Crystallographic studies on the binding of selectively deuterated LLD- and LLL-substrate epimers by isopenicillin N synthase

Article abstract of DOI:10.1016/j.bbrc.2010.06.129

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). Herein we report crystallographic studies to investigate the bi

Full text of DOI:10.1016/j.bbrc.2010.06.129

Biochemical and Biophysical Research Communications 398 (2010) 659–664
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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Crystallographic studies on the binding of selectively deuterated LLD- and
LLL-substrate epimers by isopenicillin N synthase
b,
Wei Ge a, Ian J. Clifton a, Jeanette E. Stok a,1, 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:
Received 19 June 2010
Available online 13 July 2010
Isopenicillin N synthase (IPNS) is a non-heme iron(II) oxidase which catalyses the biosynthesis of isopen-
icillin N (IPN) from the tripeptide d- -aminoadipoyl- -cysteinyl- -valine (LLD-ACV). Herein we report
crystallographic studies to investigate the binding of a truncated LLL-substrate in the active site of IPNS.
L
-a
L
D
Two epimeric tripeptides have been prepared by solution phase peptide synthesis and crystallised with
Keywords:
Antibiotics
Biosynthesis
Enzyme mechanism
Metalloenzymes
Non-heme iron oxidase
Penicillin
the enzyme. d-
same configuration as the natural substrate LLD-ACV at each of its three stereocentres; its epimer d-
aminoadipoyl- -cysteinyl-
at its third amino acid. LLL-ACV has previously been shown to inhibit IPNS turnover of its substrate LLD
ACV; the all-protiated tripeptide d- -aminoadipoyl- -cysteinyl-
L-a-Aminoadipoyl-L-cysteinyl-D-2-amino-3,3-dideuteriobutyrate (LLD-ACd2Ab) has the
L-
a
-
L
L-2-amino-3,3-dideuteriobutyrate (LLL-ACd2Ab) has the opposite configuration
-
L
-a
L
D
-2-aminobutyrate (LLD-ACAb) is a sub-
strate for IPNS, being turned over to a mixture of penam and cepham products. Comparisons between the
crystal structures of the IPNS:Fe(II):LLD-ACd2Ab and IPNS:Fe(II):LLL-ACd2Ab complexes offer a possible
rationale for the previously observed inhibitory effects of LLL-ACV on IPNS activity.
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
of b-lactam antibiotics for the clinic. The IPNS mechanism has been
investigated primarily using solution-phase incubation experi-
Isopenicillin N synthase (IPNS) is a non-heme iron(II) oxidase
(NHIO) central to the biosynthesis of penicillin and cephalosporin
antibiotics [1]. IPNS catalyses the oxidative bicyclisation of the lin-
ments [6], solution-phase spectroscopy [7], and protein crystallog-
raphy [8–14]. Incubation experiments in solution and in crystallo
involve synthesizing analogues of ACV and incubating them with
IPNS, then deducing mechanistic information by analysing the
incubation products formed using either NMR for solution-phase
experiments or crystallography for solid-phase experiments.
ear tripeptide d-L-a-aminoadipoyl-L-cysteinyl-D-valine (LLD-ACV, 1)
to isopenicillin N (IPN, 2) (Scheme 1A). This unusual transforma-
tion takes place in a single step via a putative iron(IV)-oxo interme-
diate [2,3], with one molecule of oxygen fully reduced to two
molecules of water in the process [1]. It is broadly accepted that
non-heme iron oxidases generate highly reactive iron(IV)-oxo (fer-
ryl) species in their reaction cycles [4,5].
IPNS has been studied extensively over a number of years
[1,6,7], on account of the unique transformation that it catalyses
and the importance of this reaction to the industrial preparation
Reaction of the truncated analogue d-(
teinyl- -aminobutyrate (LLD-ACAb 3) with IPNS gave three prod-
ucts: the epimeric C2-methyl penams -methyl 4 and b-methyl 5,
plus cepham 6 (Scheme 1B) in a 1:7:3 product ratio ( -methyl pe-
nam:b-methyl penam:cepham) [15,16]. LLD-ACAb analogues in
which the -aminobutyrate residue is stereospecifically deuterated
L-a-aminoadipoyl)-L-cys-
D-a
a
a
a
at its b-carbon have been incubated with IPNS, and intriguingly both
isomers give the same b-methyl penam product 7 (Scheme 1B) [17].
In contrast IPN formation from ACV is highly stereoselective with
respect to thiazolidine closure [18], presumably because the bulk
of the isopropyl side-chain restricts its ability to rotate within the
IPNS active site [2,19,20]. b-Methyl penam and cepham natural
Abbreviations: AC6FV, d-
line; ACA, d- -aminoadipoyl-cysteinyl-alanine; ACd2Ab, d-
teinyl-2-amino-3,3-dideuteriobutyrate; ACG, d- -aminoadipoyl-cysteinyl-glycine;
ACOmC, d- -aminoadipoyl-cysteine (1-carboxy-2-thiomethyl)ethyl ester; ACV,
d- -aminoadipoyl-cysteinyl-valine; IPN, isopenicillin N; IPNS, isopenicillin
a
-aminoadipoyl-cysteinyl-3,3,3,30,30,30-hexafluorova-
-aminoadipoyl-cys-
a
a
a
a
products similar to 5 and 6 but bearing a D-a-aminoadipoyl side-
a
N
chain have been isolated from fermentations of Streptomyces ACC
13285 [21], and are presumably formed by IPNS-mediated cycliza-
tion of ACAb followed by side-chain epimerisation [1,21].
ACAb 3 lacks one methyl group relative to ACV 1 – the a-amin-
obutryate residue presents an ethyl group in place of the isopropyl
synthase; NHIO, non-heme iron(II) oxidase.
* Corresponding author. Fax: +44 1865 285002.
** Corresponding author. Fax: +61 2 9351 3329.
E-mail addresses: jack.baldwin@chem.ox.ac.uk (J.E. Baldwin), peter.rutledge@
sydney.edu.au (P.J. Rutledge).
1
Present address: School of Chemistry and Molecular Biosciences, The University
group of valine – and it has been postulated that the penam:
of Queensland, Brisbane, Queensland 4072, Australia.
0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.06.129

660
W. Ge et al. / Biochemical and Biophysical Research Communications 398 (2010) 659–664
Scheme 1. (A) The reaction of IPNS with its natural substrate LLD-ACV 1 to give IPN 2; (B) IPNS-mediated turnover of LLD-ACAb 3 gives a mixture of two epimeric penams 4 and
5 plus the cepham 6; (C) the selectively deuterated ACAb analogues used in this study LLD-ACd2Ab 10 and LLL-ACd2Ab 11.
LLD-ACd2Ab 10 and d-L-a-aminoadipoyl-L-cysteinyl-L-2-amino-3,3-
dideuteriobutyrate (LLL-ACd2Ab 11).
cepham product ratio observed with ACAb is controlled by the rel-
ative energy barriers to reaction of the iron(IV)-oxo intermediate
with primary and secondary CAH bonds [22,23]. Compare the
structures of the putative intermediates 8 and 9 formed in the
reaction of IPNS with ACV 1 and ACAb 3 respectively (Scheme 1):
the iron(II)-oxo moiety must distinguish between primary and ter-
tiary CAH bonds in the first case, but between primary and second-
ary in the second. Crystallographic experiments and modelling
studies based on the crystal structures have provided a structural
rationale for the diminished product selectivity shown by IPNS in
reaction with ACAb 3 and deuterated isotopomers [24].
2. Materials and methods
2.1. Synthesis of LLD-ACd2Ab 10 and LLL-ACd2Ab 11
Commercially available 1,1-dideutereio ethanol 12 was treated
with sodium hydride and p-toluenesulfonyl chloride to afford the
corresponding O-p-toluenesulfonate, which was used to alkylate
diethyl acetamidomalonate forming 13. Acid-mediated deprotec-
tion and decarboxylation gave the free amino acid, which was pro-
tected as its benzhydryl ester 14 by reaction with p-toluenesulfonic
acid and diphenyldiazomethane [25]. Coupling of 14 with known
dipeptide 15 [20] was achieved using EDCI and HOBt in anhydrous
dichloromethane to give the fully protected tripeptide 16 as a mix-
ture of diastereoisomers [26]. Compound 16 was deprotected by
heating in refluxing TFA for 30 min, in the presence of anisole as a
cation scavenger [27]. The resultant crude mixture of the LLD- and
LLL-tripeptides was purified by reversed phase HPLC (10 mM
The selectively di-deuterated substrate analogue d-
L-a-amin-
oadipoyl- -cysteinyl- -2-amino-3,3-dideuteriobutyrate (LLD-AC-
L
D
d2Ab 10) was conceived of as a mechanistic trap to probe the
reaction of IPNS with ACAb 3 and isotopomers: by replacing both
b-hydrogen atoms with deuterium it was envisaged that the
resulting kinetic isotope effect would slow reaction at the b-posi-
tion of ACAb, increasing reaction at the c-carbon to probe cepham
formation from these substrates. However attempts to trap inter-
mediates in the reaction cycle of LLD-ACd2Ab 10 with IPNS in crys-
tallo have not been successful, returning only complex mixtures of
products at the enzyme active site. Thus attention has turned in-
stead to the stereochemistry of the third amino acid in this trun-
cated analogue, and we report herein crystal structures of IPNS
in complex with the epimeric, truncated substrate analogues
NH4HCO3, Hypersil 5
l
C18 column, 250 ꢀ 10 mm internal diame-
ter, k = 254 nm; 4 mL minꢁ1
)
to afford pure LLL-ACd2Ab 11
(Rt = 11.3 min) and LLD-ACd2Ab 10 (Rt = 11.8 min) in a 1:1 ratio.
Stereochemical assignment of the two isomers was deduced by

W. Ge et al. / Biochemical and Biophysical Research Communications 398 (2010) 659–664
661
Fig. 1. (A) The active site of anaerobic IPNS:Fe(II):LLD-ACd2Ab complex; (B) the active site of anaerobic IPNS:Fe(II):LLL-ACd2Ab complex; (C) overlay showing the relationship
between the active site regions of the LLL-ACd2Ab and LLD-ACd2Ab complexes. A 2mFo–DFc map electron density map is shown in blue at 1
r in (A) and (B); the red sphere is
iron (II).
analogy with related tripeptides, and confirmed by the X-ray crystal
structures of their respective complexes with IPNS.
microscope, then removed from the anaerobic environment, ex-
changed into a cryoprotectant solution (1:1 mixture of well buffer:
saturated lithium sulfate in 40% v/v glycerol solution), and flash-
frozen in liquid nitrogen [28,29].
2.2. Crystallization experiments
Crystals of the IPNS:Fe(II):LLD-ACd2Ab and IPNS:Fe(II):LLL-AC-
d2Ab complexes were grown under anaerobic conditions as re-
ported previously with minor modification [28,29]. Crystals
suitable for structure determination were selected using a light
2.3. Data collection and structure determination
Data were collected at the Synchrotron Radiation Source
(SRS), Daresbury, UK at 100 K, with temperature control achieved

662
W. Ge et al. / Biochemical and Biophysical Research Communications 398 (2010) 659–664
through the use of an Oxford Cryosystems Cryostream. Data pro-
cessing was carried out using MOSFLM [30] and programs from
the CCP4 suite [31]. Refinement was undertaken with REFMAC5
[32], and the program O was used for model building [33]. Initial
phases were generated by molecular replacement, using co-ordi-
nates for the protein from the IPNS:Fe(II):ACV structure published
previously [19], and manual rebuilding of protein side-chains was
performed as necessary. Crystallographic coordinates and struc-
ture factors have been deposited in the Worldwide Protein data
Bank, accession numbers 2vbp for LLL-complex, 2wo7 for LLD. The
colour (Fig. 1) was prepared using the program CCP4mg [34].
the natural substrate [35]. It was proposed at the time and in sub-
sequent work with the LLL-configured substrates d- -amin-
oadipoyl- -cysteinyl-
-3,3,3,30,30,30-hexafluorovaline (LLL-AC6FV 18)
[36] and d- -aminoadipoyl- -cysteine (1-(R)-carboxy-2-thio-
methyl)ethyl ester (LLL-ACOmC 19) [37] that LLL-substrates cannot
be turned over by IPNS because they do not fit into the active site
properly.
The novel substrate analogues LLD-ACd2Ab 10 and LLL-ACd2Ab 11
were prepared using solution phase peptide synthesis (Scheme 2)
and crystallised with IPNS. Plate-shaped crystals of IPNS:-
Fe(II):LLD-ACd2Ab 10 and IPNS:Fe(II):LLL-ACd2Ab 11 were obtained
using standard conditions [28], and the structures of the IPNS:-
Fe(II):LLD-ACd2Ab and IPNS:Fe(II):LLL-ACd2Ab complexes were
solved to 2.50 Å and 1.50 Å resolution respectively (Fig. 1, Table 1).
The crystal structure of IPNS:Fe(II):LLD-ACd2Ab (Fig. 1A) mirrors
the IPNS:Fe(II):LLD-ACAb structure published previously, as is to
be expected [24]. The substrate is bound in the active site by
L
-a
L
L
L
-a
L
3. Results and discussion
It has been reported previously that LLL-ACV 17 – epimeric to
the natural substrate LLD-ACV in the configuration of its third resi-
due (valine) – is not turned over by IPNS, but inhibits turnover of
Scheme 2. (A) Synthesis of tripeptides 10 and 11. Reagents and conditions: (i) TsCl, NaH, rt, 68%; (ii) diethyl acetamidomalonate, Na, EtOH, 42%; (iii) HBr, 49%; (iv) TsOH, H2O,
then Ph2CN2, MeCN, 50%; (v) EDCL, HOBt, Et3N, 48%; (vi) TFA, anisole, reversed phase HPLC, 20%; (B) ‘all
L’ ACV analogues previously studied: LLL-ACV 17 [35], LLL-AC6FV 18,
[36] and LLL-ACOmC 19 [37].
Table 1
X-ray data collection and crystallographic statistics for IPNS:Fe(II):LLL-ACd2Ab and IPNS:Fe(II): LLD-ACd2Ab.
IPNS:Fe(II):LLL-ACd2Ab
SRS, Daresbury, UK
1.488
2vbp
IPNS:Fe(II):LLD-ACd2Ab
SRS, Daresbury, UK
1.488
X-ray source
Wavelength (Å)
PDB acquisition code
2wo7
Resolution (Å)
1.50
2.50
Space group
P212121
P212121
Unit cell dimensions (a Å, b Å, c Å)
40.51, 74.34, 100.70
41.29, 74.76, 101.13
Resolution shell (Å)
29.05–1.50
288 319
44 669
90.8
1.58–1.50
30 679
5345
76.1
41.50–2.50
50 629
11 384
98.8
2.64–2.50
6815
1635
98.7
Total number of reflections
Number of unique reflections
Completeness (%)
Average I/
r
(I)
21.9
5.3
5.8
2.2
7.8
20.9
23.1
9.6
6.0
20.8
23.4
10.5
2.4
58.9
66.8
30.7
Rmerge (%)a
Rmeas (%)b
Rpim (%)c
Rcryst (%)d
Rfree (%)e
22.3
26.8
0.021; 1.9
18.9
25.7
RMS deviationf
0.013; 1.432
17.1; 17.9; 18.0; 13.4
304
Average B factors (Å2)g
Number of water molecules
12.1; 14.8; 20.1; 27.1
305
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 [38,39].
hkl
i
hkl
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
P
P
Rpim
¼
1=ðN ꢁ 1Þ jIiðhklÞ ꢁ IðhklÞj=
iIiðhklÞ ꢀ 100 [39].
hkl
i
hkl
P
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).
g
Average B factors in order: main chain; side-chain; substrate and iron; solvent and sulfate.

W. Ge et al. / Biochemical and Biophysical Research Communications 398 (2010) 659–664
663
coordination of the
L-cysteinyl thiol to iron and a salt bridge be-
Acknowledgments
tween the -aminoadipoyl carboxylate and Arg87. The dideute-
L-a
ro-ethyl side-chain of the third residue (d2Ab) is oriented directly
towards iron, into the putative oxygen binding site opposite
Asp216. However the metal is hexacoordinate, with two water
molecules coordinated to it, versus the single aquo ligand in the
IPNS:Fe(II):LLD-ACV complex [19]: the smaller size of the aminobu-
tyrate side-chain allows a water molecule to bind opposite Asp216,
where the valinyl isopropyl group excludes water from this site
when ACV 1 binds. It is important to note that LLD-ACAb and isotop-
omers are turned over by IPNS [15–17], so the water molecule
bound opposite Asp216 does not tightly lock this site nor exclude
oxygen binding and turnover.
We thank Dr Annaleise Howard-Jones, Prof. Chris Schofield,
Dr Victor Lee, Dr Zhihong Zhang, Dr Karl Harlos, Dr Jing He and
the scientists at SRS daresbury for help and discussions.
References
[1] J.E. Baldwin, C.J. Schofield, The biosynthesis of b-lactams, in: M.I. Page (Ed.),
The Chemistry of b-Lactams, Blackie, Glasgow, 1992, pp. 1–78.
[2] N.I. Burzlaff, P.J. Rutledge, I.J. Clifton, C.M.H. Hensgens, M. Pickford, R.M.
Adlington, P.L. Roach, J.E. Baldwin, The reaction cycle of isopenicillin
synthase observed by X-ray diffraction, Nature 401 (1999) 721–724.
N
[3] J.E. Baldwin, Recent advances in the chemistry of the b-lactam antibiotics, in:
A.G. Brown, S.M. Roberts (Eds.), Special Publication No. 52, The Royal Society of
Chemistry, London, 1985, pp. 62–85.
The IPNS:Fe(II):LLL-ACd2Ab structure (Fig. 1B) is similar to the
[4] M. Costas, M.P. Mehn, M.P. Jensen, L. Que, Dioxygen activation at mononuclear
nonheme iron active sites: enzymes, models, and intermediates, Chem. Rev.
104 (2004) 939–986.
[5] P.C.A. Bruijnincx, G. van Koten, R. Gebbink, Mononuclear non-heme iron
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[6] J.E. Baldwin, M. Bradley, Isopenicillin N synthase: mechanistic studies, Chem.
Rev. 90 (1990) 1079–1088.
LLD-ACd2Ab complex around its
residues (the -aminoadipoyl side-chain is tethered by the salt
bridge to Arg87 and the -cysteinyl sulfur to iron), but substantially
different in the region of the third residue (d2Ab). The -d2Ab side-
chain points away from iron, into a region of space near Tyr189
that is occupied by water molecules in the IPNS:Fe(II):LLD-ACd2Ab
L-a-aminoadipoyl and L-cysteinyl
L
-a
L
L
and IPNS:Fe(II):LLD-ACV structures. The
ented towards the metal, and hydrogen bonds to a water ligand
in the iron binding site opposite Asp216. The dideutero-ethyl
L
-d2Ab carboxylate is ori-
[7] R.D.G. Cooper, The enzymes involved in biosynthesis of penicillin and
cephalosporin; their structure and function, Bioorg. Med. Chem. 1 (1993) 1–17.
[8] P.L. Roach, I.J. Clifton, V. Fulop, K. Harlos, G.J. Barton, J. Hajdu, I. Andersson, C.J.
Schofield, J.E. Baldwin, Crystal structure of isopenicillin N synthase is the first
from a new structural family of enzymes, Nature 375 (1995) 700–704.
[9] J.M. Ogle, I.J. Clifton, P.J. Rutledge, J.M. Elkins, N.I. Burzlaff, R.M. Adlington, P.L.
Roach, J.E. Baldwin, Alternative oxidation by isopenicillin N synthase observed
by X-ray diffraction, Chem. Biol. 8 (2001) 1231–1237.
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elimination of hydrogen fluoride from a fluorinated substrate analogue by
isopenicillin N synthase, Biochem. J. 382 (2004) 659–666.
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Unexpected oxidation of a depsipeptide substrate analogue in crystalline
isopenicillin N synthase, ChemBioChem 7 (2006) 351–358.
side-chain of L-d2Ab is comfortably accommodated in the region
above Tyr189 that has previously been shown to accept the hexa-
fluoro-isopropyl side-chain of 18 and the methylsulfide of 19
[36,37] even without the capacity for new hydrogen bonding or
other interactions to compensate for the loss of hydrogen bonded
water molecules usually present in this region.
Smaller tripeptides such as d-
L
-a
-aminoadipoyl-
L-cysteinyl-gly-
cine (LL-ACG) and d- -aminoadipoyl-
L
-a
L-cysteinyl-
D-alanine (LLD-
[12] A.R. Howard-Jones, J.M. Elkins, I.J. Clifton, P.L. Roach, R.M. Adlington, J.E.
ACA) demonstrate a similar conformation when bound in the IPNS
active site, with the carboxylate of their third residue oriented to-
wards iron [40]. The smaller, less hydrophobic nature of these side-
chains allows extra water molecules into the region opposite
Asp216, and the glycinyl carboxylate of ACG is also tied by hydro-
gen bonding to a water ligand at the iron centre.
Baldwin, P.J. Rutledge, Interactions of isopenicillin
N synthase with
cyclopropyl-containing substrate analogues reveal new mechanistic insight,
Biochemistry 46 (2007) 4755–4762.
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Isopenicillin
N synthase mediates thiolate oxidation to sulfonate in a
depsipeptide substrate analogue: implications for oxygen binding and a link
to nitrile hydratase?, J Am. Chem. Soc. 130 (2008) 10096–10102.
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Rutledge, Structural studies on the reaction of isopenicillin N synthase with a
sterically demanding depsipeptide substrate analogue, ChemBioChem 10
(2009) 2025–2031.
4. Conclusion
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antibiotics, J. Am. Chem. Soc. 103 (1981) 7650–7651.
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J.J. Usher, Penicillin biosynthesis. On the stereochemistry of carbon–sulphur
bond formation with modified substrates, J. Chem. Soc. Chem. Commun.
(1983) 1319–1320.
[18] J.E. Baldwin, R.M. Adlington, B.P. Domayne-Hayman, H.-H. Ting, N.J. Turner,
Stereospecificity of carbon–sulphur bond formation in penicillin biosynthesis,
J. Chem. Soc. Chem. Commun. 11 (1986) 110–113.
[19] P.L. Roach, I.J. Clifton, C.M.H. Hensgens, N. Shibata, C.J. Schofield, J. Hadju, J.E.
Baldwin, Structure of isopenicillin N synthase complexed with substrate and
the mechanism of penicillin formation, Nature 387 (1997) 827–830.
[20] J.M. Elkins, P.J. Rutledge, N.I. Burzlaff, I.J. Clifton, R.M. Adlington, P.L. Roach, J.E.
Baldwin, Crystallographic studies on the reaction of isopenicillin N synthase
with an unsaturated substrate analogue, Org. Biomol. Chem. 1 (2003) 1455–
1460.
[21] D.C. Aldridge, D.M. Carr, D.H. Davies, A.J. Hudson, R.D. Nolan, J.P. Poyser, C.J.
Strawson, Antibiotics 13285-A1 and 13285-A2 – novel cepham and penam
metabolites from a Streptomyces species, J. Chem. Soc. Chem. Commun. (1985)
1513–1514.
It has been shown previously that IPNS can tolerate great varia-
tion in the third residue of its substrate ACV 1, reacting with a wide
range of D-configured amino acids in the valinyl binding pocket [6].
Numerous analogues bearing a diverse range of side-chains in place
of the valinyl isopropyl group are turned over by IPNS, including
substrates that incorporate smaller side-chains (e.g. LL-ACG, LLD
ACA and LLD-ACAb discussed above), more sterically demanding
alkyl chains (LLD-AC-isoleucine, LLD-AC-allo-isoleucine, LLD-AC-
aminopentanoate), cyclopropyl groups (LLD-AC-cyclopropylalanine,
LLD-AC-methylcyclopropylglycine), -bonds LLD-AC-vinylglycine,
-
a
-
p
(
LLD-AC-dehydrovaline, LLD-AC-allylgylcine) and heteroatoms (LLD
-
AC-O-methyl-allo-threonine, LLD-AC-S-methylcysteine) [6].
However LLL-configured analogues are not substrates for the en-
zyme, and can inhibit turnover [35]. The crystal structures of IPNS
with LLD-ACd2Ab 10 and LLL-ACd2Ab 11 reported here demonstrate
that for LLL-configured analogues like 11, the side-chain of the third
residue is oriented away from the active site metal, directed in-
stead into a region of the protein above Tyr189. As a result the iron
is unprotected: the binding site opposite Asp216 is open and coor-
dinates an extra water ligand. This water molecule is held by
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