The Interaction of Isopenicillin N Synthase with Homologated Substrate Analogues δ-(L-α-Aminoadipoyl)-L-homocysteinyl-D-Xaa Characterised by Protein Crystallography

Article abstract of DOI:10.1002/cbic.201200728

Isopenicillin N synthase (IPNS) converts the linear tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) into bicyclic isopenicillin N (IPN) in the central step in the biosynthesis of penicillin and cephalosporin antibiotics. Solution-phase incubation experiments have shown that IPNS turns over analogues with a diverse range of side chains in the third (valinyl) position of the substrate, but copes less well with changes in the second (cysteinyl) residue. IPNS thus converts the homologated tripeptides δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-valine (AhCV) and δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-allylglycine (AhCaG) into monocyclic hydroxy-lactam products; this suggests that the additional methylene unit in these substrates induces conformational changes that preclude second ring closure after initial lactam formation. To investigate this and solution-phase results with other tripeptides δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-Xaa, we have crystallised AhCV and δ-(L-α-aminoadipoyl)-L-homocysteinyl-D-S-methylcysteine (AhCmC) with IPNS and solved crystal structures for the resulting complexes. The IPNS:Fe^II:AhCV complex shows diffuse electron density for several regions of the substrate, revealing considerable conformational freedom within the active site. The substrate is more clearly resolved in the IPNS:Fe^II:AhCmC complex, by virtue of thioether coordination to iron. AhCmC occupies two distinct conformations, both distorted relative to the natural substrate ACV, in order to accommodate the extra methylene group in the second residue. Attempts to turn these substrates over within crystalline IPNS using hyperbaric oxygenation give rise to product mixtures. Loose fit: IPNS catalyses the central step in penicillin biosynthesis. Substrate analogues containing L-homocysteine in place of the natural substrate's L-cysteine residue are not converted into bicyclic products. Crystal structures for IPNS complexes with two such analogues reveal that the additional CH₂ unit affords considerable conformational freedom when these analogues bind to IPNS. Copyright

Full text of DOI:10.1002/cbic.201200728

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DOI: 10.1002/cbic.201200728
The Interaction of Isopenicillin N Synthase with
Homologated Substrate Analogues d-(l-a-Aminoadipoyl)-
l-homocysteinyl-d-Xaa Characterised by Protein
Crystallography
Adam Daruzzaman,[a] Ian J. Clifton,[a] Robert M. Adlington,[a] Jack E. Baldwin,*[a] and
Peter J. Rutledge*[b]
Isopenicillin N synthase (IPNS) converts the linear tripeptide d-
(l-a-aminoadipoyl)-l-cysteinyl-d-valine (ACV) into bicyclic iso-
penicillin N (IPN) in the central step in the biosynthesis of peni-
cillin and cephalosporin antibiotics. Solution-phase incubation
experiments have shown that IPNS turns over analogues with
a diverse range of side chains in the third (valinyl) position of
the substrate, but copes less well with changes in the second
(cysteinyl) residue. IPNS thus converts the homologated tripep-
tides d-(l-a-aminoadipoyl)-l-homocysteinyl-d-valine (AhCV)
tion-phase results with other tripeptides d-(l-a-aminoadipoyl)-
l-homocysteinyl-d-Xaa, we have crystallised AhCV and d-(l-a-
aminoadipoyl)-l-homocysteinyl-d-S-methylcysteine
(AhCmC)
with IPNS and solved crystal structures for the resulting com-
plexes. The IPNS:FeII:AhCV complex shows diffuse electron
density for several regions of the substrate, revealing consider-
able conformational freedom within the active site. The sub-
strate is more clearly resolved in the IPNS:FeII:AhCmC complex,
by virtue of thioether coordination to iron. AhCmC occupies
two distinct conformations, both distorted relative to the natu-
ral substrate ACV, in order to accommodate the extra methyl-
ene group in the second residue. Attempts to turn these sub-
strates over within crystalline IPNS using hyperbaric oxygena-
tion give rise to product mixtures.
and
d-(l-a-aminoadipoyl)-l-homocysteinyl-d-allylglycine
(AhCaG) into monocyclic hydroxy-lactam products; this sug-
gests that the additional methylene unit in these substrates in-
duces conformational changes that preclude second ring clo-
sure after initial lactam formation. To investigate this and solu-
Introduction
Isopenicillin N synthase (IPNS) is a non-heme iron enzyme that
catalyses conversion of d-(l-a-aminoadipoyl)-l-cysteinyl-d-
valine (ACV, 1, Scheme 1) into isopenicillin N (IPN, 2).[1] This
enzyme and its reaction behaviour have been studied exten-
sively, in a wide range of spectroscopic,[2–4] mechanistic,[1,5] the-
oretical[6,7] and crystallographic experiments.[8–10] It is generally
agreed that IPNS catalysis proceeds via high-valent iron-oxo in-
termediate 3.[10,11]
Of the many and various analogues of ACV (1) to have been
subjected to reaction with IPNS,[5] substrates in which the
second residue (l-cysteine) is replaced by l-homocysteine re-
vealed a particularly rich vein of unexpected oxidative chemis-
try.[12–14] The reaction of d-(l-a-aminoadipoyl)-l-homocysteinyl-
d-valine (AhCV, 4, Scheme 2) was first studied to probe the
substrate specificity of IPNS with regard to the central residue
of its substrate, and in the hope of shedding light on the then
putative monocyclic intermediate 3.[12] Incubation of 4 with
IPNS yielded the epimeric g-lactam alcohols 5 (Scheme 2),
which were shown to epimerise in aqueous solution at pH 7.5.
The corresponding reaction with d-(l-a-aminoadipoyl)-l-homo-
cysteinyl-d-allylglycine (AhCaG) gave an analogous g-lactam al-
cohol product.[12]
Repeating the reaction of 4 with IPNS under 18O2 revealed
significant 18O incorporation into the hydroxy group of the
product 5 (although levels of incorporation were lower than
seen with other hydroxylated products of IPNS incubations,
presumably because the 5-OH is readily exchanged via an acyl
iminium ion intermediate). Incubation of monodeuterated pep-
tides in which either Ha or Hb is stereospecifically replaced by
D gave the partially 5-deuterated g-lactam; levels of deuterium
loss were consistent with a degree of stereospecificity akin to
that observed during conversion of ACV (1) into IPN (2): “ca.
80% of the maximum theoretical deuterium loss expected for
a fully stereospecific event at the 4-position of the homocys-
teinyl residue” was observed with the deuterated analogues of
4.[12] A mechanism was proposed for formation of 5, through
initial formation of the monocyclic g-lactam 6, homologue of
the ACV-derived b-lactam 3. However, it seems that the
iron(IV)-oxo unit in 6 cannot execute hydrogen abstraction
from the valinyl b-carbon, for steric and/or stereoelectronic
reasons. Instead, the high-valent intermediate is quenched by
[a] Dr. A. Daruzzaman, Dr. I. J. Clifton, Dr. R. M. Adlington,
Prof. Sir J. E. Baldwin
Chemistry Research Laboratory, University of Oxford
Mansfield Rd, 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
Sydney, NSW 2006 (Australia)
E-mail: peter.rutledge@sydney.edu.au
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Scheme 1. IPNS converts the linear tripeptide ACV (1) into bicyclic IPN (2); the reaction is generally agreed to proceed via the monocyclic b-lactam intermedi-
ate 3.
Scheme 2. The reaction of AhCV (4) with IPNS gave the hydroxy g-lactam 5, presumably via monocyclic intermediate 6.
other means, leaving the enzyme-bound g-lactam to collapse
to an acyl iminium ion, which is hydrolysed to the observed
product 5. The alternative means of iron(IV)-oxo quenching
and the end fate of the sulfur atom were not resolved.
conditions), suggesting that 10 is the sole product of the enzy-
matic reaction.
Crystallography has significantly enhanced our understand-
ing of IPNS catalysis.[8–10,15] Crystal structures of IPNS with its
natural substrate and analogues have elucidated different
modes of substrate and analogue binding, and revealed the in-
fluence of active site geometry on reactivity.[9,16–21] The applica-
tion of hyperbaric oxygenation to trigger reaction within crys-
talline IPNS has rendered further detail, allowing the enzyme
mechanism to be studied step-by-step within the crystalline
protein.[10,22–26]
Attempts to answer these questions with further incubation
experiments raised more questions. d-(l-a-Aminoadipoyl)-l-ho-
mocysteinyl-d-cysteine (7) was converted into the bicyclic g-
lactam disulfide 8 by IPNS (Scheme 3A); this suggests FeꢀS
Here we report the results of a crystallographic investigation
into the reaction of IPNS with AhCV (4). Using protein crystal-
lography we have characterised the binding of AhCV (4) and
methylsulfide analogue d-(l-a-aminoadipoyl)-l-homocysteinyl-
d-S-methylcysteine (AhCmC, 12, Scheme 4) to IPNS.
Results and Discussion
Synthesis of tripeptides
AhCV (4) and AhCmC (12) were prepared from the constituent
amino acids l-a-aminoadipic acid (13), l-homocystine (14) and
d-valine or d-S-methylcysteine (Scheme 4). l-a-Aminoadipic
acid (13) was converted into the doubly-protected deriva-
tive 15 in good yield (55% over two steps) as previously re-
ported.[23] The disulfide 14 was reductively cleaved with
sodium and liquid ammonia by the du Vigneaud procedure[27]
and protected in situ to give the PMB thioether 16 in high
yield (83% over two steps). Isobutyl chloroformate-mediated
coupling of acid 15 and amine 16 gave dipeptide 17 in excel-
lent yield (90%). d-Valine and d-S-methylcysteine (obtained
from d-cystine as reported by du Vigneaud)[27] were converted
into the corresponding benzhydryl esters 18 and 19, respec-
tively,[28] and were then coupled to dipeptide 17 by diimide
methodology to afford the fully protected tripeptides 20 and
21. Global deprotection with TFA[29] gave AhCV (4) or AhCmC
(12) as their TFA salts, from which the pure tripeptides were
Scheme 3. IPNS converts: A) AhCC (7) into bicyclic disulfide 8, and B) fluori-
nated analogue 9 into thiocarboxylate 10 and carboxylate 11 products. l-
AA=d-(l-a-aminoadipoyl).
bond cleavage by the terminal cysteine thiolate group in an in-
termediate akin to 6.[13] The fluorinated analogue d-(l-a-amino-
adipoyl)-l-3,3-difluorohomocysteinyl-d-valine (9) was designed
to stabilise the enzyme-bound intermediate corresponding to
6 and prevent iminium ion formation; incubation of 9 with
IPNS gave thiocarboxylic acid 10 and carboxylic acid 11 in
a ratio of >5:1.[14] The purified thiocarboxylate 10 was shown
to decompose to carboxylate 11 at pH 7.5 (IPNS incubation
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Scheme 4. Synthesis of tripeptide analogues 4 and 12; a) NaOH, (Boc)2O, H2O/tBuOH, RT, 92%; b) PMBCl, Et3N, DMF, 388C, 60%; c) Na, NH3(l), ꢀ788C; d) PMBCl,
ꢀ788C to RT, 83% (over two steps); e) isobutyl chloroformate, Et3N, THF/H2O, 08C, 90%; f) 18 or 19, EDCI, HOBt, Et3N, CH2Cl2, RT, 66% (20)/46% (21); g) TFA,
anisole, reflux, RP-HPLC 46% (4)/12% (12); l-AA=d-(l-a-aminoadipoyl). Insert: the cyclic thioether (S)-2-amino-6-oxo-6-[((S)-2-oxotetrahydrothiophen-3-yl)ami-
no]hexanoic acid (22) was isolated as the major product from TFA-mediated deprotection of 21.[21]
obtained by reversed-phase HPLC. Deprotection of the S-meth-
ylcysteine derivative 21 also gave rise to a significant quantity
of the cyclic thioester 22,[21] which is presumably formed
through thiol-mediated cleavage of the amide bond under the
acidic conditions of the deprotection reaction.
fined. Regions of the tripeptide that are tethered to the pro-
tein—the aminoadipoyl amino and a-carboxylate groups, the
homocysteinyl thiolate group and the valinyl carboxylate
group—are clearly resolved. However the electron density de-
fining other parts of the substrate is less clear, evincing confor-
mational freedom and multiple occupancy in these regions
(which include the aminoadipoyl g-, d- and e-centres, the ho-
mocysteinyl N, carbonyl, a-, b- and g-carbons, and the valinyl
Ca and side chain). There is also evidence for partial occupan-
cy by a second water ligand at iron, in the binding site trans to
Asp216.
The structure of the IPNS:FeII:AhCV complex
The crystal structure of the IPNS:FeII:AhCV complex was solved
to 1.40 ꢁ resolution (Figure 1, Table 1). The structure of the
protein is generally well defined, and closely mirrors the struc-
IPNS:FeII:substrate
analogue
com-
It appears that AhCV (4) occupies the IPNS active site as
a mixture of several conformers, but that two of these pre-
dominate; these are discussed as conformations A and B in
more detail below. Conformation A is a skeletal arrangement
close to that observed for the natural substrate ACV (1):[9] iron
tures
of
other
plexes.[9,16–18,30,31] The active site iron is bound by the familiar
triad of protein side chains (His214, Asp216 and His270),
a water ligand opposite His214 and the homocysteinyl thiolate
group opposite His270. The substrate AhCV (4) is less well de-
Figure 1. Stereoview of the active site of the IPNS:FeII:AhCV complex, showing electron density as: 2mFoꢀDfc density[43] at 1s (cyan) and mFoꢀDfc density at
ꢁ3s [green (+3s) and magenta (ꢀ3s)], where s is the RMS value of each map over an asymmetric unit. The figure was generated using CCP4mg.[44]
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dipoyl and valinyl regions as well. The conformational freedom
is also translated to the protein itself, in the eight residues at
the protein C terminus (Leu324–Thr331): regions of all these
residues are poorly defined in the electron density map, indi-
cating multiple occupancy/conformational freedom. Mobility in
the C terminus has been linked to substrate binding and prod-
uct departure from the IPNS active site.[23] In the IPNS:FeII:AhCV
complex it appears that the conformational changes required
to accommodate the additional methylene group in the vicini-
ty of the side chain of Leu324 destabilise the conformation of
the whole protein tail relative to other anaerobic IPNS–sub-
strate complexes.
Table 1. Crystallographic data for IPNS:FeII:AhCV and IPNS:FeII:AhCmC
IPNS:FeII:AhCV
IPNS:FeII:AhCmC
X-ray source
ESRF beamline
ID14EH2
0.933
ESRF beamline
ID14EH1
0.934
wavelength [ꢁ]
PDB ID
3zku
3zky
resolution [ꢁ]
space group
1.40
P212121
1.45
P212121
unit cell dimensions a, b, c [ꢁ] 46.48, 71.10, 101.05 46.71, 71.29, 100.92
resolution shell [ꢁ]
total number of reflections
41.0–1.40 1.43–1.40 23.0–1.45 1.53–1.45
293826 10334 296581 34273
number of unique reflections 66426
3106
89.1
4.6
25.1
30.2
16.4
60499
99.9
13.7
7.8
8.6
3.4
8691
99.7
6
25.6
29.5
14.5
completeness [%]
average I/s(I)
99.4
22.6
5.1
5.7
2.5
Rmerge [%][a]
The structure of the anaerobic IPNS:FeII:AhCmC complex
Rmeas [%][41]
Rpim [%][42]
Rcryst [%][b]
16.2
18.3
0.022 ꢁ (2.08)
13.2, 16.1, 23.1, 23.9 11.5, 14.1, 17.2, 21.0
273 259
16.5
18.2
0.024 ꢁ (2.38)
The thioether analogue AhCmC (12) was conceived so as to
reduce some of the conformational freedom seen with AhCV
(4). It was envisaged that coordination of the S-methylcysteinyl
sulfur to iron would provide an extra anchor relative to AhCV
(4) because thioether ligation to iron in the IPNS active site is
well precedented.[10,25,32,33]
The crystal structure of the IPNS:FeII:AhCmC complex was
solved to 1.45 ꢁ (Figure 2, Table 1). As in the case of the AhCV
complex, the overall structure of the protein is well defined,
and mirrors other IPNS–tripeptide complexes.[9,16–18,30,31] Iron is
bound by the His214, Asp216 and His270 side chains, a water
ligand opposite His214 and the homocysteinyl thiolate.
Rfree [%][c]
RMS deviation[d]
average B factors [ꢁ2][e]
number of water molecules
[a] Rmerge =ꢀjꢀh jIh,jꢀhIhij/ꢀjꢀhhIhiꢂ100.
[b] Rcryst =ꢀj jFobs jꢀjFcalcd j j/ꢀjFobs j ꢂ100.
[c] Rfree =based on 5% of the total reflections. [d] RMS deviation from
ideal for bonds (followed by the value for angles). [e] Average B factors in
order: main chain, side chain, substrate and iron, solvent (water).
is pentacoordinate, bound by the side chains of His214,
Asp216, His270, the homocysteinyl thiolate group and the
water ligand opposite His214, and the valinyl isopropyl group
sits in the binding site trans to Asp 216. In conformation B, the
homocysteinyl carbonyl group is rotated approximately 608
“back” towards Phe211 (“back” relative to conformation A),
taking up the position of the cysteinyl-derived carbonyl group
in the IPNS:FeII:IPN and exposed IPNS:FeII:ACmC complexes.[10]
In those complexes the observed carbonyl rotation is the
result of oxidative cyclisation (i.e., b-lactam formation), which
cannot have occurred in the anaerobic IPNS:FeII:AhCV com-
plex; we propose instead that carbonyl rotation in the AhCV
complex reflects the conformational freedom of this region of
the substrate. Rotation of the homocysteinyl carbonyl towards
Phe211 moves the valinyl isopropyl group away from the iron
binding site opposite Asp216. This allows a water ligand to co-
ordinate iron in that position, and iron is hexacoordinate. As
a result, the valinyl isopropyl is rotated approximately 308 rela-
tive to conformation A, occupying a position analogous to that
observed in the IPNS:FeII:ACV:NO complex[9] and IPNS:-
FeII:thiocarboxylate complex derived from d-(l-a-aminoadipo-
yl)-l-cysteinyl d-a-hydroxyisovaleryl ester (ACOV).[22]
Relative to ACV (1), AhCV (4) includes an additional methyl-
ene unit between Ca and the side-chain thiol of its second res-
idue. IPNS can accommodate this change, and binds AhCV
well enough. However the price is greater conformational free-
dom for the enzyme-bound substrate, and some compromis-
ing of the tripeptide’s ability to reserve the iron-binding site
opposite Asp216 for the cosubstrate oxygen to bind. Distor-
tion at the homocysteinyl residue is translated along the sub-
strate backbone, and affects conformation of both the aminoa-
The analogue AhCmC (12) is indeed better defined in the
electron density map than AhCV (4), as anticipated. AhCmC
(12) occupies the active site in two conformations in a 1:1
ratio, discussed below as conformation A [similar to the ACV-
like conformation A seen with AhCV (4)] and conformation C
(different from either of the principal conformations occupied
by 4). In both conformations the substrate is tethered in the
active site by enzyme–substrate interactions similar to those
previously observed with ACV and other substrate analogues:
through the thiolate sulfur to iron, through a salt bridge to the
aminoadipoyl amino group, and with hydrogen bonds to the
carboxylate group of the third residue (S-methylcysteine).[9,22]
In addition, the S-methylcysteinyl sulfur also coordinates to
iron, as expected. The thioether sulfur sits 2.63 ꢁ from the
metal, a distance similar to those seen previously with d-(l-a-
aminoadipoyl)-l-cysteinyl-d-S-methylcysteine (ACmC, 2.69 ꢁ),[10]
d-(l-a-aminoadipoyl)-l-cysteinyl-d-thioisoleucine
(ACtI,
2.66 ꢁ)[32] and d-(l-a-aminoadipoyl)-l-cysteinyl-d-methionine
(ACM, 2.57 ꢁ).[33] As a consequence the metal is hexacoordi-
nate, in contrast to the IPNS:FeII:ACV and IPNS:FeII:AmCOV
complexes[9,22] and to conformation A of the IPNS:FeII:AhCV
complex. This additional link between AhCmC (12) and the
protein affixes this tripeptide much more rigidly in the IPNS
active site than AhCV (4) and limits the conformational free-
dom otherwise imparted by the substitution of homocysteine
for cysteine.
Conformation A of AhCmC (12, Figure 2) has a skeletal ar-
rangement akin to those observed with ACV,[9] ACOV[22] and
conformation A of AhCV (Figure 1). The S-methyl group is ac-
commodated in the valinyl pocket with the methyl group
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Figure 2. Stereoview of the active site of the IPNS:FeII:AhCmC complex, with electron density shown in cyan as 2mFoꢀDfc density[43] at 1s (where s is the
RMS value of the map over an asymmetric unit). The figure was generated by using CCP4mg.[44]
pointing between the side chains of Leu231 and Leu223, as
previously observed in the IPNS:FeII:ACmC complex.[10] The g-
methylene group of the homocysteinyl residue sits “under” the
homocysteinyl a-carbon in a position analogous to that of the
cysteinyl b-carbon atom in the ACV complex; this forces the
homocysteinyl b-carbon atom to sit “behind” the substrate
backbone pointing towards Phe211.
preserved. The l-AA Cb thus swings approximately 1808 rela-
tive to conformation A, through rotation of the CgꢀCd bond
towards Val185 and the CaꢀCb bond in an opposite sense.
This reorganisation forces the CgꢀCd bond of the l-AA residue
from a staggered to an eclipsed conformation (Figure 2).
The position of the S-methylcysteine residue is similar in
both conformations, but the iron–sulfur distances for the thio-
late and sulfide groups are significantly different in either case.
The hCys thiolate group is 2.06 ꢁ from iron in conformation A
but 2.55 ꢁ in conformation C; the mCys sulfide is 2.63 ꢁ from
the metal in conformation A, but only 2.41 ꢁ away in confor-
In conformation C, the system finds alternative means to ac-
commodate the additional methylene group of homocysteine.
The S-methylcysteinyl residue has limited freedom to move, by
virtue of the FeꢀS(CH3) linkage, so conformational change is
forced on the aminoadipoyl residue. Rotation about the C1ꢀ mation C. This reflects the overall shift in the position of the
Ca, CaꢀCb and CaꢀN bonds of homocysteine (hCys) bring the
hCys Cb towards Phe285 while maintaining its position
“behind” the substrate backbone. This repositions the hCys Cg
from “below” hCys Ca to “below” the hCys N (“below” in the
orientation shown in Figure 2). This change in the orientation
of hCys Cg shunts the whole hCys-mCys portion of the tripep-
tide approximately 0.50 ꢁ in the direction of the aminoadipoyl
carboxylate and Arg87. The aminoadipoyl (l-AA) chain moves
to accommodate these changes without compromising its
links to the protein—in particular, the salt bridge to Arg87 is
substrate towards Arg87 in conformation C relative to confor-
mation A.
Conclusions
The crystal structures of the IPNS:FeII:AhCV and IPNS:-
FeII:AhCmC complexes evince the conformational flexibility of
the second (homocysteinyl) residue in these substrates, relative
to the shorter cysteinyl side chain in the native substrate ACV
(1, Figure 3). The additional thioether tether to iron from
Figure 3. Comparison of substrate binding to iron in the complexes of IPNS with A) ACV (1, PDB ID: 1bk0), B) AhCV (4, PDB ID: 3zku), and C) AhCmC (12, PDB
ID: 3zky). The figure was generated by using CCP4mg.[44]
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8.5 Hz, 2H; 2CHAr), 6.88 (d, J=9.0 Hz, 2H; 2CHAr) 7.22 (d, J=8.5 Hz,
2H; 2CHAr), 7.26–7.41 ppm (12H; 12CHAr); 13C NMR (100.6 MHz,
CDCl3): d=17.2, 19.0, 21.2, 27.0, 28.2, 30.1, 31.8, 35.1, 35.3, 52.0,
52.9, 55.1, 55.2, 57.1, 66.8, 77.8, 79.8, 113.8, 126.8, 127.3, 127.4,
127.9, 128.1, 128.4, 129.8, 130.1, 139.3, 139.5, 155.4, 158.5, 159.6,
170.5, 171.0, 172.4, 172.6 ppm; IR (KBr disc): n˜max =3302, 2964 (m),
1742, 1715, 1643, 1514, 1249, 1174 cmꢀ1; MS (APCI+): m/z: 884 (6)
[M+H]+; HRMS: m/z calcd for C49H61N3O10SNa: 906.3975 [M+Na]+;
found: 906.3947.
AhCmC (12) counteracts this flexibility to some extent, such
that this substrate analogue occupies two distinct conforma-
tions. AhCV (4), without this additional anchor point, is even
more mobile in the enzyme active site.
It is presumably this conformational fluidity that underpins
the failure of monocyclic intermediate 6 to undergo the
second ring closure (Scheme 2). We propose that the addition-
al methylene group of the homocysteinyl residue forces subtle
reorganisation of the active site such that the valinyl side chain
is oriented away from the iron(IV)-oxo species in 6. This would
prevent reaction at valine Cb (as occurs with ACV) and favour
an alternative reaction pathway, leading to dissociation from
the protein with loss of the homocysteinyl sulfur atom. At-
tempts to trap and to characterise intermediate 6 directly were
unsuccessful: exposing crystals of either complex (IPNS:-
FeII:AhCV or IPNS:FeII:AhCmC) to high pressures of oxygen gas
(20 bar) for various time periods (5 min to 24 h) returned only
complex electron density maps that could not be resolved into
coherent product structures (data not shown). Nonetheless,
the crystal structures of the anaerobic IPNS:FeII:AhCV and
IPNS:FeII:AhCmC complexes allow a better understanding of
the reactions of IPNS with ACV analogues that incorporate ho-
mocysteine derivatives at the second position.
N-tert-Butoxycarbonyl-a-para-methoxybenzyl-d-(l-a-aminoadipoyl)-S-
para-methoxybenzyl-l-homocysteinyl-S-methyl-d-cysteine benzhydryl
ester (21): Triethylamine (0.45 mL, 3.3 mmol) was added to a stirred
solution of N-tert-butoxycarbonyl-a-para-methoxybenzyl-d-(l-a-
aminoadipoyl)-S-para-methoxybenzyl-l-homocysteine (17, 1.01 g,
1.6 mmol), S-methyl-d-cysteine benzhydryl ester para-toluenesulfo-
nate salt (19, 0.77 g, 1.6 mmol), EDCI (0.31 g, 1.6 mmol) and HOBt
(0.22 g, 1.6 mmol) in CH2Cl2 (20 mL). The reaction mixture was
stirred at room temperature for two days. Further CH2Cl2 (20 mL)
was then added, and the mixture was washed with water (20 mL),
dilute HCl (1m, 20 mL) and finally water (20 mL). The CH2Cl2 solu-
tion was concentrated in vacuo, and the residue was dissolved in
ethyl acetate (20 mL). The ethyl acetate solution was washed with
saturated aqueous sodium bicarbonate (10 mL), water (10 mL) and
saturated brine (10 mL), dried over magnesium sulfate and concen-
trated in vacuo. The crude product was purified by column chro-
matography (petroleum ether/ethyl acetate 80:20) to give 21 as
a sticky white solid. Yield: 0.68 g (46%); m.p. 73–748C; Rf =0.15
1
(CH2Cl2/ethyl acetate 90:10); [a]2D4 =+0.76 (c=0.53, CHCl3); H NMR
Experimental Section
(500 MHz, CDCl3): d=1.42 (s, 9H; (CH3)3C), 1.50–1.71 (m, 3H; 2
from NHCHCH2CH2CH2, 1 from NHCHCH2CH2CH2), 1.72–1.84 (m,
1H; 1of NHCHCH2CH2CH2), 1.85–1.97 (overlapping m and s: m, 1H;
1 from CHCH2CH2SCH2Ar; s, 3H; CH2SCH3), 2.05–2.17 (3 overlapping
Synthesis of tripeptide substrates
N-tert-Butoxycarbonyl-a-para-methoxybenzyl-d-(l-a-aminoadipoyl)-S-
para-methoxybenzyl-l-homocysteinyl-d-valine benzhydryl ester (20):
Triethylamine (0.37 mL, 2.7 mmol) was added to a stirred solution
of N-tert-butoxycarbonyl-a-para-methoxybenzyl-d-(l-a-aminoadi-
poyl)-S-para-methoxybenzyl-l-homocysteine (17, 0.82 g, 1.3 mmol),
d-valine benzhydryl ester para-toluenesulfonate salt (18, 0.61 g,
1.3 mmol), EDCI (0.26 g, 1.3 mmol) and HOBt (0.18 g, 1.3 mmol) in
CH2Cl2 (20 mL). The reaction mixture was stirred for two days. After
the stirring, further CH2Cl2 (20 mL) was added to the reaction mix-
ture, which was then washed with water (20 mL), dilute HCl (1m,
20 mL), and water again (20 mL). The CH2Cl2 solution was then
concentrated in vacuo, and the residue was dissolved in ethyl ace-
tate (20 mL). The solution was washed with saturated aqueous
sodium bicarbonate (10 mL), water (10 mL), and saturated brine
(10 mL), dried over magnesium sulfate and concentrated in vacuo.
The crude product was purified by column chromatography (petro-
leum ether/ethyl acetate 80:20) to give 20 as a sticky white solid.
Yield: 0.78 g (66%); m.p. 36–378C; Rf =0.15 (CH2Cl2/ethyl acetate
90:10); [a]2D4 =+3.05 (c=0.53, CHCl3); 1H NMR (400 MHz, CDCl3):
d=077, 0.87 (2d, J=6.9 Hz, 6H; (CH3)2CH), 1.42 (s, 9H; (CH3)3C),
m: m, 1H;
1
from CHCH2CH2SCH2Ar; m, 1H;
1
from
NHCHCH2CH2CH2; m, 1H; 1 from NHCHCH2CH2CH2), 2.39–2.52 (m,
2H; CHCH2CH2SCH2Ar), 2.87 (A of ABX, JAB =14.0 Hz, JAX =6.5 Hz,
1H; 1 from CHCH2SCH3), 3.00 (B of ABX, JBA =14.0 Hz, JBX =5.0 Hz,
1H; 1 from CHCH2SCH3), 3.68 (s, 2H; SCH2Ar), 3.78, 3.80 (2s, 6H; 3
from CH3OArCH2S and 3 from CH3OArCH2O), 4.23–4.33 (brm, 1H;
NHCHCH2CH2CH2), 4.58–4.67 (m, 1H; CHCH2CH2SCH2Ar), 4.81–4.86
(m, X of ABX, 1H; CHCH2SCH3), 5.08 (A of AB, JAB =12.0 Hz, 1H;
1 from OCH2Ar), 5.11 (B of AB, JBA =12.0 Hz, 1H; 1 from OCH2Ar),
5.19 (d, J=8.0 Hz, 1H; NHCHCH2CH2CH2), 6.25 (d, J=8.0 Hz, 1H;
NHCHCH2CH2), 6.83 (d, J=8.5 Hz, 2H; 2CHAr), 6.88 (d, J=8.5 Hz,
2H; 2CHAr), 7.02 (d, J=7.5 Hz, 1H; NHCHCH2SCH3), 7.22 (d, J=8.5,
2H; 2CHAr), 7.26–7.41 ppm (m, 12H; 12CHAr); 13C NMR (125.7 MHz,
CDCl3): d=15.8, 21.2, 27.0, 28.2, 31.1, 31.8, 35.1, 35.2, 35.9, 51.6,
51.8, 52.9, 55.2, 66.8, 78.4, 79.8, 113.8, 127.0, 127.3, 128.1, 128.2,
128.5, 129.8, 129.9, 130.1, 139.0, 139.1, 155.4, 158.5, 159.6, 169.5,
170.8, 172.4 ppm; IR (KBr disc): n˜max =3300, 2964, 1742, 1715, 1643,
1514, 1249, 1174 cmꢀ1; MS (APCI+): m/z: 902 (3) [M+H]+; HRMS:
m/z calcd for C48H59N3O10S2Na: 924.3540 [M+Na]+; found:
924.3533.
1.52–1.70 (m, 3H;
2
from NHCHCH2CH2CH2,
1
from
NHCHCH2CH2CH2), 1.78–1.89 (2 overlapping m: m, 1H; 1 from
NHCHCH2CH2CH2; m, 1H; 1 from CHCH2CH2SCH2Ar), 2.08–2.24 (3
overlapping m: m, 2H; 1 from CHCH2CH2SCH2Ar and 1 from
d-(l-a-Aminoadipoyl)-l-homocysteinyl-d-valine (4): N-tert-Butoxycar-
bonyl-a-para-methoxybenzyl-d-(l-a-aminoadipoyl)-S-para-methoxy-
benzyl-l-homocysteinyl-d-valine benzhydryl ester (20, 0.11 g,
0.12 mmol) was dissolved in TFA (5.0 mL). Anisole was added
(0.60 mL, 5.5 mmol), and the reaction mixture was heated to reflux
at 80–908C under argon with stirring. The reaction mixture was
heated under reflux for 30 min and was then allowed to cool to
room temperature, concentrated in vacuo and azeotroped with tol-
uene (2ꢂ10 mL). The crude residue was dissolved in water (20 mL),
and the resulting solution was washed with ethyl acetate (2ꢂ
NHCHCH2CH2CH2; m, 1H;
1 from NHCHCH2CH2CH2; m, 1H;
(CH3)2CH), 2.35–2.52 (m, 2H; CHCH2CH2SCH2Ar), 3.67 (A of AB, JAB
=
14.5 Hz, 1H; 1 from SCH2Ar), 3.70 (B of AB, JBA =14.0 Hz, 1H;
1 from SCH2Ar), 3.78, 3.80 (2s, 6H; CH3OArCH2S and CH3OArCH2O),
4.22–4.35 (brm, 1H; NHCHCH2CH2CH2), 4.60 (X of ABX, JXA =8.5 Hz,
J
XB =4.5 Hz, 1H; CHCH2CH2SCH2Ar), 5.08 (A of AB, JAB =12.0 Hz, 1H;
1 from OCH2Ar), 5.11 (B of AB, JBA =12.0 Hz, 1H; 1 from OCH2Ar),
5.21 (d, J=8.0 Hz, 1H; NHCHCH2CH2CH2), 6.21 (d, J=8.0 Hz, 1H;
NHCHCH2CH2), 6.81 (d, J=8.0 Hz, 1H; NHCHCH(CH3)2), 6.84 (d, J=
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 599 – 606 604

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10 mL). The combined organic phases were extracted with water
(2ꢂ10 mL), and the pooled aqueous phases were back-extracted
with ethyl acetate (10 mL). The combined aqueous extracts were
lyophilised to give a yellow crusty solid. The crude product was pu-
rified by reversed-phase HPLC (C18, 250ꢂ10 mm; ammonium bi-
carbonate (10 mm in H2O/MeOH) as eluent, running time: 0–6 min
0% MeOH, 6–13 min 25% MeOH, 13–20 min 0% MeOH; v/v,
4 mLmin-1) to yield 4 as a fluffy white solid. Yield: 12 mg (26%);
tR =9 min 30 s; 1H NMR (500 MHz, CDCl3): d=0.77, 0.81 (2d, J=
7.0 Hz, 6H; (CH3)2CH), 1.52–1.69 (m, 2H; NHCHCH2CH2CH2), 1.71–
1.86 (m, 2H; NHCHCH2CH2CH2), 1.95–2.01 (2 overlapping m: m, 2H;
PDB ID: 3kzu (AhCV structure) and PDB ID: 3kzy (AhCmC structure).
Figures 1 and 2 were prepared using CCP4mg.[40]
Abbreviations
AC-: d-(l-a-aminoadipoyl)-l-cysteinyl-, ACmC: d-(l-a-aminoadi-
poyl)-l-cysteinyl-d-S-methylcysteine, ACOmC: d-(l-a-aminoadi-
poyl)-l-cysteinyl (1-(S)-carboxy-2-thiomethyl)ethyl ester, ACtI:
d-(l-a-aminoadipoyl)-l-cysteinyl-d-thiaisoleucine, ACV: d-(l-a-
aminoadipoyl)-l-cysteinyl-d-valine, AhCV: d-(l-a-aminoadipoyl)-
l-homocysteinyl-d-valine, AhCaG: d-(l-a-aminoadipoyl)-l-ho-
mocysteinyl-d-allylglycine, AhCmC: d-(l-a-aminoadipoyl)>-l-
homocysteinyl-d-S-methylcysteine, APCI: atmospheric pressure
chemical ionisation, EDCI: 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride, HOBt: 1-hydroxybenzotriazole hy-
drate, IPN: isopenicillin N, IPNS: isopenicillin N synthase, PMB:
para-methoxybenzyl, TFA: trifluoroacetic acid.
CHCH2CH2SH;
m,
1H;
(CH3)2CH),
2.25–2.35
(m,
2H;
NHCHCH2CH2CH2), 2.42–2.60 (m, 2H; CHCH2CH2SH), 3.65 (t, J=
6.0 Hz, 1H; NHCHCH2CH2CH2), 4.01 (d, J=6.0 Hz, 1H; (CH3)2CHCH),
4.48 ppm (X of ABX, JXA =9.0 Hz, JXB =5.5 Hz, 1H; CHCH2CH2SH);
HRMS: m/z calcd for C15H26N3O6S: 376.1542 [MꢀH]ꢀ; found:
376.1538.
d-(l-a-Aminoadipoyl)-l-homocysteinyl-d-S-methyl-cysteine (12): N-
tert-Butoxycarbonyl-a-para-methoxybenzyl-d-(l-a-aminoadipoyl)-S-
para-methoxybenzyl-l-homocysteinyl-S-methyl-d-cysteine benzhy-
dryl ester (21, 0.09g, 0.10mmol) was dissolved in TFA (4.0 mL). Ani- Acknowledgements
sole was added (0.50mL, 4.6mmol), and the reaction mixture was
heated to reflux at 80–908C under argon with stirring. The mixture
We thank Dr. Nicolai Burzlaff, Dr. Jon Elkins, Professor Chris Scho-
was heated at reflux for 30min, and was then allowed to cool to
room temperature, concentrated in vacuo and azeotroped with tol-
uene (2ꢂ10mL). The crude residue was dissolved in water (20mL),
and the aqueous solution was washed with ethyl acetate (2ꢂ
10mL). The combined organic phases were extracted with water
(2ꢂ10mL), and the pooled aqueous phases were back-extracted
with ethyl acetate (10mL). The combined aqueous extracts were
lyophilised to give a yellow crusty solid. The crude product was pu-
rified by reversed-phase HPLC (see above) to yield 12 as a fluffy
field, and the scientists at the ESRF in Grenoble for help and dis-
cussions. Financial support was provided by the MRC, BBSRC and
EPSRC.
Keywords: antibiotics · biosynthesis · enzyme mechanisms ·
lactams · metalloenzymes · non-heme iron enzymes
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white solid. Yield: 4.8mg (12%); tR =5 min 40 s; H NMR (500 MHz,
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NHCHCH2CH2CH2), 1.90–2.05 (overlapping s and m: s, 3H; CH2SCH3;
m, 2H; CHCH2CH2SH), 2.31 (t, J=7.5 Hz, 2H; NHCHCH2CH2CH2),
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2.43–2.62 (m, 2H; CHCH2CH2SH), 2.75 (A of ABX, JAB =14.0 Hz, JAX
8.0 Hz, 1H; 1 from CH2SMe), 2.93 (B of ABX, JBA =14.0 Hz, JBX
=
=
4.5 Hz, 1H;
1
from CH2SMe), 3.65 (t, J=6.0 Hz, 1H;
NHCHCH2CH2CH2), 4.29 (X of ABX, JXA =8.0 Hz, JXB =4.5 Hz, 1H;
CHCH2SMe), 4.48 ppm (X of ABX, JXA =9.5 Hz, JXB =5.0 Hz, 1H;
CHCH2CH2SH); HRMS: m/z calcd for C14H24N3O6S2: 394.1107
[MꢀH]ꢀ; found 394.1103.
Crystallography and structure determination: Crystals of the
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liquid nitrogen.
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Data were collected at the European Synchrotron Radiation Source
(ESRF), Grenoble, France, with use of an Oxford Cryosystems Cryo-
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have been deposited in the Worldwide Protein Data Bank, under
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Received: November 21, 2012
Published online on March 7, 2013
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ChemBioChem 2013, 14, 599 – 606 606