CH2), 2.82 (1H, A of ABX, JAB 14.0, JAX 7.5 Hz, 1 of CH2- ford Cryosystems Cryostream. Data were processed using DENZO SH), 2.87 (1H, B of ABX, JBA 14.0, JBX 5.5 Hz, 1 of CH2SH), 3.23 (3H, s, [42], MOSFLM [43] and the CCP4 suite of programs [44], then re- + CH3O), 3.65 (1H, m, H3NCHCH2CH2CH2), 3.86 (1H, qd, J 6.5, 3.5 Hz, fined using REFMAC5 [45] and Coot for model building [46]. Initial CHOCH3), 4.13 (1H, d, J 3.5 Hz, NHCHCHOCH3), 4.52 (1H, X of ABX, phases were generated using co-ordinates for the protein from the JXA 7.5, JXB 5.5 Hz, NHCHCH2SH). previously published IPNS:Fe(II):ACV structure [10], and manual rebuilding of protein side-chains was performed as necessary. 2.2. Crystallography and structure determination The active site iron atom and water molecules were modeled in during an early stage of the refinement process. Electron density Crystals of the IPNS:Fe(II):ACmT complex were grown under for ACmT was clearly visible throughout, and modeled in sequen- anaerobic conditions as previously reported (1.8–2.0 M lithium tially at an advanced stage of refinement. Crystallographic coordi- sulfate and 100 mM Tris/HCI, pH 8.5) [40,41]. Crystals for X-ray dif- nates and structure factors for the IPNS:Fe(II):ACmT complex have fraction were selected using a light microscope, removed from the been deposited in the Protein Data Bank (PDB) under accession anaerobic environment and exchanged into cryoprotectant buffer number 3zoi. 2708 I.J. Clifton et al. / FEBS Letters 587 (2013) 2705–2709

Fig. 2. Close-up of the ligands around iron(II) in the IPNS active site: (a) IPNS:Fe(II):ACV complex; and (b) IPNS:Fe(II):ACmT complex. Key water ligands are shown: #713 opposite His214 in the ACV complex, compared to #2178 (opposite His214) and #2173 (opposite Asp216) in the ACmT structure. Figure generated using ccp4mg[53].

3. Results and discussion ACmT 3 is unusual in that despite its size, this analogue fails to exclude water from the iron binding site opposite Asp216. ACmT 3 3.1. Structure of the IPNS:Fe(II):ACmT complex is isosteric with ACI 6 and thus more sterically demanding than the natural substrate ACV, yet the IPNS:Fe(II):ACmT complex still The X-ray crystal structure of the anaerobic IPNS:Fe(II):ACmT incorporates the extra water ligand at iron. We propose that this complex was solved to 1.81 Å resolution (Table 1, Figs. 1 and 2). arrangement arises due to the greater hydrophilicity of the mThr This structure reveals an interesting binding arrangement around side-chain relative to Val, and the capacity for hydrogen bonding iron. The familiar facial triad of the protein-derived ligands is in between the methoxy group of 3 and water ligand #2173. Further, place: the side-chains of His214, Asp216 and His270 ligate to iron, we postulate that the bonding interactions between this water li- as seen previously with IPNS and other non-heme iron enzymes gand, iron and the mThr methoxy group, and the greater bulk of [1,9,10,47]. ACmT is held in the enzyme active site in a similar the mThr side-chain relative to the native substrate, combine to way to the native substrate [10]: via a salt bridge to the amin- exclude the cosubstrate O2 from the active site of the IPNS:- oadipoyl amino group, hydrogen bonds to the carboxylate of the Fe(II):ACmT complex. mThr residue, and ligation of the cysteinyl thiolate to iron in the site opposite His270. However in contrast to the IPNS:Fe(II):ACV 4. Conclusions complex, there are two water ligands bound to the metal with ACmT, meaning that iron is hexacoordinate. In the complex of IPNS Ligation of water molecule #2173 in the oxygen binding site of with its natural substrate ACV (Fig. 2a) [10] and complexes with the IPNS:Fe(II):ACmT complex provides a possible structural expla- substrate analogues containing third residues of a similar size nation for the failure of IPNS to turnover ACmT 3. Held by hydro- [22,24,48], only one water ligand is present at iron, opposite gen bonds to the substrate methoxy group and ligated to iron His214 (#713 in Fig. 2a). In addition to this usual water ligand at trans to Asp216, this water molecule and the mThr side-chain iron (#2178 in Fig. 2b), the ACmT complex contains a second water may prevent oxygen entry and thus block turnover before reaction molecule bound in the site trans to Asp216 (#2173). This second can even begin. water ligand is 2.36 Å from the iron and 2.41 Å from the methoxy oxygen of the mThr residue, ideally placed to hydrogen bond to it (Fig. 2b). Acknowledgements In the IPNS:Fe(II):ACV structure and related complexes [10,22], the site trans to Asp216 is occupied by the side-chain of the sub- We thank Dr Peter Roach, Dr Nicolai Burzlaff, Dr Charles Hens- strate D-valine residue: iron is pentacoordinate with the valinyl gens, Professor Chris Schofield, Dr Alex Parker, Dr Jon Elkins and Dr isopropyl group sitting within van der Waals contact of the metal. Karl Harlos for helpful discussions. Financial support was provided This interaction effectively reserves the sixth iron binding site for by the MRC, BBSRC and EPSRC. P.J.R. thanks the Rhodes Trust for a the co-substrate molecular oxygen [10]. Hexacoordinate iron has scholarship. previously been observed in IPNS complexes: (i) with analogues that incorporate a methyl sulfide in their third residue (e.g. AC-D- Appendix A. Supplementary data thia-allo-isoleucine (ACtaI, 9) [20] and AC-D-methionine [49]), in which the affinity of sulfur for iron means that the sulfide S coor- Supplementary data associated with this article can be found, dinates to the metal; (ii) with smaller substrate analogues (e.g. in the online version, at http://dx.doi.org/10.1016/j.febslet. AC-Gly and AC-D-Ala,[28] AC-D-a-aminobutyrate [26], AC-D-vinyl- 2013.07.016. glycine [23] and the dipeptide d-L-a-aminoadipoyl-L-homocysteine (AhC) [50]), where the smaller side-chain leaves room for a References second water ligand to bind to iron opposite Asp216; and (iii) with LLL-configured substrates (e.g. AC-L-hexafluorovaline [51] and [1] Costas, M., Mehn, M.P., Jensen, M.P. and Que Jr., L. (2004) Dioxygen activation at mononuclear nonheme iron active sites: enzymes, models, and AC-L-2-amino-3,3-dideuteriobutyrate [52]), where the different intermediates. Chem. Rev. 104, 939–986. substrate stereochemistry permits an additional water ligand at [2] Hausinger, R.P. (2004) Fe(II)/alpha-ketoglutarate-dependent hydroxylases and iron. related enzymes. Crit. Rev. Biochem. Mol. Biol. 39, 21–68. I.J. Clifton et al. / FEBS Letters 587 (2013) 2705–2709 2709

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