The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism

Article abstract of DOI:10.1021/jacs.6b08610

The final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1) is the highly unusual cytochrome P450-catalyzed, oxidative rearrangement of pentalenolactone F (2), involving the transient generation and rearrangement of a neopentyl cation. In Streptomyces arenae this reaction is catalyzed by CYP161C2 (PntM), with highly conserved orthologs being present in at least 10 other Actinomycetes. Crystal structures of substrate-free PntM, as well as PntM with bound substrate 2, product 1, and substrate analogue 6,7-dihydropentalenolactone F (7) revealed interactions of bound ligand with three residues, F232, M77, and M81 that are unique to PntM and its orthologs and absent from essentially all other P450s. Site-directed mutagenesis, ligand-binding measurements, steady-state kinetics, and reaction product profiles established there is no special stabilization of reactive cationic intermediates by these side chains. Reduced substrate analogue 7 did not undergo either oxidative rearrangement or simple hydroxylation, suggesting that the C1 carbocation is not anchimerically stabilized by the 6,7-double bond of 2. The crystal structures also revealed plausible proton relay networks likely involved in the generation of the key characteristic P450 oxidizing species, Compound I, and in mediating stereospecific deprotonation of H-3_re of the substrate. We conclude that the unusual carbocation intermediate results from outer shell electron transfer from the transiently generated C1 radical to the tightly paired heme-a€￠Fe³⁺-OH radical species. The oxidative electron transfer is kinetically dominant as a result of the unusually strong steric barrier to oxygen rebound to the neopentyl center C-1_si, which is flanked on each neighboring carbon by syn-axial substituents.

Full text of DOI:10.1021/jacs.6b08610

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Article
The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of
Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism
Lian Duan, Gerwald Jogl, and David E. Cane
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b08610 • Publication Date (Web): 02 Sep 2016
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The Cytochrome P450-Catalyzed Oxidative Rearrangement in the
Final Step of Pentalenolactone Biosynthesis: Substrate Structure
Determines Mechanism
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Lian Duan,† Gerwald Jogl,#, and David E. Cane†,#,
* *
†Department of Chemistry, Brown University, Box H, Providence, Rhode Island 02912-9108, USA
#Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island
02912, USA
ABSTRACT: The final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1) is the highly unusual
cytochrome P450-catalyzed, oxidative rearrangement of pentalenolactone F (2), involving the transient generation and
rearrangement of a neopentyl cation. In Streptomyces arenae this reaction is catalyzed by CYP161C2 (PntM), with highly
conserved orthologues being present in at least 10 other Actinomycetes. Crystal structures of substrate-free PntM, as well
as PntM with bound substrate 2, product 1, and substrate analogue 6,7-dihydropentalenolactone F (7) revealed interac-
tions of bound ligand with three residues, F232, M77, and M81 that are unique to PntM and its orthologues and absent
from essentially all other P450s. Site-directed mutagenesis, ligand-binding measurements, steady-state kinetics, and reac-
tion product profiles established there is no special stabilization of reactive cationic intermediates by these side chains.
Reduced substrate analogue 7 did not undergo either oxidative rearrangement or simple hydroxylation, suggesting that
the C1 carbocation is not anchimerically stabilized by the 6,7-double bond of 2. The crystal structures also revealed plau-
sible proton relay networks likely involved in the generation of the key characteristic P450 oxidizing species, Compound I,
and in mediating stereospecific deprotonation of H-3re of the substrate. We conclude that the unusual carbocation inter-
mediate results from outer shell electron transfer from the transiently generated C1 radical to the tightly paired heme-
•Fe3+-OH radical species. The oxidative electron transfer is kinetically dominant as a result of the unusually strong steric
barrier to oxygen rebound to the neopentyl center C-1si which is flanked on each neighboring carbon by syn-axial substit-
uents.
Introduction
We recently reported the cloning and biochemical
characterization of a pair of orthologous cytochrome
P450s, designated PntM (CYP161C2) and PenM
(CYP161C3), that catalyze the final step in the biosynthesis
of the sesquiterpenoid antibiotic pentalenolactone (1) in
Streptomyces arenae and S. exfoliatus UC5319, respectively
(Scheme 1).4 The conversion of pentalenolactone F (2) to
pentalenolactone (1) involves an oxidative rearrangement
that is completely unprecedented in the already vast rep-
ertoire of P450-catalyzed transformations. Isotopic label-
ing studies have established that the net 2-electron oxida-
tion involves the stereospecific removal of H-1si of 2, syn-
1,2-migration of the 2si methyl group (C-12), and anta-
rafacial loss of H-3re to generate the characteristic (1S)-1,2-
dimethyl-cyclopent-2-ene moiety of pentalenolactone
(1).5
Cytochrome P450 enzymes are responsible for a re-
markable range of controlled oxidations at sp3 carbon
centers that have little or no parallel in laboratory organic
chemistry, including regiospecific and stereospecific in-
sertion of oxygen into unactivated C–H bonds and oxida-
tive cleavage of C–C bonds.1 Such reactions are catalyzed
by both catabolic P450s, whose primary biological func-
tion is degradation of a broad range of foreign organic
compounds2a or mobilization of environmental organic
chemicals as sources of energy and carbon,2b as well as by
metabolic P450s that catalyze substrate-specific reactions
as part of multistep biosynthetic pathways.3
Scheme 1. Oxidative rearrangement of pentalenolactone
F (2) to pentalenolactone (1)
Cytochrome P450-catalyzed reactions normally involve
the generation of caged substrate radicals, resulting from
abstraction of a substrate hydrogen atom by the highly
reactive ferryl oxygen of porphyrin+•~Fe4+=O (Compound
I), followed by rapid oxygen rebound that transfers the
hydroxyl group to the original reacting carbon atom
(Scheme 2).6 The transition state for the abstraction of
the hydrogen atom of the substrate by the reactive oxygen
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atom of Compound I involves a linear geometry of the resolution crystal structures of wild-type PntM in both
1
reacting O--H–C array, while oxygen rebound requires a
rapid rotation of the initially-formed, Fe-bound OH group
so as to allow approach and recombination with the
paired carbon radical.7 P450-catalyzed rearrangements
are rare, with prior examples limited to the formation of
isomeric hydroxylation products derived from either 1) an
allylic radical8 or 2) a cyclopropylcarbinyl radical whose
rapid, thermodynamically-driven ring-opening generates
the corresponding 3-butenyl radical.9 Such ring opening
reactions have in fact been exploited as radical clocks that
have established the rate constants for oxygen rebound
within the initially-generated [Fe3+-OH]/carbon radical
pairs to be ~1010–1011 s-1.9 Even in such artificially engi-
neered systems, however, the proportion of ring-opened
rearrangement products is typically 1-15% or less. Func-
tionalized cyclopropane derivatives bearing substituents
that favor, and which are therefore diagnostic of, carbo-
cation-based rearrangements have also indicated that less
than 2% of the overall oxidation product mixture results
from an alternative ring opening of a cyclopropylcarbinyl
cation.10-12 Such carbocations are most likely formed by a
stepwise process involving oxidation of the initially
formed carbon radical by rapid electron transfer to the
strongly oxidizing [Fe3+-OH] species, in competition
with the usually favored oxygen rebound reaction.
substrate-free form and individually complexed with sub-
strate 2, product 1, and the reduced substrate analogue
6,7-dihydropentalenolactone F (7). We have also deter-
mined the 2.05 Å structures of four active site PntM mu-
tants with bound substrate 2. We have also determined
the steady-state kinetic parameters, substrate binding
constants, and reaction product profiles for the wild-type
and mutant proteins with native substrate 2, as well as for
wild-type PntM incubated with reduced substrate ana-
logue 7. The combined results exclude any special elec-
tronic features in either the substrate pentalenolactone F
(2) or the PntM protein itself that might favor a carbo-
cation over a radical mechanism, and also rule out any
unusual mode of substrate–protein binding. We conclude
instead that the conversion of 2 to the neopentyl carbo-
cation intermediate 6 is a consequence of tightly con-
strained, conventional binding of pentalenolactone F,
combined with the exceptional degree of steric hindrance
to attack at C-1 of 2 that prevents access of the transient-
ly-generated heme-bound hydroxyl group to the si face of
the C-1 radical, thus favoring competing electron transfer
to the paired, strongly oxidizing [Fe3+OH] radical.
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Results
Incubation
of
PntM
with
6,7-
dihydropentalenolactone F (7). We initially speculated
that the carboxylate-conjugated 6,7-double bond of the
natural substrate 2 might provide anchimeric stabilization
of a C-1 carbocation in the derived intermediate 6
(Scheme 3a). To probe this idea, we tested whether the
Scheme 2. P450-catalyzed hydrogen abstraction, oxygen
rebound, and competing electron transfer.
reduced analogue 6,7-dihydropentalenolactone
F (7)
would undergo simple hydroxylation at C1 when incubat-
ed with PntM, in competition with the usual oxidative
rearrangement. The requisite sample of 7 was readily pre-
pared by hydrogenation of pentalenolactone F methyl
ester (2-Me) followed by mild basic hydrolysis (Scheme
3b). GC-MS analysis established that the reduction was
stereospecific, generating a 12:1 mixture of diastereomers.
The major diastereomer was subsequently assigned as
(6R)-7, based on the best fit to the electron density in the
2.03 Å crystal structure of PntM complexed with 7, dis-
cussed below (Figure 4). Binding of 7 to PntM resulted in
a normal Type I P450 binding spectrum,1,4 with the char-
acteristic hypsochromic UV shift from the low-spin, lig-
and-free 420 nm absorption to the high-spin 390 nm sub-
strate-bound state (Figure S4). Analysis of the concentra-
tion dependence of the UV difference spectra gave a Kd of
170±17 ꢀM for 7, nearly 25 times weaker than binding of
the natural substrate 2 (Kd 7.2±0.8 ꢀM) (Table 1). The ana-
logue 7 was recovered completely unchanged upon ex-
tended aerobic incubation with PntM, NADPH, and the
ferredoxin/NADPH:ferredoxin oxidoreductase, as estab-
lished by capillary GC-MS analysis of the organic extract,
with no trace of the analogous products of either oxida-
tive rearrangement, 6,7-dihydropentalenolactone (8), or
direct hydroxylation, (9) (Scheme 3b).
Neopentyl radicals, such as that which would be formed
by conventional P450-catalyzed oxidation at C-1 of 2, do
not undergo skeletal rearrangement.13 By contrast, neo-
pentyl cations, typically generated by SN1 solvolysis reac-
tions, undergo extremely rapid, coupled Wagner-
Meerwein rearrangement such that they can neither be
trapped nor directly observed.14 To explain the PntM-
catalyzed oxidative rearrangement of 2, we therefore have
proposed a mechanism involving P450-mediated genera-
tion of the C-1 neopentyl cation intermediate 6 (Scheme
1).4 Circumstantial support for such a carbocationic
mechanism comes from the isolation from large-scale
fermentations of isomeric rearrangement products, pen-
talenolactones A (3), B (4), and P (5), that presumably are
formed by alternative deprotonation of the intermediates
generated by the rearrangement of the initially formed
neopentyl cation 6.15
The apparent ability of PntM to catalyze an exclusive
carbocation-based oxidative rearrangement clearly pre-
sents an intriguing mechanistic puzzle. To unravel this
conundrum, we have now determined the 2.00 – 2.28 Å
Scheme 3. 6,7-Dihydropentalenolactone F (7)
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Table 1. Binding of pentalenolactone F (2) and 6,7-
1
dihydropentalenolactone F (7) to wild-type PntM and
2
mutants.
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4
5
6
7
8
9
Protein
Protein
KD (ꢀM)
KD (ꢀM)
170±17 (7)
340±90 (2)
43±3 (2)
PntM - wt
7.2±0.8 (2)
270±85 (2)
225±45 (2)
160±12 (2)
PntM - wt
PntM-M81A
PntM-M81C
PntM-M77S
PntM-F232A
PntM-F232G
PntM-F232L
115±8 (2)
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PntM-M81C- 31±1 (2)
BME
Figure 1. Structure of substrate-free PntM. A, Schematic representation of the structure of PntM with bound bicine. Bicine is
shown as sticks with carbon atoms in pink, the heme cofactor is shown as sticks with carbon atoms in bright orange and the
heme-bound Fe3+ is shown as an orange sphere. T2 is the N-terminal amino acid and F398 is the C-terminal amino. B, Final 2mFo
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– DFc electron density map of the active site region (contoured at 1 σ). C, close-up view, including relevant water molecules
(small red spheres) and H-bonds (distances in Å) that position bicine in the active site.
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PntM Structures. The structure of substrate-free PntM
was solved to a resolution of 2.00 Å by molecular re-
placement, using as a template the polyene macrolide
epoxidase PimD (PDB ID 2X9P). By soaking individual
PntM crystals in crystallization buffer containing pen-
nearly half the circumference of the protein just below
the latitude of the heme plane, while the long B-C loop
(D55-S71) and C-Helix (A72-M81) as well as the conserved
F-Helix, (L157 – F171, G-Helix (N176 – V199), and short F-G
loop (D172 – D175) control access by substrate to the dis-
tal face of the heme cofactor. The hexacoordinate Fe3+ of
the heme cofactor is liganded on the proximal face by the
thiolate of the conserved C347, while the axial water lig-
and (W1) on the distal face is slightly displaced by ~0.9 Å
from the vertical axis by the hydroxyl group of a bound
bicine molecule, which originates from the crystallization
buffer (Figure 1C).16 Water W1 is held
talenolactone
F (2), pentalenolactone (1), or 6,7-
dihydropentalenolactone F (7), we were also able to ob-
tain structures of PntM complexed with substrate (2)
(2.03 Å), product (1) (2.28 Å), and the reduced substrate
analogue (7) (2.03 Å) (Table S1). The topology of PntM
corresponds to the classic P450 fold, characterized by the
long I-Helix from D219 to R250 that spans the distal face
of the heme cofactor. (Figure 1) The bent equatorial D-
Helix encompassing residues T88 – A129 wraps around
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Figure 2. Structure of PntM with bound substrate 2. A, stereo diagram showing heme/substrate 2-binding site. 2 is shown as
sticks with carbon atoms in light blue. B, final 2mFo – DFc electron density map of the active site region (contoured at 1 σ). C,
close-up view showing water relay 1 (W1, W2, and W3) and H-bonds positioning substrate 2. D, close-up view showing water
relay 2 (W4, W5, W6).
in place by a network of hydrogen bonds involving the
side chain hydroxyl of T236, the backbone carbonyl oxy-
gen of F232 and a second molecule of water, W2, that is
wedged into the expanded kink of the I-helix.17 The car-
boxyl group of the bicine is anchored by a pair of H-bonds
to NH2 of R240 (2.8 Å) and to water molecule W25 (2.8 Å)
which is also H-bonded to NH1 of R240 (2.8 Å), one heli-
cal turn of the I-Helix downstream of T236. The bicine is
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and ester oxygen atoms of 2 are hydrogen-bonded to both
itself buttressed by van der Waals interactions with the
1
2
3
4
5
6
7
8
side chains of F232, M77 and M81, the latter two residues
being located at the C-terminus of the C-Helix.
NH1 and NH2 of R74 at distances of 2.9 Å and 4.0 Å, re-
spectively. The C3 methylene of substrate 2 is sandwiched
between the sidechains of M77 and M81, while the C14
and C15 geminal methyl groups of 2 abut the aromatic
face of F232, which itself contacts the M81 side chain.
The topology of PntM undergoes minimal changes up-
on binding of either substrate 2, product 1, or substrate
analogue 7 (Figures 2, 3, 4, and S10), with the calculated
rmsd of the Cα carbons ranging from 0.13 – 0.22 Å com-
pared to PntM with bound bicine. Significantly, the bind-
ing of the substrate, pentalenolactone F (2), precisely ori-
ents the scissile C1–H-1si bond so that it is pointed di-
rectly towards the site that would be occupied by the
strongly oxidizing ferryl oxygen of porphyrin+•/Fe4+=O
(Compound I) (Figure 2). The bound substrate 2 is held
tightly in place by an interlocking network of ionic, hy-
drogen bond, and van der Waals interactions. The C13
carboxylate of 2 is positioned by a pair of bifurcating H-
bonds to the NH2 of R240 (2.7 Å) and to the side chain
amide NH2 of N283 (3.3 Å), while the lactonic carbonyl
Amino acid F232 forms part of a highly unusual
F
232GYET236 sequence at the I-Helix kink that replaces the
nearly universally conserved A/Gn-1GnXXTn+3 motif char-
acteristic of the vast majority of P450s. A BLAST search of
the non-redundant protein databases using PntM as the
query reveals that the 10 closest matches (sequence iden-
tity 74-89% over 398 aa; E value 0.0), each of which har-
bor the FGYET motif, are all likely PntM orthologs found
at identical loci within known or probable pentalenolac-
tone biosynthetic gene clusters (Figure S9). Each of these
10 orthologs also displays the same R74XXM77XALM81 sub-
strate-binding motif.
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Figure 3. Structure of PntM with bound product 1. A, stereo diagram showing heme/product 1-binding site. 1 is shown as sticks
with carbon atoms in white/blue. B, final 2mFo – DFc electron density map of the active site region (contoured at 1 σ). C, close-up
view showing relevant water molecules and H-bonds positioning product 1 in the active site.
I-Helix motif, while none retain the R74XXM77XALM81
By contrast, the next 88 best protein matches (sequence
identity 39-49%) all contain the canonical A/Gn-1GnXXTn+3
sequence character istic of the PntM orthologs.
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During the catalytic cycle of a prototypical P450, gener- bonded to water W3 (3.4 Å), held in place by H-bonds
1
2
3
4
5
6
7
8
ation of the key oxidant requires delivery of two protons
to the distal oxygen atom of the intermediate reduced
ferrous-dioxygen (peroxyferric) complex, followed by re-
lease of a molecule of water and formation of Compound
I. Since this oxygen atom is buried 12-15 Å from the sur-
face of the P450 protein, it is not directly accessible to the
external medium. Delivery of the requisite protons there-
fore requires a proton relay chain, with the hydroxyl
group of the conserved I-Helix threonine frequently act-
(2.4 and 3.1 Å) to the carboxylate oxygen atoms of E356. In
the actual intermediate Fe2+-O2-pentalenolactone F com-
plex, it is of course likely that water W1 is either further
shifted by the oxygen atom of the bound O2, or displaced
altogether, with the remainder of the proton relay net-
work remaining essentially intact.19 Notably, the well-
characterized proton relay networks in two other well-
studied P450s, Pseudomonas putida cytochrome P450cam
,
which catalyzes the C5 exo-hydroxylation of camphor,18,20
and Saccharopolyspora erythraea P450eryF,3b,21 which cat-
alyzes the C6-hydroxlation of 6-deoxyerythronolide B, the
parent macrolide aglycone of erythromycin A, are each
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ing as the penultimate proton donor/acceptor, as in the
3,18
.
prototypical monoxygenase P450cam
A presumptive
proton relay network (Water relay 1) is readily apparent in
the structure of substrate-bound PntM (Figure 2C). Thus
water W1, laterally displaced from the axial sixth ligand
position of the heme iron by the binding of pentalenolac-
tone F (2), is H-bonded to both the hydroxyl group of
T236 (2.7 Å) and the carbonyl oxygen of F232 (2.7 Å),
while these two oxygen atoms are each in turn H-bonded
(2.8 and 3.1 Å) to water W2, which resides in the expand-
ed groove of the I-helix. W2 is further H-bonded to the
side chain hydroxyl of T237 (2.6 Å), which is in turn H-
anchored by homologous glutamate residues, P450cam
-
E366 and P450eryF-E360, respectively, which occupy
identical sites in the corresponding L-Helix of each pro-
tein.20,21 Indeed, this glutamate is nearly universally con-
served in all P450s, being found in >90% of the top 100
BLAST matches to the PntM sequence. Although the con-
served glutamates do not appear to have a continuous
Figure 4. Structure of PntM with bound substrate analogue 7. A, stereo diagram showing heme/analogue 7-binding site. 7 is
shown as sticks with carbon atoms in cyan. B, final 2mFo – DFc electron density map of the active site region (contoured at 1 σ).
C, close-up view showing relevant water molecules and H-bonds positioning 7 in the active site.
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parison described above established that PntM harbors a
H-bonded link with bulk water at the protein surface, it
1
2
3
4
5
6
7
8
has been proposed that the requisite proton delivery may
involve concerted dynamic fluctuations of amino acid
chains and bound waters.3b,22
constellation of active site residues, F232, M77, and M81,
unique among all known P450s, that snugly cradle the
bound substrate, pentalenolactone F (2) (Figure 2). Given
the proximity of all three residues to C1, C2, and C3 of the
substrate, the loci for the generation, rearrangement, and
deprotonation of the proposed neopentyl cation interme-
diate, we examined the possibility that the side chains of
these three amino acid residues might electronically sta-
bilize the various carbocation intermediates through
quadrupole-cation or dipole-cation interactions, analo-
gous to well-established role of aromatic residues in the
active sites of terpene synthases that stabilize and chap-
erone the reactive carbocation intermediates genrerated
by the ionization and cyclization of allylic diphosphates
to complex terpenes.23
The PntM-catalyzed oxidative rearrangement of pen-
talenolactone F (2) to pentalenolactone (1) is completed
by deprotonation of the labile H-3re proton from the rear-
ranged neopentyl cation (Scheme 1). From the crystal
structure of PntM with bound substrate 2, the only suita-
ble Brønsted base is the previously cited water W4 which
is positioned 4.2 Å from C3 of 2, just off the in-line axis of
the C3- H-3re bond (Figure 2D). Water W4, which is H-
bonded to the epoxide oxygen of 2 (3.1 Å) is connected to
the external medium by another well-organized proton
relay system (water relay 2) also involving waters W5 and
W6. The latter water, which is exposed to the external
medium, is anchored by an H-bond (2.8 Å) to NH1 of the
previously mentioned R74, while W5 may also be held in
place by H-bonds to the sidechain sulfides of M77 and/or
M81. Notably, R74, M77, and M81 are themselves located
at successive turns of the C-Helix, and together play a
major role in the binding and orientation of the substrate,
as noted above. Indeed, it is conceivable that protonation
of water W6 causes repulsion of the positively charged
guanidino side chain of R74, thus displacing Helix C and
facilitating release of the product pentalenolactone (1).
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To probe the catalytic roles of F232, M81, and M77, the
F232A, F232G, F232L, F232Y, and F232H mutants of PntM,
as well as the M81A, M81C, and M77S variants, were pre-
pared by site-directed mutagenesis. Each mutant was pu-
rified to homogeneity, as monitored by SDS-PAGE (Figure
S1), and the MD of each protein was directly verified by
LC-ESI-MS (Table S4). The LC-MS analysis revealed that
the original M81C protein preparation had actually been
obtained as the disulfide conjugate with β-
mercaptoethanol (M81C-BME). The native M81C mutant
was then readily prepared by carrying out the purification
of recombinant protein using buffers that did not contain
β-mercaptoethanol. In substrate binding assays, the M81C
and M81C-BME mutants had KD values for substrate 2
that were only 4-6-fold greater than that of wild-type
PntM, while the majority of the remaining mutants
bound 2 with 15-50-fold lower affinity than did PntM, ex-
cept for F232Y and F232H, which did not bind 2 at all (Ta-
ble 1, Figures S5 and S6).
In the structure of PntM with bound product pen-
talenolactone (1) (Figure 3), the rmsd of the Cα carbons
compared to substrate-bound PntM is 0.25 Å, while there
is insignificant change in the positions of either M77, M81,
or F232 (Figure S10D). The binding of 1 is unchanged from
that of the substrate 2, other than the necessary alteration
in the conformation and substitution pattern of C1-C3 due
to the migration of the β-methyl group to C1 and the in-
troduction of the 2,3-double bond. Water W1 remains H-
bonded to both the side chain hydroxyl group of T236 (2.7
Å) and the backbone carbonyl oxygen of F232 (2.4 Å),
while the latter two oxygen atoms are H-bonded (2.6 and
2.8 Å, respectively) to W2 in the I-Helical groove (Figure
3C). On the other face of bound 1, water W4 remains H-
bonded to both the epoxide oxygen (3.0 Å) and the propi-
onate carboxyl oxygen (2.8 Å).
Table 2. Steady-state kinetic parameters for PntM and
mutants.
PntM/
mutant
kcat
kcat/Km
(min-1ꢀM-1)
Km
R2
(min-1)
(ꢀM)
Wild-type
F232A
F232L
1.8±0.3
5.0x10-2
36±14
0.9
5
0.035±0.002 8.2x10-4
43±5
0.9
9
When PntM is bound to 6,7-dihydropentalenolactone F
(7), the relative positions of the side chains of F232, M77,
M81 are unperturbed compared to PntM with the bound
natural substrate 2, as are the positions of the three
bound water molecules, W1, W2, and W4 (Figures 4 and
S10F). As a consequence of the reduction of the 6,7-
double bond, the C13-carboxylate of 7 is slightly twisted
with respect to the carboxylate of 2, while still held in
place by H-bonds to R240 (2.7 Å) and N283 (3.8 Å). The
altered conformation of 7 also results in a ca. 0.3 Å clock-
wise rotation of C1 away from the iron of the heme cofac-
tor, providing a possible rationale for the absence of any
detectable oxidative rearrangement or hydroxylation
products.
0.090±0.00
6
0.004±0.001 3.7x10-4
7.5x10-3
12.0±3
37±6
0.9
6
M81A
0.9
8
M81C
0.092±0.003 4.9x10-3
19±3
0.9
9
M77S
0.019±0.001
1x10-3
19±4
0.9
6
M81C-BME 0.085±0.25
3.0x10-4
280±110
0.9
9
Site-directed mutagenesis of active site PntM resi-
dues. The protein structural studies and sequence com-
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The steady-state kinetic parameters and product pro- anchimeric assistance by the 6,7-double bond of substrate
1
2
3
4
5
6
7
8
files for the six active PntM mutants were then deter-
mined, using a calibrated GC-SIM-MS assay to quantitate
the time-dependent formation of 2 (Table 2, Figures S7
and S8). Significantly, the F232L mutant exhibited only a
2. These crystallographic studies do establish that the
enzyme tightly controls the geometry of binding of both
the substrate and product through a complex network of
reinforcing H-bond and van der Waals interactions, so as
to precisely position the target C1-Hsi bond of the sub-
strate in line with the reactive iron-oxo intermediate,
Compound I, while providing a strategically placed water
molecule (W4) as part of a dedicated proton relay system
that can accept the labile proton from C3-Hre. The key
issue then becomes: What controls the exclusive for-
mation of the key carbocation intermediate from the ini-
tially generated carbon radical?
7-fold reduction in kcat/Km compared to wild type Pnt M,
reflecting a 20-fold decrease in kcat that was partially off-
set by a modest 3-fold reduction in Km. Importantly, GC-
MS analysis of the total product mixture indicated for-
mation of only the natural oxidative rearrangement prod-
uct, pentalenolactone (1), with no detectable products
corresponding to simple hydroxylation at C1 or any other
site. This result conclusively rules out any special orbital
stabilization of the carbocation intermediates by the aro-
matic ring F232 and indicates that the role of the aromatic
side chain is to precisely position the substrate 2 and de-
rived intermediates. Notably, the F232A mutant, which
carries the canonical I-helix Ala residue, still generates
exclusively pentalenolactone, albeit with a >60-fold lower
9
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kcat
/Km than wild type PntM. The M81C mutant showed a
modest 10-fold reduction in kcat
/Km compared to wild-
type, with exclusive formation of the oxidative rear-
rangement product 1, while the M81C-BME, M81A, and
M77S mutants were all 5-15-fold less active but with no
change in product profile.
The structures of the PntM F232L, M77S, M81A, M81C,
and M81C-BME mutants with bound pentalenolactone F
(2) were solved to resolutions of 2.06-2.12 Å (Table S2,
Figure S11). None of these five mutants exhibited notable
changes in the either the overall protein topology or the
positions of the amino acid side chains, the heme cofac-
tor, bound 2, or the key bound water molecules W1–W4,
except for the mutated residues and a few minor changes,
some of which are noted below. In PntM_F232L, the M77
side chain is now observed as a mixture two conformers,
one (55%) unchanged from the wild-type and the other
(45%) in which the sulfur atom has been rotated 2.2 Å
towards the space vacated by the replacement of the phe-
nyl ring of F232 by the isopropyl group of L232, which
itself is closely superimposed on the corresponding atoms
of the F232 side chain (Figure S11B). In both conformers,
the position of the M77 methyl remains unchanged. In
the M81C mutant, the only significant change is that a
new water molecule is found in the site previously occu-
pied by the terminal methyl of M81 (Figure S11D). Finally,
in the M81C-BME mutant, the only significant change is
that the terminal hydroxyl group of the covalently linked
β-mercaptoethanol has replaced water W5 and is hydro-
gen-bonded (2.5 Å) to water W4, which has been very
slightly displaced from its original position in the wild
type protein (Figure S11J).
Figure 5. Model showing steric hindrance to oxygen re-
bound to the C1si face of pentalenolactone F.
Ortiz de Montellano and Groves have provided a criti-
cal analysis of the use of radical clock substrates and the
apparent competition between P450-catalyzed formation
of radical rebound products and electron-transfer pro-
cesses that result in the formation of cation-derived
products.24 Drawing on the extensive prior studies by Ko-
chi25 of the kinetics, mechanism, and product profiles for
the oxidation of a wide range of organic radicals by a se-
ries of organometallic complexes of increasing reduction
potential, they have proposed that in P450-catalyzed reac-
tions there are two competing processes that follow initial
formation of the caged [Fe3+-OH]/carbon radical pair. The
first, which is characteristic of the vast majority of P450s,
involves a rapid oxygen rebound reaction, which normally
occurs at a rate of ~1010-1011 s-1, in which the iron-bound
hydroxyl group is transferred within the caged radical pair
to the substrate carbon atom, resulting in generation of
the typical hydroxylated product from the nominally un-
reactive C-H bond (Scheme 2). In competition with this
oxygen rebound process, there is also the possibility of a
thermodynamically favorable electron transfer from the
carbon radical to the strongly oxidizing heme •Fe3+-OH
species. This latter process is normally too slow to com-
pete with hydroxylation, or at best represents no more
Discussion. The combined protein structure and site-
directed mutagenesis studies conclusively rule out any
special role of the F232, M77, or M81 residues at the active
site of PntM in promoting or favoring the formation of
cationic intermediates instead of, or in addition to, radical
intermediates
in
the
oxidative
rearrangement
of pentalenolactone F (2) to pentalenolactone (1), while
the reduced substrate analogue studies do not support
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Hre bound directly toward the predicted position of the
than 1-2% of the overall reaction flux in the P450-
1
2
3
4
5
6
7
8
catalyzed oxidation of specifically engineered radical
clock substrates. As pointed out by Kochi, such an elec-
tron transfer, which has been assigned a second order rate
constant of ~108 M-1s-1, is an outer sphere process that de-
pends only on the difference between the reduction po-
tential of the oxidizing •Fe3+-OH species and the ioniza-
tion potential of the carbon radical.25 The radical ioniza-
tion potential is itself insensitive to the degree of β-
branching. Indeed, the rate of electron transfer has been
shown not to be affected by substitution adjacent to the
reactive oxo atom of Compound I in CYP105D7. Im-
portantly, al-though C1 of 1-deoxypentalenic acid is also
formally a neopentyl center, it is flanked on the targeted
re face by only one CH3 substituent, attached to C2, while
the other neighboring β-carbon, C8, carries a proton on
the re face. This reduced degree of steric hindrance at C1
of 11 compared to that on the si face of 2 is apparently
sufficient to permit normal oxygen rebound to occur dur-
ing CYP105D7-catalyzed oxidation, allowing formation of
the canonical hydroxylation product, pentalenic acid (10),
in direct contrast to the exclusive electron transfer and
oxidative rearrangement catalyzed by PntM.
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radical center 25
By contrast, Kochi has clearly demon-
.
strated that the inner sphere ligand transfer process,
which corresponds to oxygen rebound, is extremely sensi-
tive to steric effects, including those associated with β-
branching, with neopentyl radicals undergoing ligand
transfer substantially more slowly than isobutyl radicals.25
Thus steric hindrance retards oxygen rebound while having
no effect on the (normally kinetically silent) rate of elec-
tron transfer associated with oxidative formation of the
carbocation. While none of the ingenious radical clock
probes investigated by Ortiz de Montellano9 or by New-
comb10 suppress oxygen rebound by more than 2% rela-
tive to cation formation, the C1si face of pentalenolactone
F represents an extreme, naturally occurring case in
which the initially generated C1 radical is buttressed on
both sides, by the axial C2si-methyl (C-12) and the C7-
vinylidene substituents. (Figure 5). Oxygen rebound with-
in the Heme-•Fe3+-OH/pentalenolactone F-C1 radical pair
is therefore likely to be strongly retarded (probably by a
factor >104) such that the otherwise kinetically insignifi-
cant electron transfer to generate the C1 carbocation
would now become the dominant, indeed essentially ex-
clusive, kinetic process (Scheme 2).26 Rapid rearrange-
ment of the resulting C1 neopentyl cation followed by
deprotonation of H-3re gives pentalenolactone (1), with
competitive formation of the minor rearrangement prod-
ucts, pentalenolactones A (3), B (4), and P (5) (Scheme 1).
Scheme 4. CYP105D7-catalyzed formation of pentalenic
acid (10) by hydroxylation of 1-deoxypentalenic acid (11),
with insertion of oxygen insertion of oxygen into C1-H-1re.
Conclusions. The PntM-catalyzed conversion of pen-
talenolactone F (2) to pentalenolactone (1) involves the
rearrangement of an oxidatively-generated neopentyl cat-
ion that is unique among all known P450-catalyzed reac-
tions. The combined protein structural, site-directed mu-
tagenesis, ligand-binding, steady-state kinetic, and prod-
uct profile data rule out any unusual features of the pro-
tein active site, the heme cofactor, or the mode of sub-
strate binding, while also excluding any special electronic
interactions between substrate and active site residues or
within the substrate itself that might stabilize carbocation
intermediates. Severe steric inhibition of normal oxygen
rebound favors competing internal electron transfer, lead-
ing to generation of the highly unusual neopentyl carbo-
cation intermediate that undergoes rapid rearrangement
and deprotonation.
Pentalenic acid (10) is a commonly occurring shunt me-
tabolite of pentalenolactone biosynthesis that results
from hydroxylation of the early pathway intermediate, 1-
deoxypentalenic acid (11). The formation of 10 involves
stereospecific removal of H-1re with net retention of con-
figuration, in contrast to the abstraction of H-1si of 2 in
the oxidative rearrangement catalyzed by PntM.5 The re-
sponsible P450, CYP105D7 (SAV_7469) has been cloned
and characterized from S. avermitilis.27 Although the re-
cently determined structure of CYP105D7 (PDB ID 4UBS)
has two molecules of diclofenac, rather than the natural
substrate 11, bound in the active site,28 alignment with the
structure of PntM carrying bound pentalenolactone F (2)
reveals that the binding region for the carboxylate of 11
lies on the wall of the active site cavity of CYP105D7 that
is opposite to that established for carboxylate binding by
PntM. Binding of 1-deoxypentalenic acid (11) to CYP105D7
could therefore readily be accommodated by a ca. 180°
rotation with respect to the heme compared to the bind-
ing of 2 to PntM, resulting in orientation of the target C1-
Experimental Methods.
Materials. Isopropylthio-β-galactopyranoside (IPTG),
ampicillin, and kanamycin were purchased from Thermo
Scientific. Chloroform-D (D, 99.8%) was purchased from
Cambridge Isotope Laboratories, Inc. All other chemical
reagents were purchased from Sigma-Aldrich and utilized
without further purification. DNA primers were synthe-
sized by Integrated DNA Technologies. Restriction en-
zymes, Q5® high-fidelity DNA polymerase, and T4 DNA
ligase were purchased from New England Biolabs Inc
(NEB) and used according to the manufacturer’s specifi-
cations. Competent Escherichia coli 10-beta and
BL21(DE3) cloning and expression strains were purchased
from NEB. The pre-charged 5-mL HisTrapTM FF column,
HiloadTM 16/600 SuperdexTM 200 column, and HiprepTM
Q FF 16/10 column were purchased from GE Healthcare
Life Sciences. Amicon Ultra Centrifugal Filter Units
(Amicon Ultra-15 and Amicon Ultra-4 (30,000 MWCO)
and Steriflip® Vacuum-driven Filtration System (0.22 m,
50mL) were purchased from Millipore. Costar® 96-Well
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Microplates were purchased from Thermo Scientific. The
Thrombin CleanCleaveTM Kit was purchased from Sigma-
Aldrich. CrystalEX second generation Corning® 96-well
plates and Cryschem 24-well sitting drop plates were pur-
chased from Hampton Research. Plasmids pET28a-penM
and pET28a-pntM used for protein expression and the
strain S. exfoliatus ZD22 used for isolation of pentaleno-
lactone F were prepared by Dr. Dongqing Zhu, as previ-
ously described.4
off and eluted with benzene to recover purified 2-Me (170
1
mg) which was identical to authentic 2-Me by H and 13C
1
2
3
4
5
6
7
8
NMR and GC-MS.
6,7-Dihydropentalenolactone F methyl ester (7-
Me). Purified 2-Me (100 mg) was dissolved in anhydrous
methanol (50 mL) in a 250-mL high-pressure hydrogena-
tion bottle. After adding 20 mg of 5% Pd/C powder, the
bottle was filled with hydrogen gas (22 psi) and shaken for
24 h. The mixture was filtered through Celite and then
concentrated under reduced pressure. The hydrogenated
products were separated on a silica gel column eluted
9
Methods. General methods were as previously de-
scribed.29 Growth media and conditions used for E. coli
and Streptomyces strains and standard methods for han-
dling strains were those described previously, unless oth-
erwise noted.30 All DNA manipulations performed follow-
ing standard procedures.30b DNA sequencing was carried
out at the U. C. Davis Sequencing Facility, Davis, CA or by
Genewiz. All proteins were handled at 4 °C, unless other-
wise stated. Protein concentrations were determined ac-
cording to the method of Bradford,31 using a Tecan Infi-
nite M200 Microplate Reader with bovine serum albumin
as the standard. Protein purity and size was estimated
using both SDS-PAGE gel electrophoresis and an ATKA
FPLC System. Accurate protein molecular weight was
determined by ESI-MS on an Agilent 6530 Accurate-Mass
Q-TOF LC/MS equipped with a Phenomenex Jupiter C4
column (50 mm x 2.00 mm, 5 ꢀm). Substrate binding as-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
with ~900 mL benzene:ethyl acetate (15:1). 7-Me H NMR
(600 MHz, CDCl3) δ 1.03 (s, 3H), 1.15 (s, 3H), 1.41 (dd, J =
13.0, 7.5 Hz, 1H), 1.65 (dd, J = 13.0, 8.2 Hz, 1H), 1.74 (d, J =
13.9 Hz, 1H), 1.85 (d, J = 13.9 Hz, 1H), 2.04 (m, 1H), 2.24 (m,
1H), 2.32 (m, 1H), 2.81 (q, J = 8.7 Hz, 1H), 3.00 (m, 1H), 3.01
(d, J = 5.1 Hz, 1H), 3.11 (d, J = 5.1 Hz, 1H), 3.75 (s, 3H), 4.18
(dd, J = 12.0, 7.4 Hz, 1H), 4.64 (dd, J = 12.0, 6.2 Hz, 1H); 13C
NMR (150 MHz, CDCl3) δ 29.56, 30.56, 35.34, 41.26, 45.13,
47.50, 48.69, 50.32, 51.05, 51.13, 52.27, 56.57, 58.25, 68.43,
170.39, 174.22. HR-ESI-MS [M+H]+ 7-Me m/z 295.1548
(calculated for C16H23O5 : 295.1545). (Figures S2 and S3).
Hydrolysis of methyl esters to pentalenolactone
(1),
pentalenolatone
F
(2),
and
6,7-
dihydropentalenolactone F (7). The methyl esters 1-
Me, 2-Me, and 7-Me were each dissolved in 4:1 THF:H2O
and 2 eq. LiOH was added to the solution. After 30 min at
0 °C, the reaction mixture was warmed to room tempera-
ture and incubated an additional 2 h. After acidification
with 2 N HCl to pH 2, the quenched reaction mixture was
extracted 3X with CHCl3 and the combined organic ex-
tracts were concentrated to dryness in vacuo. The typical
yields were quantitative and the resulting carboxylic acids
1, 2, and 7 were dissolved in buffer and directly used with-
out further purification.
1
says were carried out on the Tecan Microplate Reader. H
and 13C NMR spectra were obtained on a Bruker Avance
III HD Ascend 600 MHz spectrometer. GC-MS analyses
were carried out using an Agilent Technologies 5977A
MSD with an Agilent Technologies 7890B GC system.
Isolation of pentalenolactone F (2). Streptomyces ex-
foliatus ZD22, from which the penM gene had been delet-
ed, was grown on Mannitol soya flour (MS) agar for 6
days, as previously described.4 The spores were harvested
and used to inoculate a seed culture in a liquid medium
containing 2.5% Pharmamedia and 2.5% bactodextrose.
After incubation at 28 °C for 3 days, 10 mL of seed culture
was added to 1 L fermentation medium containing 0.2%
NaCl, 0.5% CaCO3, 1% corn gluten meal, 0.1125% bacto-
dextrose, 0.2% blackstrap molasses, and 2% corn starch,
pH 7.2. After another 3 days shaking at 28 °C, the culture
was centrifuged and the cells were washed with water and
then discarded. The combined aqueous layers were acidi-
fied to pH 2.6 with HCl and extracted with chloroform.
The organic extract was dried over Na2SO4, concentrated
in vacuo, and the crude product was dissolved in 3 mL of
methanol and 7 mL of benzene. For methylation, 0.5 mL
trimethylsilyldiazomethane (TMS-CHN2) was added and
the solution was stirred at room temperature for 30 min.
A mixture of pentalenolactone F methyl ester (2-Me) and
9-epi- pentalenolactone F methyl ester (~2:1) was ob-
tained after flash silica gel column chromatography of the
crude methyl esters (600 mL benzene:ethyl acetate/10:1).
The concentrated mixture of isomeric methyl esters (260
mg) was applied to a 20 cm x 20 cm Silica gel 60 F254 TLC
plate which was developed with benzene:ethyl:acetate
(5:1). The band containing 2-Me (Rf 0.55-0.65) was scraped
Substrate binding assays with wild-type PntM and
mutants. Wild-type or mutant PntM (17 mg/mL) was
diluted 20x in 50 mM Tris-HCl (pH 8.0) to a final concen-
tration of 19 ꢀM. For binding assays with wild-type PntM,
2 (72 mM in THF) or 7 (86 mM in THF) were each dilut-
ed into the same buffer to final concentrations of 50 ꢀM,
25 ꢀM, 10 ꢀM, 2.5 ꢀM, and 1 ꢀM for 2 and 200 ꢀM, 100 ꢀM,
50 ꢀM, 25 ꢀM, 12.5 ꢀM, and 3.13 ꢀM for 7. For binding
assays with mutants of PntM, the dilution series for 2 en-
compassed 2 mM, 1 mM, 500 ꢀM, 250 ꢀM and 200 ꢀM.
Diluted protein (135 ꢀL) and diluted 2 or 7 (15 ꢀL) were
mixed in 96-well plates. At least 3 sets of UV-Vis absorp-
tion spectra (350-500 nm) were recorded for each assay
concentration and UV difference spectra were obtained
for ligand-bound compared to ligand-free protein (Fig-
ures S4 and S5). Saturation curves were generated from
the absorption maxima at 390 nm (substrate bound) and
at 420 nm (substrate free) and used to calculate KD by
non-linear squares fitting to the equation ꢁA
=
ꢁAmax•([L]/([L] + KD)) (n=3) (Figure S6). The reported
errors are the standard deviations calculated by fitting
program.
Steady-state kinetic analysis of PntM and mutants.
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with 4 mL dichloromethane and the combined organic
a) GC-MS calibration curve for 1-Me. A reference se-
1
2
3
4
5
6
7
8
ries of increasing concentrations of 1-Me (4.8, 9.7, 14.5,
19.4, and 48.4 ꢀM) was mixed with 10 ꢀM farnesol as in-
ternal standard and analyzed by GC-MS using an Agilent
5977A GC-MS and Agilent HP-5MS column with a tem-
perature program from 40 °C - 240 °C with an increase of
5 °C per min. For data collection, farnesol was quantitated
by Selected Ion Monitoring (SIM), up to 35 min using SIM
Group A, (consisting of mass fragments m/z 41.10, 55.05,
61.00, 69.05, 81.00, 93.00, 107.00). 1-Me was quantitated
after 35 min using SIM Group B (consisting of mass frag-
ments m/z 77.00, 91.00, 105.00, 115.00, 127.00, 128.00,
129.00, 130.00, 131.00, 143.00, 145.00, 157.00, 171.00, 202.00,
290.00). The SIM peak areas for the farnesol internal
standard and 1-Me were individually summed and then
used to calculate the calibration curve of normalized peak
area versus concentration for 1-Me (Figure S7).
extracts were dried over Na2SO4. The concentrated ex-
tract was dissolved in 200 ꢀL methanol and treated with
trimethylsilydiazomethane (4 ꢀL, TMS-CHN2, 2 M solu-
tion in hexane) to generate the corresponding methyl
esters. GC-MS analysis using an Agilent 5977A GC-MS
and Agilent HP-5MS column and temperature program
from 40 - 240 °C with an increase of 5 °C per min showed
the same product profile as that for oxidation of 2 by
wild-type PntM, with the exclusive product being 1-Me.
No additional peaks with [M+]+ m/z 308 or [M-H2O]+ m/z
290 corresponding to any hydroxylated derivative of 2-Me
were detected.
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Incubation of dihydopentalenolactone F (7) with
PntM and GC-MS product analysis. Freshly prepared
and purified PntM (10 ꢀL, 360 ꢀM final concentration)
was added to 20 mM Tris-HCl buffer (pH 8.0, 2.5% glyc-
erol) containing 7 (1.1 mM), 20 ꢀL spinach ferredoxin-
NADPH oxidoreductase (12.5 U/mL), 50 ꢀL spinach ferre-
doxin (2.2 mg/mL) in total volume of 4 mL. The reaction
was initiated by addition of 100 ꢀL NADPH (50 mM) and
incubated for 2 h at 30 °C. The reaction was quenched by
addition of 20 ꢀL 2 N HCl and the mixture was extracted
(3X) with 2 mL dichloromethane. The combined organic
layers were dried over anhydrous Na2SO4, concentrated
b) Steady-state kinetics. Kinetic assays were carried
out at 30 °C using wild-type PntM (3.8 ꢀM), and mutants
F232A (4.4 ꢀM), F232G (15.2 ꢀM), F232L (5.5 ꢀM), M81A
(12.4 ꢀM), M81C (7.2 ꢀM), M81MBE (12 ꢀM) or M77S (10.3
ꢀM) in 20 mM Tris-HCl buffer (pH 8.0) containing 2.5%
glycerol, 20 ꢀg/mL spinach ferredoxin, 0.05 U/mL spinach
ferredoxin-NADP reductase, pentalenolactone F (2) (14.4 -
144 ꢀM) and farnesol (1 ꢀM) as internal standard in a total
volume of 1.00 mL. The reactions were initiated by addi-
tion of 1 mM NADPH, incubated for 20 min at 30 °C, then
quenched with 5 ꢀL of 2 N HCl to give a final pH ~2. The
products were extracted 2X with 1 mL dichloromethane
and the combined organic extracts were dried over
Na2SO4. The concentrated extract was dissolved in 100 ꢀL
methanol and treated with trimethylsilydiazomethane (1
ꢀL, TMS-CHN2, 2 M solution in hexane) to generate the
corresponding methyl ester, 1-Me. Samples were analyzed
and quantitated by GC/MS using the above-described
SIM method, using farnesol as internal standard and the
calibration curve for 1-Me to obtain the observed initial
velocities of formation of 1-Me for each incubation. The
steady-state kinetic parameters were calculated by direct
non-linear least squares fitting to the Michaelis-Menten
equation (SigmaPlot 12.5 (Table 2, Figure S8). The record-
ed error limits represent the statistical deviations calcu-
lated by the data fitting program (n=4, except for F232A
for which n=1).
and
dissolved
in
100
ꢀL
methanol.
Tri-
methylsilydiazomethane (10 ꢀL) was added to generate
the methyl esters and the products were analyzed by GC-
MS using the column and temperature program described
above.
Preparation of PntM mutants. Site-directed muta-
genesis was carried out with the Quikchange II XL site-
directed mutagenesis kit (Agilent) using plasmid DNA
pET28a/PntM as template and PCR primer pairs listed in
Table S3. The expression and purification steps were same
as those used for wild-type PntM. The mutant M81C-BME
was generated during purification of PntM M81C using
the standard buffer containing β-mercaptoethanol. To
obtain unmodified PntM M81C, only buffers without β-
mercaptoethanol used. The DNA sequences of all mutant
constructs were verified directly. The purity of each mu-
tant protein was verified by SDS-PAGE (Figure S1) and the
protein MW determined by LC-ESI-HRMS (Table S4).
Purification of PntM for X-ray crystallography.
Fresh clones of E. coli BL21 containing plasmid pET28a-
pntM were grown on LB agar media with 50 ꢀg/mL kan-
amycin. LB media (5 mL) with 50 ꢀg/mL kanamycin was
seeded with a single colony and incubated overnight. The
seed culture (5 mL) was added to 500 mL Terrific Broth
(TB) containing 50 ꢀg/mL kanamycin and incubated at 37
°C until an OD >0.6, then induced with 0.4 mM IPTG.
The cells were further grown at 18 °C for 24 h, harvested
by centrifugation and then suspended in lysis buffer (100
mM Tris-HCl, 500 mM NaCl, 0.1 mM DTT, 2.7 mM β-
mercaptoethanol, 10 mM imidazole, pH 8.0) containing 10
mg/L pepstatin, 10 mg/L PMSF, and 0.2 mg/mL benzami-
dine. The cells were broken using a French press at 10000
psi and separated by centrifugation at 15000g for 1 h. The
Preparative incubation of PntM and mutants with
2 or 7 and GC-MS analysis of products. Preparative-
scale incubations were carried out using wild-type PntM
(3.8 ꢀM), or with mutants F232A (4.4 ꢀM), F232G (15.2
ꢀM), F232L (5.5 ꢀM), M81A (12.4 ꢀM), M81C (7.2 ꢀM),
M81MBE (12 ꢀM) or M77S (10.3 ꢀM) in 20 mM Tris-HCl
buffer (pH 8.0) containing 2.5% glycerol, 20 ꢀg /mL spin-
ach ferredoxin, 0.05 U/mL spinach ferredoxin-NADP re-
ductase, pentalenolactone F (14.4 - 144 ꢀM and farnesol (1
ꢀM) as internal standard in a total volume of 4.00 mL.
The reactions were initiated by adding 1 mM NADPH,
then incubated for 4 h at 30 °C to maximize substrate
consumption. The reaction was quenched with 16 ꢀL of 2
N HCl to a final pH ~2. The products were extracted 2X
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supernatant was applied to a Ni-NTA column which was Å, and c = 83.51 Å. Diffraction data to 2.07 Å for PntM-
1
2
3
4
5
6
7
8
first washed with buffer A (100 mM Tris-HCl, 20 mM im-
idazole, 2.7 mM β-mercaptoethanol pH 8.0) and then
eluted with buffer B (100 mM Tris-HCl, 500 mM imidaz-
ole, 2.7 mM β-mercaptoethanol pH 8.0. The collected
His6-tag-PntM protein was concentrated and diluted with
thrombin digestion buffer (50 mM Tris-HCl, 10 mM CaCl2,
pH 8.0), then incubated with thrombin-agarose (1 mL)
overnight (10 h) at 4 °C. The digested protein was reload-
ed onto Ni-NTA, and the flow-through protein lacking
the His6-tag was then applied to a size exclusion column
(Hiload 16/600, Superdex 200 pg) for further purification.
The purified protein was concentrated to 16.8 mg/mL and
the molecular weight and purity confirmed by LC-ESI-MS
(Table S4).
M81A with bound 2 were collected in space group P21212
with cell dimensions a = 44.62 Å, b = 163.86 Å, and c =
82.22 Å. Diffraction data to 2.12 Å for PntM-M81C with
bound 2 were collected in space group P21212 with cell
dimensions a = 44.56 Å, b = 164.23 Å, and c = 82.97 Å. Dif-
fraction data to 2.06 Å for PntM-M81C-BME with bound 2
were collected in space group P21212 with cell dimen-
sions a = 44.57 Å, b = 163.23 Å, and c = 83.29 Å. A single
crystal was used for each data set. The diffraction images
were integrated with the XDS package32 and scaled with
pointless/aimless from the CCP4 suite.33 The data pro-
cessing statistics are summarized in Tables S1 and S2.
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Structure determination and refinement. The PntM
structure was solved by molecular replacement with the
program Phaser34 from the Phenix program suite35 in
space group P21212 using the polyene macrolide epoxidase
PimD (Pdb code 2X9P) as search model (data set PntM).
The model was further rebuilt with ARP/wARP36 and
manually checked and completed with Coot.37 Final crys-
tallographic refinement was performed with the program
phenix.refine.35 The other structures were subsequently
solved by molecular substitution. Geometry restraints for
ligands 1, 2 and 7 were obtained from PRODRG.38 The
crystallographic R/Rfree factors are 0.14/0.17, 0.14/0.18,
0.16/0.19, 0.18/0.22, 0.15/0.19, 0.16/0.19, 0.15/0.18, 0.16/0.20
and 0.16/0.19 for the nine data sets: PntM, PntM with 2,
PntM with 7, PntM with 1, PntM-F232L with 2, PntM-
M77S with 2, PntM-M81A with 2, PntM-M81C with 2 and
PntM-M81C-BME with 2, respectively. The Ramachandran
statistics (most favored/outliers) are 98%/0%, 99%/0%,
98%/0%, 97%/0%, 98%/0%, 98%/0%, 98%/0%, 98%/0%
and 98%/0%, for PntM, PntM with 2, PntM with 7, PntM
with 1, PntM-F232L with 2, PntM-M77S with 2, PntM-
M81A with 2, PntM-M81C with 2, and PntM-M81C-BME
with 2, respectively. The refinement statistics are summa-
rized in Tables S1 and S2. Protein figures were generated
using Pymol.39
Protein crystallization – wild-type PntM. Crystals of
PntM were obtained at 15 °C using the sitting drop vapor
diffusion method. The reservoir solution contained 1.2 M
sodium citrate, 10% glycerol, 100 mM bicine, pH 9.0. Pro-
tein solution (1 ꢀL, 16.8 mg/mL PntM, 10 mM Tris-HCl, 15
mM NaCl, and 10% glycerol) was mixed with 1 ꢀL of reser-
voir solution. After growing for 7 days, crystals were flash-
frozen in liquid nitrogen and stored. PntM with bound 2,
7, or 1 was obtained by soaking PntM crystals for 2 h into
a 2-ꢀL drop containing mother liquor with 2 mM 2, 7, or 1
and 5% DMSO.
Protein crystallization – PntM mutants. A cross-
microseeding technique was used to get larger crystals for
mutants. Small pieces of PntM crystals were crushed into
tiny seeds in 500 ꢀL mother buffer to make the seeding
solution. Protein solution (1 ꢀL, 10 mM Tris-HCl, 15 mM
NaCl, and 10% glycerol) was mixed with 1 ꢀL of seeding
solution. After growing for 4 days, crystals were flash-
frozen in liquid nitrogen and stored. Mutant PntM pro-
teins complexed with 2 were obtained by soaking crystals
for 2 h in a 2-ꢀL drop containing mother liquor with 2
mM 2 and 5% DMSO .
X-ray crystallographic data collection. X-ray diffrac-
tion data for all the crystals were collected with a Rigaku
Saturn 944+ CCD detector on a Rigaku FR-E Superbright
rotating anode source at the Brown University Structural
Biology Core Facility. All data were collected at a wave-
length of 1.542 Å and 100 K. Diffraction data for PntM in
space group P21212 were collected to 2.00 Å resolution
with cell dimensions a = 44.46 Å, b = 164.59 Å, and c =
83.30 Å. Diffraction data to 2.03 Å for PntM with 2 were
collected in space group P21212 with cell dimensions a =
44.75 Å, b = 164.48 Å, and c = 83.29 Å. Diffraction data to
2.03 Å for PntM with 7 were collected in space group
P21212 with cell dimensions a = 44.53 Å, b = 164.37 Å,
and c = 82.04 Å. Diffraction data to 2.28 Å for PntM with 1
were collected in space group P21212 with cell dimen-
sions a = 44.33 Å, b = 163.76 Å, and c = 81.34 Å. Diffraction
data to 2.08 Å for PntM-F232L with bound 2 were collect-
ed in space group P21212 with cell dimensions a = 44.60
Å, b = 164.15 Å, and c = 82.54 Å. Diffraction data to 2.08 Å
for PntM-M77S with bound 2 were collected in space
group P21212 with cell dimensions a = 44.68 Å, b = 164.60
ASSOCIATED CONTENT
Supporting Information.
Tables of crystallographic statistics, H and 13C NMR spectra
1
for 7-Me, , UV difference spectra for ligand binding by wild-
type PntM and mutants, ESI-MS data for PntM and mutants,
steady-state kinetic plots, and GC-MS calibration plots, and
additional protein structural figures. This material is availa-
ble free of charge via the Internet at http://pubs.acs.org. The
coordinates for substrate-free PntM (PDB ID 5L1R), PntM
with bound 2 (5L1O), PntM with bound 1 (5L1P), PntM with
bound 7 (5L1Q), and the PntM mutants F232L (5L1S), M77S
(5L1T), M81A (5L1U), M81C (5L1V), and M81C-BME (5L1W,
each with bound 2, are available from the RCSB Protein Data
Bank (http://www.rcsb.org/pdb).
AUTHOR INFORMATION
Corresponding Author
*To whom correspondence should be addressed: David E.
Cane, Department of Chemistry, Brown University, Box H,
Providence, Rhode Island 02912-9108, USA; Gerwald Jogl,
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Journal of the American Chemical Society
Department of Molecular Biology, Cell Biology and Biochem-
istry, Brown University, Providence, Rhode Island 02912, USA
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(14) Keating, J. T.; Skell, P. S. Carbonium Ions 1970, 2, 573-653.
Author Contributions
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(15) a) Cane, D. E.; Sohng, J. K.; Williard, P. G. J. Org. Chem.
1992, 57, 844-852. b) Strictly speaking, the isolation of 3, 4, and 5
from cultures of S. exfoliatus is simply consistent with, but does
not prove, that these minor shunt metabolites are formed by
PenM. Since individually they represent less than 0.1% of the
total pentalenolactones produced by S. exfoliatus, they have not
been detected by GC-MS analysis of in vitro PenM- or PntM-
catalyzed oxidation of 2.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
ACKNOWLEDGMENT
This work was supported by a grant from the U. S. National
Institutes of Health, GM022172, to D.E.C.
(16) Attempts to remove bound bicine by soaks in bicine-free
buffers under a variety of conditions resulted in collapse of the
protein crystals.
9
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