Isopenicillin N Synthase Binds δ-(L-α-Aminoadipoyl)-L-Cysteinyl-D-Thia-allo-Isoleucine through both Sulfur Atoms

Article abstract of DOI:10.1002/cbic.201100149

Isopenicillin N synthase (IPNS) catalyses the synthesis of isopenicillin N (IPN), the biosynthetic precursor to penicillin and cephalosporin antibiotics. IPNS is a non-heme iron(II) oxidase that mediates the oxidative cyclisation of the tripeptide δ-L-α-a

Full text of DOI:10.1002/cbic.201100149

DOI: 10.1002/cbic.201100149
Isopenicillin N Synthase Binds d-(l-a-Aminoadipoyl)-l-
Cysteinyl-d-Thia-allo-Isoleucine through both Sulfur Atoms
Ian J. Clifton,[a] Wei Ge,[a] Robert M. Adlington,[a] Jack E. Baldwin,*[a] and Peter J. Rutledge*[b]
Isopenicillin N synthase (IPNS) catalyses the synthesis of iso-
penicillin N (IPN), the biosynthetic precursor to penicillin and
cephalosporin antibiotics. IPNS is a non-heme iron(II) oxidase
that mediates the oxidative cyclisation of the tripeptide d-l-a-
aminoadipoyl-l-cysteinyl-d-valine (ACV) to IPN with a concomi-
tant reduction of molecular oxygen to water. Solution-phase
incubation experiments have shown that, although IPNS can
turn over analogues with a diverse range of hydrocarbon side
chains in the third (valinyl) position of its substrate, the
enzyme is much less tolerant of polar residues in this position.
Thus, although IPNS converts d-l-a-aminoadipoyl-l-cysteinyl-d-
isoleucine (ACI) and AC-d-allo-isoleucine (ACaI) to penam prod-
ucts, the isosteric sulfur-containing peptides AC-d-thiaisoleu-
cine (ACtI) and AC-d-thia-allo-isoleucine (ACtaI) are not turned
over. To determine why these peptides are not substrates, we
crystallized ACtaI with IPNS. We report the synthesis of ACtaI
and the crystal structure of the IPNS:FeII:ACtaI complex to
1.79 ꢀ resolution. This structure reveals direct ligation of the
thioether side chain to iron: the sulfide sulfur sits 2.66 ꢀ from
the metal, squarely in the oxygen binding site. This result artic-
ulates a structural basis for the failure of IPNS to turn over
these substrates.
Introduction
Isopenicillin N synthase (IPNS) mediates the oxidative bicyclisa-
tion of d-l-a-aminoadipoyl-l-cysteinyl-d-valine (ACV, 1) to iso-
penicillin N (IPN, 2), the central step in penicillin biosynthesis
(Scheme 1).[1] IPNS is a non-heme iron(II) oxidase (NHIO) and
erant of polar side chains and heteroatoms in this position.[6]
Thus, whereas d-l-a-aminoadipoyl-l-cysteinyl-d-isoleucine (ACI,
4) and AC-d-allo-isoleucine (ACaI, 5) are converted by IPNS to
the penam products 6 and 7 respectively (Scheme 2),[10–12] the
isosteric sulfur-containing peptides AC-d-thiaisoleucine (ACtI,
8) and AC-d-thia-allo-isoleucine (ACtaI, 9) are not turned
over.[6] To investigate the failure of IPNS to turn over such sub-
strates, we have synthesised the tripeptide ACtaI 9, crystallised
it with IPNS, and solved the structure of the resulting complex.
Results and Discussion
Synthesis of ACtaI 9
ACtaI 9 was prepared from d-threonine (10) and protected di-
peptide 15 (Scheme 3). d-Threonine was converted to (2S,3R)-
Scheme 1. The reaction of IPNS with its natural substrate ACV (1) to give
IPN (2), via the high-valent iron intermediate 3. a) IPNS, FeII, O2.
methyl
3-(acetylthio)-2-(tert-butoxycarbonylamino)butanoate
(11) as reported by Gross and co-workers.[13] The key step in
this sequence is nucleophilic displacement of a tosylate leaving
group by thioacetate. The thioester of 11 was cleaved by trans-
esterification in alkaline methanol, and the resulting thiolate
reduces one molecule of oxygen to water while oxidizing ACV
to IPN. NHIOs are thought to generate highly reactive iron(IV)-
oxo (ferryl) species in their reaction cycles,[2,3] and there is
good evidence that the conversion of ACV to IPN proceeds via
an iron(IV)-oxo intermediate, 3.[4,5] IPNS has attracted much at-
tention over many years, due to the unique chemistry of the
reaction that it catalyses and the clinical significance of the
product that it forms.[1,5–9]
[a] Dr. I. J. Clifton, Dr. W. Ge, Dr. R. M. Adlington, Prof. Sir J. E. Baldwin
Chemistry Research Laboratory, University of Oxford
Mansfield Road, Oxford, OX1 3TA (UK)
E-mail: jack.baldwin@chem.ox.ac.uk
[b] Dr. P. J. Rutledge
School of Chemistry F11, The University of Sydney
NSW 2006 (Australia)
Fax: (+61)2-9351-3329
Solution-phase incubation studies show that IPNS tolerates
considerable variation in the third residue of its tripeptide sub-
strate ACV (1):[6] analogues incorporating a wide variety of
hydrocarbon side chains in place of the isopropyl group of d-
valine are turned over by IPNS. However IPNS is much less tol-
E-mail: peter.rutledge@sydney.edu.au
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100149.
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P. J. Rutledge et al.
aqueous HCl) afforded the amino acid 13 in 13% yield over six
steps from d-threonine. Protection of 13 as its benzhydryl
ester 14[14] and coupling to the previously reported dipeptide
15[15] gave the fully protected tripeptide 16. Global deprotec-
tion with trifluoroacetic acid[16] gave ACtaI 9 as its TFA salt,
from which the pure tripeptide was obtained by reversed-
phase HPLC.
Structure of the IPNS:FeII:ACtaI complex
Crystals of IPNS:FeII:ACtaI were grown according to the previ-
ously reported procedure.[17,18] The structure of the complex
was solved to 1.79 ꢀ resolution (Figures 1 and 2, Table 1). As is
to be expected, the overall structure of the protein is not sig-
nificantly different from that of the IPNS:FeII:ACV complex,[9]
and the substrate analogue ACtaI binds to the active-site
region in a similar orientation to the native substrate ACV and
other substrate analogues.[5,9,15,19–24] Thus, the tripeptide is
bound to iron through its cysteinyl thiolate, and tethered by
interactions between its amino and carboxylate groups and
the protein. The aminoadipoyl terminus forms a salt bridge
from its carboxylate to protein residue Arg87, and hydrogen
Scheme 2. A) The reaction of ACI (4) with IPNS gives penam 6, while its
epimer ACaI 5 gives the epimeric penam 7.[10,11] (ACI also gives rise to small
amounts of two epimeric 2-methyl-cepham products, in ca. 10% abundance
relative to penam 6.[12]); a) IPNS, FeII, O2. B) In contrast, the isosteric sulfur-
containing peptides ACtI (8) and ACtaI (9) are not turned over by IPNS.[6]
was trapped with methyl iodide to afford the methyl sulfide
12. Two-step deprotection (neat TFA followed by 2.5 molLꢀ1
Scheme 3. Synthesis of tripeptide 9. Reagents and conditions: a) CH3OH, HCl(g), 08C to RT, 23 h, 100%; b) (Boc)2O, Et3N, CH2Cl2, RT, 48 h, 77%; c) TsCl, DMAP,
pyridine, 0–48C, 48 h, 68%; d) CH3COSK, DMF, RT, 48 h, 78%; e) CH3OH, 0.2m NaOH(aq), CH3I, RT, 2 h, 67%; f) TFA, RT, 30 min, then 2.5m HCl(aq), reflux, 4 h, then
DOWEX ion exchange, 48%; g) i: TsOH·H2O, H2O, RT, 5 min; ii: Ph2CN2, CH3CN/Et2O, RT, 4 h, 86%; h) Et3N, EDCI, HOBt, CH2Cl2, RT, 48 h, 99%; i) TFA/anisole,
reflux, 60 min, quantitative, then RP-HPLC. For details of steps a–d, see ref. [13], for details of steps e–i, see the Supporting Information.
Figure 1. Stereo image showing the active site of the anaerobic IPNS:FeII:ACtaI complex. The large dark-grey sphere is iron(II); a 2mFoꢀDFc electron-density
map is shown around the substrate analogue at 1s. A colour version of this figure is available in the Supporting Information.
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Isopenicillin N Synthase Binding Mode
Figure 2. Stereo images showing close-ups of the iron binding environment in A) the IPNS:FeII:ACtaI complex, and B) the IPNS:FeII:ACV complex The large
dark-grey sphere is iron(II). A colour version of this figure is available in the Supporting Information.
bonds from its amino group to Thr331. At the other end of the
tripeptide, the thia-allo-isoleucine carboxylate is well posi-
tioned to hydrogen bond to several protein side chains, in par-
ticular Tyr189, Arg279 and Ser281.
ester (ACOmC).[26] The iron centre is also hexacoordinate in
IPNS complexes with less sterically demanding substrate ana-
logues such as AC-Gly, AC-d-Ala and AC-d-a-aminobuty-
rate,[27,28] in which the smaller side chain allows a water mole-
cule to bind iron in this site. Several lll-configured substrate
analogues have also recently been shown to bind in the IPNS
active site in a way that allows an additional water ligand at
iron, which is consequently hexacoordinate.[29–31]
However the active-site metal in the IPNS:FeII:ACtaI structure
is hexacoordinate, a marked contrast to the IPNS:FeII:ACV com-
plex in which iron is pentacoordinate. There are three ligands
from the protein, His214, Asp216 and His270 (the defining “2-
His-1-carboxylate” motif of enzymes in the NHIO family)[25] and
a single water molecule opposite His214, plus two ligands
from the substrate, the cysteinyl thiolate trans to His270, and
the methylsulfide opposite Asp216, coordinated to iron at a
distance of 2.66 ꢀ (compared to 2.40 ꢀ for the thiolate). In the
IPNS:FeII:ACV complex, the valinyl isopropyl group sits in van
der Waals contact with the metal, held by interactions with the
side chains of Leu231, Val272, Pro283 and Leu223.[9] The valinyl
isopropyl group thereby masks the site opposite Asp216, effec-
tively reserving it for the dioxygen cosubstrate to bind and ini-
tiate the oxidative reaction.
Conclusions
It is evident that the affinity of sulfur for iron overrides any
steric pressure caused by the branching methyl group on the
thia-allo-isoleucine side chain, such that this side chain can
twist as required to bring the second sulfur close to the metal.
The branching methyl group on the b-carbon of this residue
sits in a space that is occupied by the b-hydrogen atom of d-
valine in the IPNS:FeII:ACV complex.[9] The methyl sulfide side
chain of ACtaI 9 binds to iron in the putative oxygen binding
site opposite Asp216. Presumably the additional ligand locks
into this coordination site and blocks the cosubstrate from
binding, preventing reaction under normal turnover condi-
Sulfide ligation to iron has previously been observed in the
IPNS complexes with AC-d-S-methylcysteine (ACmC)[5] and d-l-
a-aminoadipoyl-l-cysteine
(1-(S)-carboxy-2-thiomethyl)ethyl
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P. J. Rutledge et al.
anaerobic environment and exchanged into cryoprotectant buffer
(a 1:1 mixture of well buffer and saturated lithium sulfate in 40%
glycerol, v/v), then flash-frozen in liquid nitrogen.
Table 1. X-ray data collection and crystallographic statistics.
X-ray source
SRS, Daresbury, UK
wavelength [ꢀ]
PDB ID
resolution [ꢀ]
space group
unit cell dimensions
a [ꢀ]
b [ꢀ]
1.488
2y6f
1.79
Data were collected at the Synchrotron Radiation Source (SRS),
Daresbury, UK, and the temperature was maintained at 100 K by
using an Oxford Cryosystems Cryostream. Data were processed by
using Denzo[33] and the CCP4 suite of programs,[34] then refined by
using REFMAC5[35] and Coot for model building.[36] Initial phases
were generated by using co-ordinates for the protein from the pre-
viously published IPNS:FeII:ACV structure,[9] and manual rebuilding
of protein side chains was performed as necessary. Crystallographic
coordinates and structure factors have been deposited in the Pro-
tein Data Bank under accession number 2y6f. Figures 1 and 2 were
prepared with CCP4mg.[37]
P212121
47.40
73.35
c [ꢀ]
102.48
19.9–1.79
55650
22362
91.3
7.4
11.8
15.2
17.80
resolution shell [ꢀ]
total number of reflections
number of unique reflections
completeness [%]
average I/s[I]
1.89–1.79
7838
3256
93.9
2.6
28.7
Rmerge [%][a]
Abbreviations: AC-=d-l-a-aminoadipoyl-l-cysteinyl-, ACtI=d-l-a-
aminoadipoyl-l-cysteinyl-d-thiaisoleucine, ACtaI=d-l-a-aminoadi-
poyl-l-cysteinyl-d-thia-allo-isoleucine, ACmC=d-l-a-aminoadipoyl-
l-cysteinyl-d-S-methylcysteine, ACOmC=d-l-a-aminoadipoyl-l-cys-
teinyl (1-(S)-carboxy-2-thiomethyl)ethyl ester, ACV=d-a-aminoadi-
poyl-cysteinyl-valine, DMAP=4-dimethylaminopyridine, EDCI=1-
(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, HOBt
=1-hydroxybenzotriazole hydrate, IPN=isopenicillin N, IPNS=iso-
penicillin N synthase, NHIO=non-heme iron(II) oxidase, TFA=tri-
fluoroacetic acid, Ts=tosyl.
Rmeas [%][b]
37.7
Rcryst [%][c]
Rfree [%][d]
22.01
RMS deviation[e]
average B factors [ꢀ2][f]
number of water molecules
0.033, 1.9
19.1, 22.1, 13.3, 12.9
296
pN¯ /¯ (Nꢀ1)
¯ ¯
¯
¯
[a] Rmerge =ꢀjꢀh jIh,jꢀ<Ih >j/ꢀjꢀh <Ih >ꢂ100; [b] Rmeas =ꢀhkl
ꢀi jIi
(hkl)ꢀI (hkl)j/ꢀhklꢀIIi(hkl)ꢂ100;[38,39] [c] Rcryst =ꢀjjFobs jꢀjFcalcd jj/ꢀjFobs jꢂ
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).
¯
¯ ¯ ¯¯
Acknowledgements
We thank Dr. Nicolai Burzlaff, Dr. Jon Elkins, Dr. Charles Hens-
gens, Dr. John Keeping, Dr. Peter Roach, Prof. Chris Schofield, and
the scientists at SRS Daresbury and DESY Hamburg for help and
discussions. Financial support was provided by the MRC, BBSRC
and EPSRC. P.J.R. thanks the Rhodes Trust for a scholarship.
tions. This result provides a structural basis to explain the fail-
ure of IPNS to turn over substrate analogues such as ACtI 8
and ACtaI 9 that include polar valine analogues in the third
position.
Keywords: antibiotics · biosynthesis · enzyme mechanisms ·
metalloenzymes · non-heme iron oxidase · penicillin
Experimental Section
Synthesis of d-(l-a-aminoadipoyl)-l-cysteinyl-d-thia-allo-isoleu-
cine 9: d-Threonine (10) was converted to d-thia-allo-isoleucine
((2S,3R)-2-amino-3-(methylthio)butanoic acid, 13) in six steps
(Scheme 3); for details of steps a–d see ref. [13], for details of
steps e and f see the Supporting Information. The amino acid 13
was protected as its benzhydryl ester 14 following Wolfe’s proto-
col[14] and coupled to known dipeptide 15[15] by using EDCI and
HOBt.[32] TFA-mediated deprotection[16] and HPLC purification (octa-
decylsilane 250ꢂ10 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 mLminꢀ1; l=254 nm, five absorbance units
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2H; CHCH2CH2CH2), 1.78–1.90 (m, 2H; CHCH2CH2CH2), 2.08 (s, 3H;
SCH3), 2.38 (t, J=7.0 Hz, 2H; CHCH2CH2CH2), 2.88 (A of ABX, JAB
14.0 Hz, JAX =7.0 Hz, 1H; 1 of CH2SH), 2.93 (B of ABX, JBA =14.0 Hz,
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=
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Received: March 6, 2011
Published online on June 15, 2011
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