Enzymatic catalysis of anti-Baldwin ring closure in polyether biosynthesis.
ABSTRACT Polycyclic polyether natural products have fascinated chemists and biologists alike owing to their useful biological activity, highly complex structure and intriguing biosynthetic mechanisms. Following the original proposal for the polyepoxide origin of lasalocid and isolasalocid and the experimental determination of the origins of the oxygen and carbon atoms of both lasalocid and monensin, a unified stereochemical model for the biosynthesis of polyether ionophore antibiotics was proposed. The model was based on a cascade of nucleophilic ring closures of postulated polyepoxide substrates generated by stereospecific oxidation of all-trans polyene polyketide intermediates. Shortly thereafter, a related model was proposed for the biogenesis of marine ladder toxins, involving a series of nominally disfavoured anti-Baldwin, endo-tet epoxide-ring-opening reactions. Recently, we identified Lsd19 from the Streptomyces lasaliensis gene cluster as the epoxide hydrolase responsible for the epoxide-opening cyclization of bisepoxyprelasalocid A to form lasalocid A. Here we report the X-ray crystal structure of Lsd19 in complex with its substrate and product analogue to provide the first atomic structure-to our knowledge-of a natural enzyme capable of catalysing the disfavoured epoxide-opening cyclic ether formation. On the basis of our structural and computational studies, we propose a general mechanism for the enzymatic catalysis of polyether natural product biosynthesis.
- Journal of The American Chemical Society - J AM CHEM SOC. 04/2002; 105(11).
- [show abstract] [hide abstract]
ABSTRACT: The structure of the unique 'red tide' dinoflagellate neurotoxin, brevetoxin-B is presented and the experimental data supporting the chemical structure is discussed. A brief account of the other brevetoxins and their structural relationships is also presented. A biosynthetic scheme for the natural formation of the brevetoxin skeleton is proposed. Studies of the most toxic of the three pure brevetoxins, brevetoxin-A, indicate a skeleton differing from that of brevetoxin-B.Toxicon 02/1985; 23(3):473-9. · 2.92 Impact Factor
Article: Rules for ring closure.[show abstract] [hide abstract]
ABSTRACT: The ease of formation of cyclic structures shows some regularity which can be described by a set of 'rules' for ring closure. The restrictions on the endocyclic transfer of groups due to SN2 displacements having linear transition states are exemplified by studied on a biogenetic type synthesis of penicillin and, more generally, on the endocyclic attack of a nucleophile attached by a linking chain to an enone or polarized double bond. The formation of five-memebered rings is considered. In base, enones such as 4-hydroxy- and 4-amino-2-methylenebutanoates close exocyclically to give the corresponding five-membered lactones and lactams, respectively. Hydroxymethyl vinyl ketones, however, do not cyclize in base. In strong acid, hydroxy-enones close endocyclically to five-membered ring, as in the formation of cyclic ethylene ketals from acetone and ethanediol. The endocyclic closure of nucleophilic centres to polarized double bonds to give five-membered rings is impeded by restrictions of geometry; these restrictions can be overcome in acid through the generation of oxonium or protonated forms.Ciba Foundation symposium 02/1978;
Enzymatic catalysis of anti-Baldwin ring closure in
Kinya Hotta1*, Xi Chen1*, Robert S. Paton2, Atsushi Minami3, Hao Li1, Kunchithapadam Swaminathan1, Irimpan I. Mathews4,
Kenji Watanabe5, Hideaki Oikawa3, Kendall N. Houk6& Chu-Young Kim1
biologists alike owing to their useful biological activity, highly
complex structure and intriguing biosynthetic mechanisms.
Following the original proposal for the polyepoxide origin of
lasalocid and isolasalocid1and the experimental determination
of the origins of the oxygen and carbon atoms of both lasalocid
of polyether ionophore antibiotics was proposed2. The model was
based on a cascade of nucleophilic ring closures of postulated
polyepoxide substrates generated by stereospecific oxidation of
all-trans polyene polyketide intermediates2. Shortly thereafter, a
related model was proposed for the biogenesis of marine ladder
toxins, involving a series of nominally disfavoured anti-Baldwin,
endo-tet epoxide-ring-opening reactions3–5. Recently, we identified
Lsd19 from the Streptomyceslasaliensis genecluster as the epoxide
hydrolase responsible for the epoxide-opening cyclization of
bisepoxyprelasalocid A6to form lasalocid A7,8. Here we report the
X-ray crystal structure of Lsd19 in complex with its substrate and
product analogue9to provide the first atomic structure—to our
knowledge—of a natural enzyme capable of catalysing the dis-
favoured epoxide-opening cyclic ether formation. On the basis of
our structural and computational studies, we propose a general
Epoxide-opening cascades, including those using the disfavoured
anti-Baldwin cyclization (Fig. 1a), have emerged as an important
synthetic strategy in the field of organic chemistry10. Whereas little is
polyethers (Fig. 1b) has been studied extensively11. Although most
cyclic ethers in ionophore polyethers are the favoured exo-cyclization
products (Fig. 1b, in blue), several compounds, including lasalocid A,
contain six-membered rings presumably formed via a disfavoured
endo-tet cyclization (Fig. 1b, in red).
tary Table 1). Lsd19 consists of two highly homologous domains
(Fig. 2a) that belong to the nuclear transport factor 2 (NTF2)-like
superfamily12, which includes D5-3-ketosteroid isomerases (KSIs)13
and limonene-1,2-epoxide hydrolase (LEH)14. Each domain consists
of three a-helices and a cone-like six-stranded b-sheet whose cavity
forms a deep substrate-binding pocket. The amino- and carboxy-
terminal domains (Lsd19A and Lsd19B, respectively) are linked by a
short loop and are oriented in a head-to-tail fashion, resembling the
homodimer structure of single-domain NTF2-like proteins13. Lsd19A
and Lsd19B have identical backbone conformations; their respective
117core Ca atoms have a root-mean-squared deviation of just 0.79A˚.
There are two significant differences between Lsd19A and Lsd19B.
First, Lsd19B has an additional 18-residue-long loop–helix–loop near
the active site of Lsd19A contains an uncyclized bisepoxide substrate
(Fig. 2b), whereas the active site of Lsd19B contains a doubly cyclized
tetrahydrofuran–tetrahydrofuran (THF–THF) product. This differ-
ence in active sites is consistent with our previously reported results
that indicated that Lsd19A catalyses a single round of cyclization of
the bisepoxide substrate, whereas Lsd19B cyclizes the initially
formed THF ether/monoepoxide intermediate, predominantly to a
tetrahydrofuran–tetrahydropyran (THF–THP) product11.
In Lsd19A, the uncyclized substrate analogue is seen in the active
site.Thisobservation isconsistentwith the fact that Lsd19isinhibited
by the low pH of the crystallization buffer (Supplementary Fig. 1a).
*These authors contributed equally to this work.
1National University of Singapore, Departmentof Biological Sciences, 14 Science Drive 4, 117543 Singapore.2Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.
Bisepoxyprelasalocid A ( R = X )
Substrate analogue ( R = Y )
( R = X )
( R = X )
Figure 1 | Polyethernatural products and proposed stepsin the cyclic ether
formation. a, An example of a cascade epoxide ring closure implicated in the
biosynthesis of ladder polyether. b, Structures of representative ionophore
leading to the formation of lasalocid A, isolasalocid A and the product
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the oxazolidinone portion of the substrate analogue is ill-defined and
thereforeomitted from the finalmodel(Fig.2b).Asthe oxazolidinone
is not present in the natural substrate, disorder in this region is not
unexpected. In contrast, the portion of the substrate that contains the
two epoxide groups that eventually undergo cyclization is clearly
defined in the electron density map (Fig. 2b). The terminal 22,23-
epoxide sits at the bottom of the pocket and is surrounded by hydro-
phobic residues, protected from potential non-specific nucleophilic
attack. The putative nucleophilic 15-hydroxyl oxygen is hydrogen
bonded to Asp38, whereas the oxygen atom of the electrophilic
18,19-epoxide that undergoes ring opening lies within hydrogen-
bonding distance to Tyr14 and Glu65. Arg54 is hydrogen-bonded
to the catalytic Asp38 via a water molecule, presumably promoting
acid of Asp38 for ensuing proton abstraction from the 15-hydroxyl
group. This active-site configuration is homologous to that of KSI13
and LEH14, whichperform similar acid/basecatalysis (Fig. 3b). Unlike
intramolecular cyclizations on a flexible substrate. Additional inter-
actions between Lsd19A and the 3-methylsalicylate side of the native
substratecanhelpfixthe ligandconformationforthe ensuing cycliza-
forms a hydrogen bond with the carbonyl oxygen of the bound sub-
strate analogue (Fig. 2d). An equivalent oxygen atom is present in the
natural substrate, bisepoxyprelasalocid A.
In Lsd19B, an isolasalocid-like THF–THF compound is seen in the
active-site pocket instead of the expected lasalocid-like THF–THP
reaction product. This unexpected reaction compound is presumably
a consequence of the acidic crystallization condition (Supplementary
Fig. 1b). We have identified His146, Asp170, Glu197 and Tyr251 as
primary candidates for the catalytic residues in Lsd19B (Fig. 2e). Like
in Lsd19A,a hydrogen bond betweenAsp170and His186could raise
the pKaof His186. The 13-carbonyl oxygen is hydrogen bonded to
Arg177, providing an additional enzyme–substrate interaction. The
binding pocket of Lsd19B is shallower than that of Lsd19A due to the
presence of Met253 at the bottom of the pocket (Fig. 2g). This allows
orientation, and promotes the critical substrate selectivity in these
highly structurally similar domains. Although the shallower binding
pocket in Lsd19B affords less substrate-binding energy available for
confining the flexible substrate into the catalytically relevant con-
formation17, the additional loop–helix–loop structure (Fig. 2a, e, in
green) seems to compensate for the lost substrate-binding surface.
Analysis of known polyether biosynthetic gene clusters reveals that
two copies of polyether epoxide hydrolase (PEH)-encoding genes are
present within each cluster: monBI, monBII (monensin)18and nigBI,
carries a single copy of PEH gene tmnB, presumably because tetrono-
mycin contains only one epoxide-derived THF ring. Sequence align-
ment of PEHs indicates that the catalytic residues identified in Lsd19
are highly conserved (Supplementary Fig. 2). This alignment also
reveals that PEHs can be divided into two groups: PEH-A (Lsd19A-
like) and PEH-B (Lsd19B-like). Only PEH-B has a bulky residue at
Figure 2 | Crystal structure of Lsd19. a, The overall fold of Lsd19. The extra
loop–helix–loop in Lsd19B andthecorresponding insertion site in Lsd19A are
showningreen. b, c, Lsd19A(b) andLsd19B(c) electrondensityfor the ligand
(simulated-annealing omit map contoured at 2.0s, green mesh) and protein
side chains (2Fo2Fcmap contoured at 1.5s, blue mesh). Carbon atoms of the
bound ligands and protein are shown in yellow and orange, respectively.
Oxygen, nitrogen and sulphur atoms are shown in red, blue and green,
respectively. Green broken lines represent hydrogen bonds. d, e, Lsd19A
(d) and Lsd19B (e) active-site hydrogen-bonding interactions between the
to clarify the view. f, g, Lsd19A (f) and Lsd19B (g) molecular surface cross-
section, showing residues that determine the pocket size and shape.
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contains a loop–helix–loop insertion that provides additional ligand-
To extend the structural knowledge gained from Lsd19, homology
models of other PEHs were constructed. In these models, a direct
correlation is observed between the depth of the binding pocket and
the length of the substrate chain (Supplementary Fig. 3), leading us to
propose a general mode of cyclic ether formation in polyether bio-
synthesis. With the deeper binding pockets, PEH-As are probably
responsible for the formation of internal cyclic ethers. Interestingly,
PEH-As that act on longer substrates, such as nigericin, carry extra
PEH-A-specific C-terminal residues (Supplementary Fig. 2) positioned
at the opening of the binding pocket (Supplementary Fig. 3a), possibly
providing additional binding surface for the longer substrates. In con-
trast, PEH-Bs with shallower binding pockets probably catalyse the
formation of terminal cyclic ethers. As seen in Lsd19B, the extra PEH-
B-specific loop–helix–loop domain can provide the binding surface for
the portion of the substrate protruding out from the shallow substrate-
As the current Lsd19B structure does not address the formation of
THP directly, we used computational studies to understand how this
plementary Tables 2–4). Quantum mechanical density functional
conditions using model substrates, and to calculate the energy differ-
ence between the two processes (Fig. 3a and Supplementary Table 2).
nucleophilic attack at themore substituted epoxide terminus,as wellas
the more product-like transition structure with shorter forming O–C
favoured. This provides an indication of how Lsd19B may achieve
the six-membered ring formation. We then explored how Lsd19B
would influence the regioselectivity of the ring-opening transition
state. Docking studies on the Lsd19B structure indicated that
Asp170, Glu197 and Tyr251 maintain close contact with the bound
ligand. Similarities between the active site organization of Lsd19B and
KSI13(Fig. 3b) further support the idea that Asp170 acts as a general
base, whereas Glu197 and Tyr251 act as general acids stabilizing
the developing transition structure oxyanion. This arrangement is
also remarkably similar to the ‘theozyme’ derived from computations
for the anti-Baldwin-cyclization antibody23, which was eventually
foundto beclosely relatedto theantibody structure24. To test this idea
quantitatively, competing 5-exo and 6-endo transition structures were
constrained to the crystal structure coordinates. From this model,
values of the activation energy and Gibbs free energy changes for ring
closure were computed. These predict a preference for the 6-endo
pathway of 2.5kcalmol21, enough to achieve nearly 100:1 selectivity
(Fig.3c).Thepositioningof the catalyticgeneral acid and general base
thermodyamically more stable THP product.
of the biosynthetic pathway involved in producing polyether natural
products using only a pair of epoxide hydrolases. Furthermore, compu-
erence for the Lsd19-catalysed cyclization. Active-site pre-organization
and general-base catalysis provides enzymatic control to overcome
otherwise disfavoured chemical transformations. Similarly, stereo-
ΔE‡ = 18.0
ΔErxn = −6.6
ΔE‡ = 20.5
ΔErxn = −2.0
ΔΔE‡ = 2.5
ΔΔErxn = 4.6
Figure 3 | Computational studies of the Lsd19-catalysed epoxide-opening
cyclization reactions. a, Acid- versus base-catalysed cyclization of a model
system via 5-exo or 6-endo cyclization, and the respective calculated lowest-
energy competing transition structures. B2PLYP/6-31111G(d,p)//B2PLYP/
6-31G(d) activation free energies (kcalmol21) and forming/breaking bond
geometries in Lsd19B and KSI (PDB accession 1E3V24). c, ‘Theozyme’
calculations, optimizing the competing 5-exo-tet and 6-endo-tet transition
structures surrounded byfixed catalyticresidues.Relativeenergiesobtainedby
B2PLYP/6-31111G(d,p)/M06-2X/6-31G(d) (kcalmol21) and forming/
breaking bond distances are given.
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a simple polyepoxide substrate by Lsd19B-like EHs that can form
templating THP unit(s) that facilitate the subsequent cascade of
step-wise endo-tet-selective epoxide-opening cyclizations.
Lsd19 was produced using a variation of the published procedure8. Lsd19 was
purified using metal affinity, anion exchange and size-exclusion chromatography
steps to .95% purity and yielding approximately 2mg protein per litre of culture.
from the Hampton Crystal Screen. Subsequent optimization resulted in a crystal
that diffracted to 1.59A˚resolution at the Stanford Synchrotron Radiation
Lightsource. Data were indexed and integrated using XDS25. The structure of
Lsd19 was determined by molecular replacement and refined by CNS26and
REFMAC5 (CCP4)27. Computational studies on the reaction mechanism were
performed with B2PLYP/6-31111G(d,p) calculated energies on B2PLYP/6-
31G(d) or M06-2X/6-31G(d) optimized geometries using the Gaussian 09 pro-
gram28. Docking Studies were performed with Autodock Vina29.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 4 May 2011; accepted 13 January 2012.
Published online 4 March 2012.
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Acknowledgements This work was supported by the Royal Commission for the
Exhibition of 1851 and Fulbright-AstraZeneca Research Fellowship (R.S.P.), the Japan
Society for the Promotion of Science (No. LS103) (K.W.), the National Institutes of
Health grant GM075962 (K.N.H.), the MEXT research grant on innovative area
22108002 (H.O.), and the National University of Singapore Life Sciences Institute
Young Investigator Award (C.-Y.K.). Data collection was performed at the Stanford
Synchrotron Radiation Lightsource. We thank D. W. Christianson, D. Hilvert and
C. Khosla for critical reading and discussion of the manuscript.
purified Lsd19. X.C. and H.L. purified and crystallized Lsd19. X.C. and I.I.M. collected
K.S. provided assistance for crystallography. K.N.H. prepared and analysed models of
Lsd19 homologues. R.S.P. and K.N.H. performed the computational study. C.-Y.K.
conceived and supervised the project. C.-Y.K. prepared the manuscript with
contributions from all co-authors.
Author Information The coordinates and structure factors have been deposited with
the Protein Data Bank under accession number 3RGA. Reprints and permissions
information is available at www.nature.com/reprints. Reprints and permissions
information is available at www.nature.com/reprints. The authors declare no
competing financial interests. Readers are welcome to commenton the online version
of this article at www.nature.com/nature. Correspondence and requests for materials
should be addressed to C.-Y.K. (firstname.lastname@example.org).
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Lsd19 expression and purification. Production of Lsd19 was performed partly
according to the procedure described elsewhere8. Briefly, pCold-based pKW620
were introducedintoEscherichiacoliBL21(DE3)strain. Theculturewasgrownin
Luria Broth medium to OD600nmof 0.6, and the production of N-terminal His-
tagged Lsd19 was induced by 100mM isopropyl-b-D-galactoside, 0.5mgml21
L-arabinose and 5ngml21tetracycline. The culture was incubated for another
20h at 15uC. Cells were harvested by centrifugation, resuspended in 50mM
sodium phosphate pH 7.4, 300mM sodium chloride, 10mM imidazole, 10%
(v/v) glycerol and lysed by sonication. After centrifugation at 15,000g for
45min, the cleared supernatant was mixed with cobalt-agarose beads (Thermo
Fisher Scientific). The mixture was incubated for 1h at 4uC with moderate
shaking. After incubation, the mixture was loaded onto a column and washed
with a wash buffercontaining 50mMsodiumphosphatepH7.4, 300mMsodium
chloride, 10mM imidazole and 10% (v/v) glycerol. Lsd19 was eluted with a wash
buffer supplemented with 150mM imidazole. Fractions containing Lsd19 were
pooled and exchanged into a buffer containing 20mM Tris pH 8.5, 1mM EDTA
and 15mM b-mercaptoethanol. Lsd19 was further purified by anion exchange
chromatography using a HiTrapQ XL column followed by gel filtration on a
Superdex200 10/300 GL column (GE Healthcare Life Sciences) in 50mM
potassium phosphate pH 7.0, 1mM EDTA and 15mM b-mercaptoethanol. The
the yield was approximately 2mg per litre of culture. The sample was further
concentrated to 12mgml21for storage.
Crystallization, data collection and structure determination. Crystals were
obtained bythe hanging-drop vapourdiffusionmethod. The bisepoxide substrate
substrate analogue (100mM in methanol) at 12.5:1 ratio (v/v) on ice, the sample
ive 1,5-diaminopentane dihydrochloride in the presence of 200–700ml Al’s oil in
the well. Before flashfreezing,Lsd19crystalsweretransferredforabout1mintoa
SLAC National Accelerator Laboratory). Data collection was performed on a
single crystal at 100K using a monochromatic X-ray at a wavelength of
0.9795A˚. Data were indexed and integrated using XDS25. Structure factor was
B KSI structure (PDB accession 1DMN)37as a searchmodel. The model was built
by Coot32and ARP/wARP33and refined by CNS26and REFMAC5 (CCP4)27.
allowed backbone conformation with 98.2% within the favoured conformation.
Other relevant data and refinement statistics are given in Supplementary Table 1.
Computation. Quantum chemical calculations were performed using Gaussian
09,revision A.228. All geometries were fully optimized. Transition structures were
identified by a single imaginary harmonic vibrational frequency. Optimizations
the harmonic approximation at 298K and 1atm, with unscaled zero-point vibra-
tional energies. Docking Studies were performed with Autodock Vina29.
Single point energy calculations were performed to ensure basis set convergence
at the B2PLYP/6-31111G(d,p)//B2PLYP/6-31G(d) level. Calculations were per-
formed at the ‘double-hybrid’ density functional level of theory with the B2PLYP
a non-local correlation energy expression that uses the Kohn–Sham orbitals in
second-order perturbation theory and delivers improved energetics over hybrid
M06-2X calculations are in accord, both qualitatively and quantitatively, over the
of the model systems. Absolute barriers showed some variations; however, the
energy differences between the two cyclization modes were consistent. Changing
(Supplementary Table 2).
gas-phase geometries using a conductor-like polarizable continuum (CPCM)
model35of water (r578.36)anddichloromethane (r58.93), defining the solute
cavity by UFF radii. As for gas-phase values, B2PLYP and M06-2X results are in
accord over preferred reaction pathway (Supplementary Table 3).
‘Theozyme’ calculations were performed using a truncated model of the active
are optimized. Electrostatic effects of the protein interior were described with a
A variety of methods show consistent energy differences between 5-exo and
dinates for all species in the text are given in the Supplementary Data. Absolute
gas-phase energies and free energies (1atm, 298K) from B2PLYP/6-31G(d)
including CPCM solvation (UAKS solute radii) are all given in kcalmol21.
Imaginary harmonic vibrations are in cm21.
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