The crystal structure of an isopenicillin N synthase complex with an ethereal substrate analogue reveals water in the oxygen binding site

Article abstract of DOI:10.1016/j.febslet.2013.07.016

Isopenicillin N synthase (IPNS) is a non-heme iron oxidase central to the biosynthesis of β-lactam antibiotics. IPNS converts the tripeptide δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to isopenicillin N while reducing molecular oxygen to water. The substrate analogue δ-(l-α-aminoadipoyl)-l-cysteinyl-O-methyl-d-threonine (ACmT) is not turned over by IPNS. Epimeric δ-(l-α-aminoadipoyl)-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 β-carbon atom of their third 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 an additional water molecule bound to the active site metal, held by hydrogen-bonding to the ether oxygen atom of the substrate analogue.

Full text of DOI:10.1016/j.febslet.2013.07.016

FEBS Letters 587 (2013) 2705–2709
journal homepage: www.FEBSLetters.org
The crystal structure of an isopenicillin N synthase complex with an
ethereal substrate analogue reveals water in the oxygen binding site
b,
Ian J. Clifton a, Wei Ge a, Robert M. Adlington a, Jack E. Baldwin a, , Peter J. Rutledge
⇑
⇑
a Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK
b School of Chemistry, The University of Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Isopenicillin N synthase (IPNS) is a non-heme iron oxidase central to the biosynthesis of b-lactam
antibiotics. IPNS converts the tripeptide d-( -aminoadipoyl)- -cysteinyl- -valine (ACV) to isopeni-
cillin N while reducing molecular oxygen to water. The substrate analogue d-( -aminoadipoyl)-
cysteinyl-O-methyl- -threonine (ACmT) is not turned over by IPNS. Epimeric d-( -aminoadipoyl)-
-cysteinyl-O-methyl- -allo-threonine (ACmaT) is converted to a bioactive penam product. ACmT
Received 10 May 2013
Revised 4 July 2013
Accepted 4 July 2013
Available online 13 July 2013
L-
a
L
D
L-
a
L-a
L-
D
L
D
and ACmaT differ from each other only in the stereochemistry at the b-carbon atom of their third
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
an additional water molecule bound to the active site metal, held by hydrogen-bonding to the ether
oxygen atom of the substrate analogue.
Edited by Peter Brzezinski
Keywords:
Non-heme iron oxidase
Penicillin
Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Biosynthesis
Antibiotic
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
iron oxidase family of enzymes [1–3]. IPNS uses an iron(II) cofactor
and molecular oxygen to catalyse cyclisation of its tripeptide sub-
ACV. ACmT 3 is not turned over by IPNS but the epimeric tripeptide
d-(L
-a
-aminoadipoyl)-L-cysteinyl-O-methyl-D-allo-threonine (AC-
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
studied using spectroscopy [5–8], crystallography [9–11], compu-
tational modeling [12,13], and turnover experiments with sub-
strate analogues [4,14]. It is generally agreed that the IPNS
reaction cycle proceeds via initial b-lactam formation and a high-
valent iron-oxo intermediate [11,15].
This result stands in contrast to incubation experiments with
the isosteric tripeptides that contain D-isoleucine (ACI, 6) and D-
allo-isoleucine (ACaI, 7) (Scheme 2b). ACI 6 and ACaI 7 are both
cyclized to penicillin products by IPNS, with retention of configu-
ration in the C–S bond-forming step [18,19]. However neither of
the corresponding sulfur-containing compounds AC-
D-thioisoleu-
A diverse array of tripeptide analogues have been incubated
with IPNS in the parallel effort to access new antibiotic structures
and characterize the enzyme mechanism [4,14]. The ACV analogue
cine (ACtI, 8) and AC- -thio-allo-isoleucine (ACtaI, 9) is turned
D
over by the enzyme [14,20]. It was concluded in 1989 that the
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
the methoxy group and an active site group restricts rotation
around C(2)–C(3) in the intermediate . . . so the [intermediate] de-
rived from the allo-threonine peptide can be cyclised, but that de-
rived from the threonine [peptide] is restrained in a conformation
in which the b-hydrogen atom cannot be attacked by the active
species’’ [21].
Abbreviations: AC-, d-
ACI, AC- -isoleucine; ACmaT, AC-O-methyl-
-threonine; ACtaI, AC- -thio-allo-isoleucine; ACtI, AC-
L
-
a
-aminoadipoyl-
L
-cysteinyl-; ACaI, AC-
-allo-threonine; ACmT, AC-O-methyl-
-thioisoleucine; ACV, AC-
D-allo-isoleucine;
D
D
D
D
D
D-
valine; IPN, isopenicillin N; IPNS, isopenicillin N synthase
⇑
Corresponding authors. Fax: +44 1865 285002 (J.E. Baldwin), +61 2 9351 3329
(P.J. Rutledge).
E-mail addresses: jack.baldwin@chem.ox.ac.uk (J.E. Baldwin), peter.rutledge@-
sydney.edu.au (P.J. Rutledge).
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

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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 cephalosporin antibiotics (
AA = d-( -aminoadipoyl)).
L-
L-a
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-
strate complex [10] paint a detailed structural picture of the en-
zyme active site. Time-resolved studies using high-pressure
oxygenation to initiate reaction within crystalline IPNS have en-
abled step-by-step elucidation of the reaction mechanism on a
structural level [11,22–25], while structures of the enzyme with
ACV analogues reveal a range of binding modes and the interplay
between steric and electronic effects at the IPNS active site [26–
32]. Seeking. a structural explanation for the failure of ACmT 3 to
react with IPNS, we have crystallised the protein with this sub-
strate analogue and solved the crystal structure of the IPNS:-
Fe(II):ACmT complex.
Benoiton (Scheme 3) [34]. Although the percentage conversion in
this etherification reaction is low (24–32% in our hands) unreacted
starting material (45–49%) is readily recovered and recycled; the
product 11 and residual starting material were isolated together
after work-up and could be separated by partitioning the resultant
oil between small volumes of DCM and water. N-Boc-O-methyl-D-
threonine 11 was isolated from the DCM phase and further purified
by crystallisation from chloroform.
The Boc group was removed using toluenesulfonic acid [35] and
the resulting carboxylic acid converted to the benzhydryl ester 12
using diphenyldiazomethane [36] (Scheme 3). Protected amino
acid 12 was coupled to previously reported dipeptide 13 [23] using
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) and 1-
hydroxybenzotriazole hydrate (HOBt) [37] under standard condi-
tions to give protected tripeptide 14. Global deprotection with tri-
fluoroacetic acid [38] gave the tripeptide 3 which was collected as
the trifluoroacetate salt by precipitation from ether. Purification by
reversed-phase HPLC (octadecylsilane 250 ꢀ 10 mm; k = 254 nm, 5
AUFS; 4 mL/min, 10 mM NH4HCO3 in water/methanol as eluant:
running time 0–5 min, 0% methanol; 5–12 min, gradient of 0–
25% methanol; 13–18 min, 2.5% methanol; 18–20 min 0% metha-
2. Materials and methods
2.1. Synthesis of d-(L-a-aminoadipoyl)-L-cysteinyl-O-methyl-D-
threonine 3
D
-Threonine 10 (Aldrich) was N-Boc-protected [33] then con-
verted to N-Boc-O-methyl- -threonine 11 using sodium iso-prop-
oxide and methyl iodide in THF as described by Chen and
D
Scheme 3. Synthesis of target tripeptide 3; (i) NaOH, H2O/tBuOH, (Boc)2O, rt, 23 h, 100%; (ii) NaOiPr, 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)
PDB acquisition code
1.5418
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
92909
27150
98.2
14.3
6.4
1.91–1.81
Total number of reflections
Number of unique reflections
Completeness (%)
10764
3414
92.1
2.9
Average I/
r
(I)
Rmerge (%)a
33.5
Rmeas (%)b
Rcryst (%)c
Rfree (%)d
7.6
16.2
20.2
40.6
RMS deviatione
0.020 (1.8)
18.6 21.0 16.3 24.6
233
Average B factors (Å2)f
Number of water molecules
P P
P P
a
b
c
d
e
f
Rmerge
Rmeas
¼
P
hjIh;j—hIhij=
hhIhi ꢀ 100.
j
j
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
P
P
P
¼
N=ðN ꢁ 1Þ jIiðhklÞ ꢁ IðhklÞj=
lIiðhklÞ ꢀ 100 [54,55].
hkl
i
hkl
P
Rcryst
¼
jjFobsj ꢁ jFcalcjj= jFobsj ꢀ 100.
Rfree = based on 5% of the total reflections.
RMS deviation from ideality for bonds (followed by the value for angles).
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 2mFoꢁDfc electron density map (cyan) contoured at 1.5
ACmT; and (ii) a mFoꢁDFc omit map (pink) contoured at 4
max) added to remaining coordinates, then re-refined). Figure generated using ccp4 mg [53].
r
around the substrate
r
around key water molecules (calculated from a model in which all waters were removed, random noise (0.2 Å
nol v/v; the tripeptide was treated with tris(2-carboxyethyl)phos-
phine hydrochloride for 20 min prior to injection onto the HPLC
column to reduce any cysteine disulfides [39]) gave tripeptide 3
as a fine white powder; Rt 11 min 40 s; dH (500 MHz, D2O): 1.05
(3H, d, J 6.5, CHCH3), 1.52–1.70 (2H, m, +H3NCHCH2CH2CH2),
(1:1 mixture of well buffer and saturated lithium sulfate in 40%
v/v glycerol), then flash-frozen in liquid nitrogen [40,41].
Data were collected at 100 K using Cu-K radiation from a Rig-
a
aku rotating anode generator and a MAR Research 300 mm image
plate detector at The Department of Molecular Biophysics, Oxford
University. The temperature was maintained at 100 K using an Ox-
ford Cryosystems Cryostream. Data were processed using DENZO
[42], MOSFLM [43] and the CCP4 suite of programs [44], then re-
fined using REFMAC5 [45] and Coot for model building [46]. Initial
phases were generated using co-ordinates for the protein from the
previously published IPNS:Fe(II):ACV structure [10], and manual
rebuilding of protein side-chains was performed as necessary.
The active site iron atom and water molecules were modeled in
during an early stage of the refinement process. Electron density
for ACmT was clearly visible throughout, and modeled in sequen-
tially at an advanced stage of refinement. Crystallographic coordi-
nates and structure factors for the IPNS:Fe(II):ACmT complex have
been deposited in the Protein Data Bank (PDB) under accession
number 3zoi.
+
1.73–1.88 (2H, m, +H3NCHCH2CH2CH2), 2.33 (2H, t, J 7.5, H3-
NCHCH2CH2CH2), 2.82 (1H, A of ABX, JAB 14.0, JAX 7.5 Hz, 1 of CH2-
SH), 2.87 (1H, B of ABX, JBA 14.0, JBX 5.5 Hz, 1 of CH2SH), 3.23 (3H, s,
CH3O), 3.65 (1H, m, +H3NCHCH2CH2CH2), 3.86 (1H, qd, J 6.5, 3.5 Hz,
CHOCH3), 4.13 (1H, d, J 3.5 Hz, NHCHCHOCH3), 4.52 (1H, X of ABX,
JXA 7.5, JXB 5.5 Hz, NHCHCH2SH).
2.2. Crystallography and structure determination
Crystals of the IPNS:Fe(II):ACmT complex were grown under
anaerobic conditions as previously reported (1.8–2.0 M lithium
sulfate and 100 mM Tris/HCI, pH 8.5) [40,41]. Crystals for X-ray dif-
fraction were selected using a light microscope, removed from the
anaerobic environment and exchanged into cryoprotectant buffer

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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 ccp4 mg [53].
ACmT 3 is unusual in that despite its size, this analogue fails to
exclude water from the iron binding site opposite Asp216. ACmT 3
is isosteric with ACI 6 and thus more sterically demanding than the
natural substrate ACV, yet the IPNS:Fe(II):ACmT complex still
incorporates the extra water ligand at iron. We propose that this
arrangement arises due to the greater hydrophilicity of the mThr
side-chain relative to Val, and the capacity for hydrogen bonding
between the methoxy group of 3 and water ligand #2173. Further,
we postulate that the bonding interactions between this water li-
gand, iron and the mThr methoxy group, and the greater bulk of
the mThr side-chain relative to the native substrate, combine to
exclude the cosubstrate O2 from the active site of the IPNS:-
Fe(II):ACmT complex.
3. Results and discussion
3.1. Structure of the IPNS:Fe(II):ACmT complex
The X-ray crystal structure of the anaerobic IPNS:Fe(II):ACmT
complex was solved to 1.81 Å resolution (Table 1, Figs. 1 and 2).
This structure reveals an interesting binding arrangement around
iron. The familiar facial triad of the protein-derived ligands is in
place: the side-chains of His214, Asp216 and His270 ligate to iron,
as seen previously with IPNS and other non-heme iron enzymes
[1,9,10,47]. ACmT is held in the enzyme active site in a similar
way to the native substrate [10]: via a salt bridge to the amin-
oadipoyl amino group, hydrogen bonds to the carboxylate of the
mThr residue, and ligation of the cysteinyl thiolate to iron in the
site opposite His270. However in contrast to the IPNS:Fe(II):ACV
complex, there are two water ligands bound to the metal with
ACmT, meaning that iron is hexacoordinate. In the complex of IPNS
with its natural substrate ACV (Fig. 2a) [10] and complexes with
substrate analogues containing third residues of a similar size
[22,24,48], only one water ligand is present at iron, opposite
His214 (#713 in Fig. 2a). In addition to this usual water ligand at
iron (#2178 in Fig. 2b), the ACmT complex contains a second water
molecule bound in the site trans to Asp216 (#2173). This second
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).
4. Conclusions
Ligation of water molecule #2173 in the oxygen binding site of
the IPNS:Fe(II):ACmT complex provides a possible structural expla-
nation for the failure of IPNS to turnover ACmT 3. Held by hydro-
gen bonds to the substrate methoxy group and ligated to iron
trans to Asp216, this water molecule and the mThr side-chain
may prevent oxygen entry and thus block turnover before reaction
can even begin.
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-
strate D-valine residue: iron is pentacoordinate with the valinyl
isopropyl group sitting within van der Waals contact of the metal.
This interaction effectively reserves the sixth iron binding site for
the co-substrate molecular oxygen [10]. Hexacoordinate iron has
previously been observed in IPNS complexes: (i) with analogues
We thank Dr Peter Roach, Dr Nicolai Burzlaff, Dr Charles Hens-
gens, Professor Chris Schofield, Dr Alex Parker, Dr Jon Elkins and Dr
Karl Harlos for helpful discussions. Financial support was provided
by the MRC, BBSRC and EPSRC. P.J.R. thanks the Rhodes Trust for a
scholarship.
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- -methionine [49]), in
D
which the affinity of sulfur for iron means that the sulfide S coor-
dinates to the metal; (ii) with smaller substrate analogues (e.g.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.febslet.
2013.07.016.
AC-Gly and AC-
D
-Ala,[28] AC-
D
-a
-aminobutyrate [26], AC-D-vinyl-
glycine [23] and the dipeptide d-
L-
a
-aminoadipoyl- -homocysteine
L
(AhC) [50]), where the smaller side-chain leaves room for a
References
second water ligand to bind to iron opposite Asp216; and (iii) with
[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
intermediates. Chem. Rev. 104, 939–986.
LLL-configured substrates (e.g. AC-
L-hexafluorovaline [51] and
AC- -2-amino-3,3-dideuteriobutyrate [52]), where the different
L
substrate stereochemistry permits an additional water ligand at
iron.
[2] Hausinger, R.P. (2004) Fe(II)/alpha-ketoglutarate-dependent hydroxylases and
related enzymes. Crit. Rev. Biochem. Mol. Biol. 39, 21–68.

I.J. Clifton et al. / FEBS Letters 587 (2013) 2705–2709
2709
[3] Bruijnincx, P.C.A., van Koten, G. and Gebbink, R. (2008) Mononuclear non-
truncated substrate analogues d-(
L
-
a
-aminoadipoyl)-
L
-cysteinyl-glycine and
heme iron enzymes with the 2-His-1-carboxylate facial triad: recent
developments in enzymology and modeling studies. Chem. Soc. Rev. 37,
2716–2744.
d-( -aminoadipoyl)- -cysteinyl-D-alanine. Biochemistry 44, 6619–6628.
L-
a
L
[29] Howard-Jones, A.R., Elkins, J.M., Clifton, I.J., Roach, P.L., Adlington, R.M.,
Baldwin, J.E. and Rutledge, P.J. (2007) Interactions of isopenicillin N synthase
with cyclopropyl-containing substrate analogues reveal new mechanistic
insight. Biochemistry 46, 4755–4762.
[30] Stewart, A.C., Clifton, I.J., Adlington, R.M., Baldwin, J.E. and Rutledge, P.J. (2007)
A cyclobutanone analogue mimics penicillin in binding to isopenicillin N
synthase. ChemBioChem 8, 2003–2007.
[31] Ge, W., Clifton, I.J., Stok, J.E., Adlington, R.M., Baldwin, J.E. and Rutledge, P.J.
(2010) The crystal structure of an LLL-configured depsipeptide substrate
analogue bound in the active site of isopenicillin N synthase. Org. Biomol.
Chem. 8, 122–127.
[32] Daruzzaman, A., Clifton, I.J., Adlington, R.M., Baldwin, J.E. and Rutledge, P.J.
(2013) The interaction of isopenicillin N synthase with homologated substrate
[4] Baldwin, J.E. and Schofield, C.J. (1992) The Biosynthesis of b-Lactams in: The
Chemistry of b-Lactams (Page, M.I., Ed.), pp. 1–78, Blackie, Glasgow.
[5] Chen, V.J., Orville, A.M., Harpel, M.R., Frolik, C.A., Surerus, K.K., Munck, E. and
Lipscomb, J.D. (1989) Spectroscopic studies of isopenicillin N synthase – a
mononuclear nonhaem Fe2+oxidase with metal coordination sites for small
molecules and substrate. J. Biol. Chem. 264, 21677–21681.
[6] Hadfield, A. and Hajdu, J.A. (1993)
A fast and portable microspectro-
photometer for protein crystallography. J. Appl. Crystallogr. 26, 839–842.
[7] Scott, R.A., Wang, S., Eidsness, M.K., Kriauciunas, A., Frolik, C.A. and Chen, V.J.
(1992) X-ray absorption spectroscopic studies of the high-spin iron(II) active
site of isopenicillin N synthase: evidence for iron-sulfur interaction in the
enzyme-substrate complex. Biochemistry 31, 4596–4601.
analogues d-(
L-a-aminoadipoyl)-L-homocysteinyl-D-Xaa characterised by
[8] Randall, C.R., Zang, Y., True, A.E., Que Jr., L., Charnock, J.M., Garner, C.D.,
Fujishima, Y., Schofield, C.J. and Baldwin, J.E. (1993) X-Ray absorption studies
protein crystallography. ChemBioChem 14, 599–606.
[33] Keller, O., Keller, W.E., van Look, G. and Wersin, G. (1985) tert-
Butoxycarbonylation of amino acids and their derivatives: N-tert-
of the ferrous active site of isopenicillin
complexes. Biochemistry 32, 6664–6673.
N synthase and related model
butoxycabonyl-
[34] Chen, F.M.F. and Benoiton, N.L. (1979) Simple preparations of N-(tert-
butyloxycarbonyl)-O-methyl- -serine and N-(tert-butyloxycarbonyl)-O-
methyl- -threonine by direct methylation. J. Org. Chem. 44, 2299–2300.
L-phenylalanine. Org. Synth. 63, 160.
[9] Roach, P.L., Clifton, I.J., Fulop, V., Harlos, K., Barton, G.J., Hajdu, J., Andersson, I.,
Schofield, C.J. and Baldwin, J.E. (1995) Crystal structure of isopenicillin N
synthase is the first from a new structural family of enzymes. Nature 375,
700–704.
[10] Roach, P.L., Clifton, I.J., Hensgens, C.M.H., Shibata, N., Schofield, C.J., Hadju, J.
and Baldwin, J.E. (1997) Structure of isopenicillin N synthase complexed with
substrate and the mechanism of penicillin formation. Nature 387, 827–830.
[11] Burzlaff, N.I., Rutledge, P.J., Clifton, I.J., Hensgens, C.M.H., Pickford, M.,
Adlington, R.M., Roach, P.L. and Baldwin, J.E. (1999) The reaction cycle of
isopenicillin N synthase observed by X-ray diffraction. Nature 401, 721–724.
[12] Wirstam, M. and Siegbahn, P.E.M. (2000) A mechanistic study of isopenicillin
N formation using density functional theory. J. Am. Chem. Soc. 122, 8539–
8547.
L
L
[35] Ferguson, A.C., Adlington, R.M., Martyres, D.H., Rutledge, P.J., Cowley, A. and
Baldwin, J.E. (2003) Total synthesis of a novel 2-thiabicyclo[3.2.0]heptan-6-
one analogue of penicillin N. Tetrahedron 59, 8233–8243.
[36] Wolfe, S. and Jokinen, M.G. (1979) Total synthesis of d-(
L-a-aminoadipoyl)-L-
cysteinyl- -valine (ACV), biosynthetic precursor of penicillins and
D
a
cephalosporins. Can. J. Chem. 1978, 1388–1396.
[37] Sheehan, J.C., Preston, J. and Cruickshank, P.A. (1965) A rapid synthesis of
oligopeptide derivatives without isolation of intermediates. J. Am. Chem. Soc.
87, 2492–2493.
[38] Bodanzky, M. and Bodanzky, A. (1984) The Practice of Peptide Synthesis in:
Reactivity and Structure Concepts in Organic Chemistry (Hafner, K., Rees, C.W.,
Trost, B.M., Lehn, J.M., Schleyer, P.v.R. and Zahruchik, R., Eds.), pp. 172–173,
Springer-Verlag, Berlin.
[39] Burns, J.A., Butler, J.C., Moran, J. and Whitesides, G.M. (1991) Selective
reduction of disulfides by tris(2-carboxyethyl)phosphine. J. Org. Chem. 56,
2648–2650.
[13] Lundberg, M., Siegbahn, P.E.M. and Morokuma, K. (2007) The mechanism for
isopenicillin
N synthase from density-functional modeling highlights the
similarities with other enzymes in the 2-His-1-carboxylate family.
Biochemistry 47, 1031–1042.
[14] Baldwin, J.E. and Bradley, M. (1990) Isopenicillin N synthase: mechanistic
studies. Chem. Rev. 90, 1079–1088.
[15] Baldwin, J.E. (1985) Recent advances in the chemistry of the b-lactam
antibiotics in: (Brown, A.G. and Roberts, S.M., Eds.), pp. 62–85, The Royal
Society of Chemistry, London. Special Publication No. 52.
[16] Baldwin, J.E., Adlington, R.M., Basak, A., Flitsch, S.L., Petursson, S., Turner, N.J.
and Ting, H.-H. (1986) Enzymatic synthesis of a new type of penicillin. J. Chem.
Soc., Chem. Commun., 975–976.
[40] Roach, P.L., Clifton, I.J., Hensgens, C.M.H., Shibata, N., Long, A.J., Strange, R.W.,
Hasnain, S.S., Schofield, C.J., Baldwin, J.E. and Hajdu, J. (1996) Anaerobic
crystallisation of an isopenicillin
N
synthaseꢂFe(II)ꢂsubstrate complex
demonstrated by X-ray studies. Eur. J. Biochem. 242, 736–740.
[41] Rutledge, P.J., Burzlaff, N.I., Elkins, J.M., Pickford, M., Baldwin, J.E. and Roach,
P.L. (2002) The use of high-pressure oxygenation to promote reaction in
enzyme crystals. Anal. Biochem. 308, 265–268.
[17] Petursson, S. and Baldwin, J.E. (1998) Synthesis of d-(
cysteinyl-(O-methyl)- -allothreonine a substrate for isopenicillin-N synthase
and its O-methyl- -threonine epimer. Tetrahedron 54, 6001–6010.
L-aminoadipoyl)-L-
D
[42] Otwinowski, Z. and Minor, W. (1997) Processing of X-ray diffraction data
collected in oscillation mode. Methods Enzymol. 276, 307–326.
[43] Leslie, A.G.W. (1999) Integration of maromolecular diffraction data. Acta
Crystallogr. D 55, 1696–1702.
D
[18] Bahadur, G., Baldwin, J.E., Field, L.D., Lehtonen, E.-M.M., Usher, J.J., Vallejo, C.A.,
Abraham, E.P. and White, R.L. (1981) Direct 1H NMR observation of the cell-
free conversion of d-(
aminoadipyl)- -cysteinyl-
Chem. Commun., 917–919.
L
-
a
-aminoadipyl)-
L
-cysteinyl-
D
-valine and d-(
L
-
a
-
[44] Collaborative Computational Project Number 4, The CCP4 Suite: Programs for
protein crystallography. Acta Crystallogr. D 50 (1994) 760–763.
[45] Murshudov, G.N., Vagin, A.A. and Dodson, E.J. (1997) Refinement of
macromolecular structures by the maximum-likelihood method [Refmac].
Acta Crystallogr. D 53, 240–255.
L
D-(-)-isoleucine into penicillins. J. Chem. Soc.,
[19] Bahadur, G.A., Baldwin, J.E., Usher, J.J., Abraham, E.P., Jayatilake, G.S. and
White, R.L. (1981) Cell-free biosynthesis of penicillins. Conversion of peptides
into new b-lactam antibiotics. J. Am. Chem. Soc. 103, 7650–7651.
[46] Emsley, P. and Cowtan, K. (2004) Coot: model-building tools for molecular
graphics. Acta Crystallogr. D 60, 2126–2132.
[20] Clifton, I.J., Ge, W., Adlington, R.M., Baldwin, J.E. and Rutledge, P.J. (2011)
Isopenicillin N synthase binds d-(
L
-
a
-aminoadipoyl)-
L
-cysteinyl-
D
-thia-allo-
[47] Hegg, E.L. and Que Jr., L. (1997) The 2-His-1-carboxylate facial triad – an
emerging structural motif in mononuclear non-heme iron(II) enzymes. Eur. J.
Biochem. 250, 625–629.
[48] Ge, W., Clifton, I.J., Howard-Jones, A.R., Stok, J.E., Adlington, R.M., Baldwin, J.E.
and Rutledge, P.J. (2009) Structural studies on the reaction of isopenicillin N
isoleucine through both sulfur atoms. ChemBioChem 12, 1881–1885.
[21] Baldwin, J.E., Adlington, R.M., Basak, A., Imming, P., Ponnamperuma, K.,
Ronneberg, H., Schofield, C.J., Ting, H.-H. and Turner, N.J. (1989) Penicillin
biosynthesis: active substrates derived by methoxy substitution in the valinyl
residue of the natural substrate. J. Chem. Soc., Chem. Commun., 802–804.
[22] Ogle, J.M., Clifton, I.J., Rutledge, P.J., Elkins, J.M., Burzlaff, N.I., Adlington, R.M.,
Roach, P.L. and Baldwin, J.E. (2001) Alternative oxidation by isopenicillin N
synthase observed by X-ray diffraction. Chem. Biol. 8, 1231–1237.
synthase with
a sterically demanding depsipeptide substrate analogue.
ChemBioChem 10, 2025–2031.
[49] Clifton, I.J., Ge, W., Adlington, R.M., Baldwin, J.E. and Rutledge, P.J. (2011) The
crystal structure of isopenicillin
N synthase with d-(L-a-aminoadipoyl)-L-
[23] Elkins, J.M., Rutledge, P.J., Burzlaff, N.I., Clifton, I.J., Adlington, R.M., Roach, P.L.
and Baldwin, J.E. (2003) Crystallographic studies on the reaction of
cysteinyl- -methionine reveals thioether coordination to iron. Arch. Biochem.
D
Biophys. 516, 103–107.
isopenicillin
Biomol. Chem. 1, 1455–1460.
[24] Daruzzaman, A., Clifton, I.J., Adlington, R.M., Baldwin, J.E. and Rutledge, P.J.
N
synthase with an unsaturated substrate analogue. Org.
[50] Daruzzaman, A., Clifton, I.J., Adlington, R.M., Baldwin, J.E. and Rutledge, P.J.
(2013) The crystal structure of isopenicillin N synthase with a dipeptide
substrate analogue. Arch. Biochem. Biophys. 530, 48–53.
(2006) Unexpected oxidation of
a
depsipeptide substrate analogue in
[51] Howard-Jones, A.R., Rutledge, P.J., Clifton, I.J., Adlington, R.M. and Baldwin, J.E.
crystalline isopenicillin N synthase. ChemBioChem 7, 351–358.
[25] Ge, W., Clifton, I.J., Stok, J.E., Adlington, R.M., Baldwin, J.E. and Rutledge, P.J.
(2008) Isopenicillin N synthase mediates thiolate oxidation to sulfenate in a
depsipeptide substrate analogue: implications for oxygen binding and a link
to nitrile hydratase? J. Am. Chem. Soc. 130, 10096–10102.
(2005) Unique binding of
synthase. Biochem. Biophys. Res. Commun. 336, 702–708.
[52] Ge, W., Clifton, I.J., Stok, J.E., Adlington, R.M., Baldwin, J.E. and Rutledge, P.J.
(2010) Crystallographic studies on the binding of selectively deuterated LLD
and LLL-substrate epimers by isopenicillin N synthase. Biochem. Biophys. Res.
Commun. 398, 659–664.
[53] McNicholas, S., Potterton, E., Wilson, K.S. and Noble, M.E.M. (2011) Presenting
your structures: the CCP4mg molecular-graphics software. Acta Crystallogr. D
67, 386–394.
a non-natural LLL-substrate by isopenicillin N
-
[26] Long, A.J., Clifton, I.J., Roach, P.L., Baldwin, J.E., Schofield, C.J. and Rutledge, P.J.
(2003) Structural studies on the reaction of isopenicillin N synthase with the
substrate analogue d-(L-a-aminoadipoyl)-L-cysteinyl-D-a-aminobutyrate.
Biochem. J. 372, 687–693.
[27] Grummitt, A.R., Rutledge, P.J., Clifton, I.J. and Baldwin, J.E. (2004) Active-site-
mediated elimination of hydrogen fluoride from fluorinated substrate
analogue by isopenicillin N synthase. Biochem. J. 382, 659–666.
[28] Long, A.J., Clifton, I.J., Roach, P.L., Baldwin, J.E., Rutledge, P.J. and Schofield, C.J.
(2005) Structural studies on the reaction of isopenicillin N synthase with the
[54] Diederichs, K. and Karplus, P.A. (1997) Improved R-factors for diffraction data
analysis in macromolecular crystallography. Nature Struct. Biol. 4, 269–275.
[55] Weiss, M.S. (2001) Global indicators of X-ray data quality. J. Appl. Cryst. 34,
130–135.
a