The crystal structure of isopenicillin N synthase with δ-(l-α- aminoadipoyl)-l-cysteinyl-d-methionine reveals thioether coordination to iron

Article abstract of DOI:10.1016/j.abb.2011.09.014

Isopenicillin N synthase (IPNS) catalyses cyclization of δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to isopenicillin N (IPN), the central step in penicillin biosynthesis. Previous studies have shown that IPNS turns over a wide range of substrate analogues in which the valine residue of its natural substrate is replaced with other amino acids. IPNS accepts and oxidizes numerous substrates that bear hydrocarbon sidechains in this position, however the enzyme is less tolerant of analogues presenting polar functionality in place of the valinyl isopropyl group. We report a new ACV analogue δ-(l-α-aminoadipoyl)-l-cysteinyl-d-methionine (ACM), which incorporates a thioether in place of the valinyl sidechain. ACM has been synthesized using solution phase methods and crystallized with IPNS. A crystal structure has been elucidated for the IPNS:Fe(II):ACM complex at 1.40 resolution. This structure reveals that ACM binds in the IPNS active site such that the sulfur atom of the methionine thioether binds to iron in the oxygen binding site at a distance of 2.57 . The sulfur of the cysteinyl thiolate sits 2.36 from the metal.

Full text of DOI:10.1016/j.abb.2011.09.014

Archives of Biochemistry and Biophysics 516 (2011) 103–107
Contents lists available at SciVerse ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
The crystal structure of isopenicillin N synthase with
d-(L-a-aminoadipoyl)-L-cysteinyl-D-methionine reveals thioether
coordination to iron
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 Rd., Oxford OX1 3TA, UK
b School of Chemistry, F11, 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) catalyses cyclization of d-(L-a-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to
Received 13 September 2011
and in revised form 28 September 2011
Available online 6 October 2011
isopenicillin N (IPN), the central step in penicillin biosynthesis. Previous studies have shown that IPNS
turns over a wide range of substrate analogues in which the valine residue of its natural substrate is
replaced with other amino acids. IPNS accepts and oxidizes numerous substrates that bear hydrocarbon
sidechains in this position, however the enzyme is less tolerant of analogues presenting polar function-
Keywords:
Antibiotics
Biosynthesis
Enzyme mechanism
Metalloenzymes
Non-heme iron oxidase
Penicillin
ality in place of the valinyl isopropyl group. We report a new ACV analogue d-(
L-a-aminoadipoyl)-L-cys-
teinyl- -methionine (ACM), which incorporates a thioether in place of the valinyl sidechain. ACM has
D
been synthesized using solution phase methods and crystallized with IPNS. A crystal structure has been
elucidated for the IPNS:Fe(II):ACM complex at 1.40 Å resolution. This structure reveals that ACM binds in
the IPNS active site such that the sulfur atom of the methionine thioether binds to iron in the oxygen
binding site at a distance of 2.57 Å. The sulfur of the cysteinyl thiolate sits 2.36 Å from the metal.
Ó 2011 Elsevier Inc. All rights reserved.
Introduction
Incubation experiments in solution have demonstrated that
IPNS turns over a diverse range of analogues variant in the third
Isopenicillin N synthase (IPNS) catalyses the key step in penicil-
lin biosynthesis: conversion of the linear tripeptide d-( -amin-
oadipoyl)- -cysteinyl-
-valine (ACV, 1)1 to bicyclic isopenicillin N
(IPN, 2) (Scheme 1) [1]. IPNS is a non-heme iron(II) oxidase (NHIO)
[2,3], and is thought to generate a highly reactive iron(IV)-oxo (fer-
ryl) species 3 during conversion of ACV to IPN [4,5].
IPNS has been the subject of considerable research interest
due to the unique chemistry that it catalyses and to the clinical
utility of b-lactam antibiotics [1]. A combination of solution-
phase turnover experiments [6], spectroscopy [7] and more
recently protein crystallography [5,8–10] has been applied to
build a detailed picture of the IPNS active site and reaction
mechanism.
position of the ACV tripeptide [6]. Analogues incorporating various
hydrocarbon sidechains in place of the valinyl isopropyl group are
L-a
L
D
substrates for IPNS, including AC-
[11], AC- -isoleucine (ACI, 5) [12], AC-
[13] and AC- -vinylglycine [14]. In contrast, IPNS is much less
accepting of polar sidechains in this position: none of AC- -Ser 6,
AC- -Cys 7 (both isosteres of ACAb) [6], AC- -O-methylthreonine
-Asn [6] are turned
D-
a-aminobutyrate (ACAb, 4)
D
D-methylcyclopropylglycine
D
D
D
D
8 (isosteric with ACI) [15], AC-D-Glu or AC-D
over by the enzyme (Fig. 1).
d-(L-a-Aminoadipoyl-L-cysteinyl-D-methionine (ACM, 9) bears
the thioether of methionine in place of the valinyl isopropyl group
of the natural substrate ACV 1; the interaction of ACM with IPNS
has not previously been investigated. In this paper we report the
synthesis of ACM 9, crystallization of this new tripeptide with IPNS,
and the crystal structure of the IPNS:Fe(II):ACM complex.
⇑
Corresponding authors. Fax: +61 2 9351 3329 (P.J. Rutledge).
E-mail address: peter.rutledge@sydney.edu.au (P.J. Rutledge).
1
Abbreviations used: AC-, d-(L-
-aminobutyrate; ACI, d-(L-
D-isoleucine; ACM, d-(L- -aminoadipoyl)-L-cysteinyl-D-methionine; ACmC,
d-(L- -aminoadipoyl)-L-cysteinyl-D-S-methylcysteine; ACOmC, d-(L- -amin-
oadipoyl)-L-cysteinyl (1-(S)-carboxy-2-thiomethyl)ethyl ester; ACtaI, d-(L- -amin-
oadipoyl)-L-cysteinyl-D-thia-allo-isoleucine; ACV, d-(L- -aminoadipoyl)-L-cysteinyl-
a
-aminoadipoyl)-L-cysteinyl-; ACAb, d-(L-
a-amin-
Results and discussion
oadipoyl)-L-cysteinyl-D-
a
a
-aminoadipoyl)-L-cysteinyl-
a
a
a
a
Synthesis of ACM 9
a
ACM 9 was prepared from
dipeptide 11 [16] (Scheme 2). -Methionine 10 was first converted
to its benzhydryl ester 12 using p-toluenesulfonic acid to mask the
D-methionine 10 and protected
D-valine; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride;
HOBt, 1-hydroxybenzotriazole hydrate; IPN, isopenicillin N; IPNS, isopenicillin N
synthase; NHIO, non-heme iron(II) oxidase.
D
0003-9861/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2011.09.014

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I.J. Clifton et al. / Archives of Biochemistry and Biophysics 516 (2011) 103–107
Scheme 1. The reaction of IPNS with its natural substrate ACV 1 to give IPN 2, via a high-valent iron intermediate 3.
Fig. 1. Some of the many ACV analogues that have been incubated with IPNS in solution (AC-
D-a-aminobutyrate (ACAb, 4) [11], AC-D-isoleucine (ACI, 5) [12], AC-D-Ser 6, AC-
D
-Cys 7 [6] and AC- -O-methylthreonine 8 [15]), and the tripeptide used in this study ACM 9.
D
Scheme 2. Synthesis of tripeptide 9. Reagents and conditions: (i) TsOH, H2O, then Ph2CN2, Et2O/MeCN, rt 3 h, 83%; (ii) EDCI, HOBt, Et3N, CH2Cl2, rt 20 h, quant.; and (iii) TFA,
anisole, reflux 1 h, quant.; reversed phase HPLC. Full experimental procedures and characterization data are available in the Supporting information, along with the NMR
spectra of compounds 9, 12 and 13.
Fig. 2. Stereo image showing the active site of the anaerobic IPNS:Fe(II):ACM complex. A 2mFo–DFc electron-density map is shown around the substrate analogue at 1r.

I.J. Clifton et al. / Archives of Biochemistry and Biophysics 516 (2011) 103–107
105
Fig. 3. Stereo image showing a close-up view of the iron binding environment in the IPNS:Fe(II):ACM complex.
Table 1
the overall structure of the protein is similar to the complexes of
IPNS with its natural substrate [9] and substrate analogues
[5,16,22–26]. ACM 9 binds to the protein in a similar manner to
ACV 1 and analogues. The cysteinyl thiolate of the tripeptide li-
gates to iron opposite His270, and ACM is held by interactions with
its amino and carboxylate groups: a salt bridge between the amin-
oadipoyl carboxylate and Arg87, and hydrogen bonding between
the aminoadipoyl amino group and Thr331. The methionine car-
boxylate forms hydrogen bonds with the adjacent sidechains of
Tyr189, Arg279 and Ser281.
The active site iron center is hexacoordinate. It is bound by the
familiar three ligands from the protein (His214, Asp216 and
His270, the defining ‘2-His-1-carboxylate’ motif of the NHIO en-
zyme family [27]), a water ligand trans to His214, and two sub-
strate-derived ligands. The cysteinyl thiolate of ACM ligates to
iron opposite His270, and the methionine thioether binds trans
to Asp216. The sulfur atoms are 2.36 Å (thiolate) and 2.57 Å (thio-
ether) from iron, respectively.
X-ray data collection and crystallographic statistics.
X-ray source
BW7B DESY, Hamburg
0.8345
2y60
1.40
P212121
Wavelength (Å)
PDB acquisition code
Resolution (Å)
Space group
Unit cell dimensions
a (Å)
46.52
70.88
b (Å)
c (Å)
100.93
20.33–1.40
137,704
55,964
83.8
9.4
7.9
9.8
14.70
Resolution shell (Å)
Total number of reflections
Number of unique reflections
Completeness (%)
1.48–1.40
19,908
8213
85.1
1.9
43.0
Average I/(
Rmerge (%)a
Rmeas (%)b
Rcryst (%)c
Rfree (%)d
r
I)
53.7
19.93
RMS deviatione
0.024, 2.1
11.6, 14.4, 12.0, 25.7
386
Average B factors (Å2)f
Number of water molecules
In complexes of IPNS with ACV and related substrate analogues
[9,22,24,26], iron is pentacoordinate: the valinyl isopropyl group
sits within van der Waals contact of the metal and effectively re-
serves the site trans to Asp216 for oxygen (O2, the co-substrate)
[9]. Hexacoordinate iron has previously been observed in several
IPNS:Fe(II):analogue complexes, in three general contexts. With
P P
P P
a
b
c
d
e
f
Rmerge
Rmeas
Rcryst
=
h|Ih,j ꢀ hIhi|/
hhIhi ꢁ 100.
j
j
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
P
P
P
=
N=ðN ꢀ 1Þ i|Ii (hkl) ꢀ IðhklÞ|/
Ii (hkl) ꢁ 100 [40,41].
hkl
hkl
I
P
P
=
||Fobs| ꢀ |Fcalc||/ |Fobs| ꢁ 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; sidechain; substrate and iron; solvent
less bulky substrate analogues such as AC-Gly, AC-
D-Ala [28], AC-
(water).
D-
a
-aminobutyrate [29] and AC- -vinylglycine [16], a second
D
water ligand can enter and bind to iron in the site opposite
Asp216. LLL-Configured peptides and depsipeptides also bind to
IPNS in a manner that permits an additional water ligand at iron,
which is therefore hexacoordinate [30–32]. And sulfide ligation
amine and diphenyldiazomethane to trap the carboxylic acid [17].
Carbodiimide-promoted peptide coupling of 11 and 12 using
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) and 1-
hydroxybenzotriazole hydrate (HOBt) [18] gave the fully protected
tripeptide 13, and global deprotection with trifluoroacetic acid [19]
afforded the target compound 9. Purification by reversed-phase
HPLC was required to generate material of sufficient purity for
crystallization with the protein.
to iron has been observed in the complexes of IPNS with AC-
D-S-
methylcysteine (ACmC, 14) [5], AC-(1-(S)-carboxy-2-thio-
methyl)ethyl ester (ACOmC, 15) [33] and AC-
(ACtaI, 16) [34].
D
-thia-allo-isoleucine
-car-
ACM 9 bears an additional methylene group between the
a
bon of its third residue and the methyl sulfide when compared to
ACmC 14, ACOmC 15 and ACtaI 16 (Fig. 4). Nonetheless IPNS easily
accommodates the additional carbon atom of the methionine side-
chain, which occupies a single conformation in which the two
methylene groups are twisted back ‘behind’ the sulfur atom (‘be-
hind’ in the orientation shown in Fig. 3). Although the thioether
sulfur is not as tightly bound to iron as the free thiolate (sitting
2.57 Å from the metal, compared to 2.36 Å for the thiolate), the
The structure of the IPNS:Fe(II):ACM complex
ACM 9 was crystallized with IPNS and iron(II) under anaerobic
conditions following the previously reported procedure [20,21].
The crystal structure of the IPNS:Fe(II):ACM complex was solved
to 1.40 Å resolution (Figs. 2 and 3, Table 1). Not unexpectedly,

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I.J. Clifton et al. / Archives of Biochemistry and Biophysics 516 (2011) 103–107
Fig. 4. ACV analogues that incorporate thioether functionality in their third position: ACM 9 (this study), AC-D-S-methylcysteine (ACmC, 14) [5], AC-(1-(S)-carboxy-2-
thiomethyl)ethyl ester (ACOmC, 15) [33], AC-D-thia-allo-isoleucine (ACtaI, 16) [34].
additional methylene unit between
a
-carbon and methyl sulfide
temperature was maintained at 100 K using an Oxford Cryosys-
tems Cryostream. Data were processed using MOSFLM [35] and
the CCP4 suite of programs [36], then refined using REFMAC5
[37] and Coot for model building [38]. Initial phases were gener-
ated using co-ordinates for the protein from the previously pub-
lished IPNS:Fe(II):ACV structure [9], and manual rebuilding of
protein sidechains was performed as necessary. Crystallographic
coordinates and structure factors have been deposited in the
Worldwide Protein Data Bank under accession numbers 2y60.
Figs. 2 and 3 were prepared using CCP4mg [39].
allows closer approach of thioether to metal (2.57 Å) than is seen
in the crystal structures of IPNS with ACmC 14, ACOmC 15 (both
2.69 Å) [5,33] and ACtaI 16 (2.66 Å) [34].
Conclusions
The new tripeptide ACM 9 binds in the active site of IPNS, its
cysteinyl thiolate tethered to iron in the site opposite His270, its
methionyl thioether ligated opposite Asp216. This second sub-
strate-derived ligand sits in the oxygen binding site, and renders
the metal hexacoordinate. It is probable that the thioether would
block oxygen from binding, preventing reaction under normal
turnover conditions. This result offers further structural evidence
for the failure of IPNS to turn over substrate analogues such as
Acknowledgments
We thank Dr. Peter Roach, Dr. Nicolai Burzlaff, Dr. Jon Elkins,
Professor Chris Schofield, Dr. John Keeping and the scientists at
SRS Daresbury and DESY Hamburg for help and discussions, and
Dr. Elizabeth McGuinness for high resolution NMR spectra. Finan-
cial support was provided by the MRC, BBSRC and EPSRC. P.J.R.
thanks the Rhodes Trust for a scholarship.
AC-D-Ser 6, AC-D-Cys 7 and AC-D-O-methylthreonine 8, which in-
clude polar amino acids in place of the valine residue of ACV 1.
Experimental
Synthesis of d-(
L-
a-aminoadipoyl)-
L
-cysteinyl-
D
-methionine 9
Appendix A. Supplementary data
D
-Methionine 10 was protected as the benzhydryl ester 12
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.abb.2011.09.014.
using p-toluenesulfonic acid and diphenyldiazomethane [17],
then coupled to previously reported dipeptide 11 [16] using
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) and 1-
hydroxybenzotriazole hydrate (HOBt) under standard conditions
[18]. Global deprotection with trifluoroacetic acid [19] and purifi-
cation by reversed-phase HPLC [octadecylsilane 250 mm ꢁ 10 mm;
10 mM NH4HCO3 in water/methanol as eluant, running time: 0–
6 min, 2.5%; 6–14 min, 25%; 14–20 min, 2.5% methanol, v/v;
4 mL minꢀ1; k = 254 nm, five absorbance units full scale (AUFS);
retention time 10.5–12 min] gave purified tripeptide 9; dH
(500 MHz, D2O): 1.54–1.65 (2H, m, +H3NCHCH2CH2CH2), 1.72–
1.81 (2H, m, +H3NCHCH2CH2CH2), 1.84–1.92 (1H, m, 1 of CH2SCH3),
1.98 (3H, s, SCH3), 2.00–2.07 (1H, m, 1 of CH2SCH3), 2.29 (2H, td, J
7.0, 2.5, +H3NCHCH2CH2CH2), 2.40 (1H, A of ABX, J 13.5, 7.5, 1 of
CH2SH), 2.47 (1H, B of ABX, J 13.5, 5.0, 1 of CH2SH), 2.79 (2H, dd,
J 7.5, 2.0, CHCH2CH2SCH3), 3.64 (1H, t, J 6.0, +H3NCHCH2CH2CH2),
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