The crystal structure of an LLL-configured depsipeptide substrate analogue bound to isopenicillin N synthase

Article abstract of DOI:10.1039/b910170e

Isopenicillin N synthase (IPNS) is a non-heme iron(II) oxidase, which catalyses the biosynthesis of isopenicillin N (IPN) from the tripeptide δ-L-α-aminoadipoyl-L-cysteinyl-D-valine (LLD-ACV) in a remarkable oxidative bicyclisation reaction. The natural substrate for IPNS is the LLD-configured tripeptide. LLL-ACV is not turned over by the enzyme, but inhibits turnover of the LLD-tripeptide. The mechanism by which this inhibition takes place is not fully understood. Recent studies have employed a range of LLD-configured depsipeptide substrate analogues in crystallographic studies to probe events preceding β-lactam closure in the IPNS reaction cycle. Herein, we report the first crystal structure of IPNS in complex with an LLL-configured depsipeptide analogue, δ-L-α-aminoadipoyl-L-cysteine (1-(-R)-carboxy-2-thiomethyl)ethyl ester (LLL-ACOmC). This report describes the crystal structure of the IPNS:Fe(II):LLL-ACOmC complex to 2.0 A resolution, and discusses attempts to oxygenate this complex at high pressure in order to probe the mechanism by which LLL-configured substrates inhibit IPNS catalysis. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b910170e

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www.rsc.org/obc | Organic & Biomolecular Chemistry
The crystal structure of an LLL-configured depsipeptide substrate analogue
bound to isopenicillin N synthase
Wei Ge,a Ian J. Clifton,a Jeanette E. Stok,a,c Robert M. Adlington,a Jack E. Baldwin*a and Peter J. Rutledge*b
Received 22nd May 2009, Accepted 29th September 2009
First published as an Advance Article on the web 29th October 2009
DOI: 10.1039/b910170e
Isopenicillin N synthase (IPNS) is a non-heme iron(II) oxidase, which catalyses the biosynthesis of
isopenicillin N (IPN) from the tripeptide d-L-a-aminoadipoyl-L-cysteinyl-D-valine (LLD-ACV) in a
remarkable oxidative bicyclisation reaction. The natural substrate for IPNS is the LLD-configured
tripeptide. LLL-ACV is not turned over by the enzyme, but inhibits turnover of the LLD-tripeptide. The
mechanism by which this inhibition takes place is not fully understood. Recent studies have employed a
range of LLD-configured depsipeptide substrate analogues in crystallographic studies to probe events
preceding b-lactam closure in the IPNS reaction cycle. Herein, we report the first crystal structure of
IPNS in complex with an LLL-configured depsipeptide analogue, d-L-a-aminoadipoyl-L-cysteine
(1-(R)-carboxy-2-thiomethyl)ethyl ester (LLL-ACOmC). This report describes the crystal structure of
˚
the IPNS:Fe(II):LLL-ACOmC complex to 2.0 A resolution, and discusses attempts to oxygenate this
complex at high pressure in order to probe the mechanism by which LLL-configured substrates inhibit
IPNS catalysis.
analogues with the enzyme,4 and X-ray crystallography.6–8 The
Introduction
consensus mechanism for non-heme iron-dependent oxidases
involves highly-reactive Fe(IV)-oxo (ferryl) intermediates,9–12 and
a range of evidence has been put forward for the involvement of
such a species in IPNS catalysis.6,13–16
Isopenicillin N synthase (IPNS) is a non-heme iron(II)-dependent
oxidase with an essential role in the biosynthesis of all penicillin
and cephalosporin antibiotics.1 IPNS mediates conversion of the
linear tripeptide d-L-a-aminoadipoyl-L-cysteinyl-D-valine (LLD-
ACV, 1) to bicyclic isopenicillin N (IPN, 2) in a single step
(Scheme 1). LLD-ACV is biosynthesised in bacteria and fungi from
the constituent amino acids L-a-aminoadipic acid, L-cysteine and
L-valine by the enzyme ACV synthetase (ACVS), a nonribosomal
peptide synthase (NRPS). In the course of the ACVS-mediated
condensation, the stereochemistry of the valine residue is inverted
to afford the LLD-tripeptide that is the natural substrate for
IPNS.2,3
The oxidative bicyclisation reaction mediated by IPNS is unique
in nature and of key commercial importance, and has been the
subject of extensive research interest over many years.1,4–6 Three
principal strategies have been used to investigate IPNS catalysis:
spectroscopic studies,5 solution-phase incubation of substrate
In 1979, Konomi et al. reported solution-phase incubation
studies using the all-L configured tripeptide LLL-ACV 3 (an epimer
of the native substrate 1; Fig. 1).17 This compound is not turned
over by IPNS, but inhibits turnover of the correctly configured
substrate LLD-ACV 1. It was assumed that LLL-ACV is not turned
over because it does not fit properly into the active site; however,
the exact mechanism of inhibition was not determined. In 2005
we reported the first crystal structure of IPNS with an LLL-
configured substrate, the tripeptide analogue d-L-a-aminoadipoyl-
L-cysteinyl-L-3,3,3,3¢,3¢,3¢-hexafluorovaline (LLL-AC6FV, 4; PDB
ID for IPNS : Fe(II):LLL-AC6FV complex: 2bu9).18
The structure of the IPNS:Fe(II):LLL-AC6FV complex revealed
that tripeptide 4 does in fact bind in the IPNS active site. It does
so with the L-aminoadipoyl and L-cysteinyl residues adopting very
similar conformations to those seen for LLD-configured substrates.
However, the altered stereochemistry of the third amino acid
orients its side-chain away from the iron and ‘up’ into a region
of the active site pocket occupied by four water molecules in
the IPNS:Fe(II):LLD-ACV complex. The carboxylate group of the
L-hexafluorovaline residue is oriented towards iron, and hydrogen-
bonded to an iron-bound water molecule and adjacent tyrosine
residue (Tyr189). As a result, the iron is ligated by an additional
aChemistry Research Laboratory, University of Oxford, Mansfield Road,
Oxford, UK OX1 3TA. E-mail: jack.baldwin@chem.ox.ac.uk; Fax: +44
1865 285002; Tel: +44 1865 275670
bSchool of Chemistry F11, The University of Sydney, NSW 2006, Australia.
E-mail: p.rutledge@chem.usyd.edu.au; Fax: +61 2 9351 3329; Tel: +61 2
9351 5020
cSchool of Chemistry and Molecular Biosciences, The University of Queens-
land, Brisbane, Queensland, 4072, Australia
Scheme 1 IPNS-mediated conversion of LLD-ACV 1 to IPN 2 using an iron(II) cofactor and molecular oxygen as cosubstrate.
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Fig. 1 a. ‘All-L’ substrate analogues studied previously LLL-ACV 317 and LLL-AC6FV 4;18 b. the epimeric depsipeptides LLD-ACOmC 5 and
LLL-ACOmC 6.
water ligand in the site opposite Asp216 (generally accepted to be
the oxygen binding site), held in place by a network of hydrogen
Results and discussion
bonds. It was proposed that this water ligand, held in place by
strong hydrogen bonding, blocks oxygen entry and inhibits the
oxidative turnover reaction before it can even begin.18
As part of ongoing studies using protein crystallography to
investigate the mechanism of IPNS catalysis,6,14,15,19–25 the sub-
strate analogue d-L-a-aminoadipoyl-L-cysteine (1-(R)-carboxy-2-
thiomethyl)ethyl ester (LLL-ACOmC, 6) was prepared in five steps
from L-a-aminoadipic acid derivative 9, S-p-methoxybenzyl-L-
cysteine 10 and tert-butyl acrylate 11 (Scheme 2).16
LLL-ACOmC 6 was crystallised with IPNS following the
previously reported procedure.26 Crystals of IPNS:Fe(II):LLL-
ACOmC were observed to form more rapidly and at a wider
range of conditions (well and drop buffer concentrations, and
concentrations of seed crystals added) than for IPNS:Fe(II):LLD-
ACOmC. Large plate-shaped crystals of IPNS:Fe(II):LLL-ACOmC
grew in ca. two days compared to ca. four days required for
IPNS:Fe(II):LLD-ACOmC.
The current study tests this hypothesis by crystallising IPNS
with another LLL-configured substrate analogue and attempting
to force oxygen into the crystalline complex at high pressure.
We recently reported co-crystallisation of the LLD-depsipeptide
d-L-a-aminoadipoyl-L-cysteine (1-(S)-carboxy-2-thiomethyl)ethyl
ester (LLD-ACOmC, 5) with IPNS and oxidative turnover of the
crystalline IPNS:Fe(II):LLD-ACOmC complex to an unexpected
sulfenate product.19 Herein, we detail the crystal structure of
IPNS complexed with the LLL-depsipeptide d-L-a-aminoadipoyl-
L-cysteine (1-(R)-carboxy-2-thiomethyl)ethyl ester (LLL-ACOmC,
6). (Note that compounds 5 and 6 are abbreviated as ACOmC
to highlight their structural relationship to the analogues d-(L-
a-aminoadipoyl)-L-cysteinyl-S-methyl-D-cysteine (LLD-ACmC)6
and d-(L-a-aminoadipoyl)-L-cysteine D-a-hydroxyisovaleryl ester
(LLD-ACOV).14)
The crystal structure the anaerobic IPNS:Fe(II):LLL-ACOmC
˚
complex was solved to 2.0 A resolution (Table 1), affording
the first structure of IPNS complexed with an LLL-configured
depsipeptide (Fig. 2a,b). In this structure, the L-a-aminoadipoyl
Scheme 2 Synthesis of LLL-ACOmC 6. i. 9, iso-butylchloroformate, Et3N, THF, -12 ◦C, 30 min, then 10, Et3N, H2O, 0 ◦C–RT, 90 min, 82%; ii. m-CPBA,
DCM, reflux, 48 h; iii. NaSCH3, NaHCO3, H2O, 0 ◦C, 1 h, 80% (over two steps) of a 1 : 2 mixture of 13 : 14; iv. PPh3, DEAD, THF, 0 ◦C, sonication
1 h, then RT 20 h, 63% of a 1 : 1 mixture of LLL : LLD-15; v. TFA, anisole, reflux, 30 min, then reversed phase HPLC, 43% of LLD-ACOmC 5 and 36% of
LLL-ACOmC 6.
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Table 1 X-Ray data collection and crystallographic statistics for
IPNS:Fe(II):LLL-ACOmC
It is interesting to note that the methylsulfide side-chain of LLL-
ACOmC 6 is accommodated satisfactorily in the region above
Tyr189, even without the potential for significant new hydrogen
bonding interactions to ameliorate the loss of those usually present
in this region. It was observed that several fluorine atoms of LLL-
AC6FV were well positioned to interact with Tyr189 and water
molecules in this area, and that the resulting hydrogen bonding
interactions would mitigate energy penalties incurred by loss of
the hydrogen-bonded network of water molecules from this area.18
In contrast, the methylsulfide side-chain of LLL-ACOmC 6 cannot
hydrogen bond to Tyr189 or adjacent water molecules in the same
way, but this residue is accommodated in this region nonetheless.
Two alternative explanations for the inhibitory effects of LLL-
configured substrates were put forward on the basis of the
IPNS:Fe(II):LLL-AC6FV crystal structure.18 The most likely ex-
planation is that hydrogen bonding between the hexafluorovalinyl
carboxylate, iron-bound water, Tyr189 and other water molecules
prevents dioxygen from displacing the metal-ligated water and
initiating turnover. Alternatively, it was postulated that oxygen
might still bind but then be unable to react because the cysteinyl
b-carbon and ‘valinyl’ nitrogen have moved in the LLL-AC6FV
X-Ray source
Wavelength/A
Rotating Anode, LMB, Oxford, UK
1.5418
˚
PDB acquisition code
Resolution/A
2vbd
2.0
˚
Space group
P212121
46.53, 71.31, 100.65
152 677
23 160
˚
Unit cell dimensions (a, b, c/A)
Total number of reflections
Number of unique reflections
Completeness (%)
97.7
Completeness in outer shell (%)
85.9
9.0
18.8
21.7
Rmerge (%)a
Rcryst (%)b
Rfree (%)c
RMS deviationd
Average B factors /A
Number of water molecules
0.007 (1.0)
13.4, 15.4, 19.1, 19.0
213
e
2
˚
ꢀ ꢀ
ꢀ ꢀ
ꢀ
b
a Rmerge
=
h|Ih,j - ꢀIhꢁ|/
hꢀIhꢁ ¥ 100. Rcryst
=
ꢂFobs| -
j
j
ꢀ
c
|Fcalcꢂ/ |Fobs| ¥ 100. Rfree = based on 5% of the total reflections.
d RMS deviation from ideality for bonds (followed by the value for angles).
e Average B factors in order: main chain; side chain; substrate and iron;
solvent.
˚
complex relative to LLD-ACV 1 (by 0.1 and 0.77 A respectively).
To test these alternative hypotheses, crystals of IPNS:Fe(II):LLL-
ACOmC were exposed to the co-substrate oxygen at high pressure
in an attempt to force oxygen binding and substrate turnover.
After exposure of the IPNS:Fe(II):LLL-ACOmC complex to high-
pressure oxygen for time spans equivalent to those required to
bring about turnover of LLD-depsipeptide analogues (20 bar for
1 min; 20 bar for 2 min; 40 bar for 10 min),14–16,19 the crystal
structure showed no change relative to the ‘time 0’ electron density
map (data not shown). Exposure to higher oxygen pressures for
longer times (40 bar for 60 min; 40 bar for 100 min; 40 bar
for 300 min) brought changes in the active site electron density
that indicate incomplete substrate occupancy—i.e. departure of
the substrate (or product(s)). However, these extremely forcing
conditions have been shown to bring about a range of non-specific
oxidative transformations in previous studies.6,20
and L-cysteinyl residues occupy equivalent positions to those
observed for LLD-ACV8 and complexes with other LLD-configured
substrates.6,14,15,19–24 LLL-ACOmC 6 is tethered by a salt bridge to
Arg87 through the L-a-aminoadipoyl carboxylate, and to iron by
the cysteinyl sulfur. The L-configured third residue occupies a very
different region of space from that observed with LLD-ACV 1
or LLD-ACOmC 5, as would be expected. The side-chain of the
third residue is positioned away from the active site metal and
above Tyr189, into a region occupied by four water molecules in
the IPNS:Fe(II):LLD-ACV complex.8 The OmC-carboxylate points
towards the iron and is linked by hydrogen bonding to a metal-
bound water molecule that is ligated in the site opposite Asp216.
The other oxygen atom of this carboxylate is hydrogen bonded to
the side-chain phenol of Tyr189.
This arrangement mirrors the binding of LLL-AC6FV 4 to
IPNS,18 which ligates to the protein through a similar hydrogen
bonding network around the carboxylate of its third residue
(Fig. 2c). A similar substrate conformation is also seen in com-
plexes of IPNS with the smaller tripeptides d-L-a-aminoadipoyl-
L-cysteinyl-glycine (LL-ACG, 7) and d-L-a-aminoadipoyl-L-
cysteinyl-D-alanine (LLD-ACA, 8; Fig. 3).23 The less sterically
demanding and less hydrophobic side-chains of these analogues
allow additional water molecules into the normally hydrophobic
region of the active site opposite Asp216, as now observed in
the IPNS complex with LLL-ACOmC 6. Furthermore, the glycinyl
carboxylate in the IPNS:Fe(II):LL-ACG complex is also linked by
hydrogen bonding to an additional water ligand at the iron centre.
Conclusion
Previous research has demonstrated that IPNS tolerates consid-
erable variation in the valinyl section of its substrate LLD-ACV 1,
and that many compounds variant at this position are efficiently
turned over by the enzyme.4 The IPNS active site appears well able
to accommodate—and react with—a large range of D-amino acids
in the binding pocket close to the iron centre. However, the crystal
structure of the IPNS:Fe(II):LLL-ACOmC complex reported here
confirms that for LLL-configured analogues such as LLL-ACOmC
6, the side-chain of the third residue cannot get near the active site
iron and sits instead above Tyr189. This leaves the iron unprotected
Fig. 3 LL-ACG 7 and LLD-ACA 8, less sterically demanding tripeptide substrates, which allow additional water molecules into the active site region
when bound to IPNS.23
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˚
Fig. 2 Active site of the anaerobic IPNS:Fe(II):LLL-ACOmC complex to 2.0 A resolution (PDB ID 2vbd). a. Overview of the whole active site region,
showing the full extent of LLL-ACOmC binding to the protein. b. Closer view of the third (L-OmC) residue of the depsipeptide substrate analogue showing
hydrogen bonding to Tyr189, Ser281 and an adjacent water molecule (A2213). c. Overlay figure showing superimposition of IPNS-bound LLL-ACOmC
with LLL-AC6FV (PDB ID 2bu9). The 2mFo–DFc electron density maps are shown in green and contoured at 1s.
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and liable to coordinate with an extra water ligand, which blocks
oxygen binding and turnover.4
CCP4 suite of programs.29 REFMAC5 was used to refine data,30
the program O to build models.31 The initial phases were generated
from the IPNS:Fe(II):ACV structure published previously8 by
molecular replacement. Manual rebuilding of side-chains was un-
dertaken where required. Crystallographic coordinates and struc-
ture factors have been deposited in the Worldwide Protein Data
Bank, accession number 2vbd. The programs MOLSCRIPT,32
BOBSCRIPT33 and Raster3D34 were used to prepare the colour
figure (Fig. 2).
This study demonstrates that although the LLL-ACOmC 6 binds
to IPNS and sits comfortably in the active site, the conformation
of the compound does not facilitate turnover by IPNS. When
LLD-ACV 1 binds to IPNS,8 it displaces the water molecule that is
ligated to iron opposite Asp216 in the ‘substrate free’ IPNS:Mn(II)
crystal structure.7 So, with LLD-ACV 1 anchored in the active
site, the iron is penta-coordinate. In contrast, the equivalent water
molecule remains bound to the IPNS:Fe(II):LLL-ACOmC complex
and appears to be locked into this site by hydrogen bonding.
This leaves the metal hexa-coordinate with a water molecule in
the oxygen binding site. The hydrogen bonding network appears
sufficiently robust to prevent oxygen from entering the active
site, so the turnover reaction is blocked before it can begin.
Furthermore, the observed hydrogen bonding around the OmC-
carboxylate potentially imparts greater stability to the complex
and explains why IPNS crystallises more readily with LLL-ACOmC
than with LLD-ACOmC 5. This may also explain why LLL-ACV
3 can compete with LLD-ACV 1 and act as an inhibitor of IPNS
catalysis.17
Abbreviations
AC6FV (d-a-aminoadipoyl-cysteinyl-3,3,3,3¢3¢3¢-
hexafluorovaline)
ACA
ACG
ACmC
(d-a-aminoadipoyl-cysteinyl-alanine)
(d-a-aminoadipoyl-cysteinyl-glycine)
(d-a-aminoadipoyl-cysteinyl-S-methyl-cysteine);
ACOmC (d-a-aminoadipoyl-cysteine (1-carboxy-2-thiomethyl)-
ethyl ester)
ACOV
ACV
IPN
(d-a-aminoadipoyl-cysteine a-hydroxyisovaleryl ester)
(d-a-aminoadipoyl-cysteinyl-valine)
(isopenicillin N)
IPNS
(isopenicillin N synthase)
Experimental
Acknowledgements
Synthesis of LLL-ACOmC 6
We thank Dr Annaleise Howard-Jones, Prof. Chris Schofield, Dr
Victor Lee, Dr Zhihong Zhang, Dr Edward Lowe and Dr Jing He
for help and discussions.
LLL-ACOmC 6 was prepared in five steps from the doubly-
protected L-a-aminoadipic acid derivative 9, S-p-methoxybenzyl-
L-cysteine 10 and tert-butylacrylate 11 (Scheme 2).16 The
crude depsipeptide product (a mixture of the LLL- and LLD-
diastereomers) was purified by reversed-phase HPLC (10 mM
NH4HCO3, Hypersil 5 m C18 column, 250 ¥ 10 mm internal
diameter; l = 254 nm; 4 mL min-1) to afford pure LLL-ACOmC
6 (Rt = 12.0 min) and LLD-ACOmC 5 (Rt = 13.5 min). Data for
6: dH (200 MHz, D2O) 1.50–1.70 (2H, m, NHCHCH2CH2CH2),
1.70–1.85 (2H, m, NHCHCH2CH2CH2), 2.07 (3H, s, SCH3), 2.31
(2H, t, J 7.0 Hz, NHCHCH2CH2CH2), 2.82–3.02 (4H, m, CH2SH
and CH2SCH3), 3.64 (1H, t, J 5.5 Hz, NH2CH L-AA), 4.63–4.72
(1H, m, NHCH L-Cys), 4.94 (1H, dd, J 7.0 Hz, 5.0 Hz, OCH
L-Omc); data for 5 is presented in ref. 16.
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