Generation of complexity in fungal terpene biosynthesis: Discovery of a multifunctional cytochrome P450 in the fumagillin pathway

Article abstract of DOI:10.1021/ja500881e

Fumagillin (1), a meroterpenoid from Aspergillus fumigatus, is known for its antiangiogenic activity due to binding to human methionine aminopeptidase 2. 1 has a highly oxygenated structure containing a penta-substituted cyclohexane that is generated by oxidative cleavage of the bicyclic sesquiterpene β-trans-bergamotene. The chemical nature, order, and biochemical mechanism of all the oxygenative tailoring reactions has remained enigmatic despite the identification of the biosynthetic gene cluster and the use of targeted-gene deletion experiments. Here, we report the identification and characterization of three oxygenases from the fumagillin biosynthetic pathway, including a multifunctional cytochrome P450 monooxygenase, a hydroxylating nonheme-iron-dependent dioxygenase, and an ABM family monooxygenase for oxidative cleavage of the polyketide moiety. Most significantly, the P450 monooxygenase is shown to catalyze successive hydroxylation, bicyclic ring-opening, and two epoxidations that generate the sesquiterpenoid core skeleton of 1. We also characterized a truncated polyketide synthase with a ketoreductase function that controls the configuration at C-5 of hydroxylated intermediates.

Full text of DOI:10.1021/ja500881e

Article
pubs.acs.org/JACS
Generation of Complexity in Fungal Terpene Biosynthesis: Discovery
of a Multifunctional Cytochrome P450 in the Fumagillin Pathway
Hsiao-Ching Lin,† Yuta Tsunematsu,‡ Sourabh Dhingra,§ Wei Xu,† Manami Fukutomi,‡ Yit-Heng Chooi,⊥
David E. Cane,¶ Ana M. Calvo,§ Kenji Watanabe,‡ and Yi Tang*,†,#
†Department of Chemical and Biomolecular Engineering, and #Department of Chemistry and Biochemistry, University of California,
Los Angeles, California 90095
‡Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan
§Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115, United States
⊥Research School of Biology, Australian National University, Canberra, ACT 0200, Australia
¶Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912, United States
S
* Supporting Information
ABSTRACT: Fumagillin (1), a meroterpenoid from Aspergillus
fumigatus, is known for its antiangiogenic activity due to binding to
human methionine aminopeptidase 2. 1 has a highly oxygenated
structure containing a penta-substituted cyclohexane that is generated
by oxidative cleavage of the bicyclic sesquiterpene β-trans-bergamo-
tene. The chemical nature, order, and biochemical mechanism of all
the oxygenative tailoring reactions has remained enigmatic despite the
identification of the biosynthetic gene cluster and the use of targeted-
gene deletion experiments. Here, we report the identification and
characterization of three oxygenases from the fumagillin biosynthetic
pathway, including a multifunctional cytochrome P450 monooxyge-
nase, a hydroxylating nonheme-iron-dependent dioxygenase, and an ABM family monooxygenase for oxidative cleavage of the
polyketide moiety. Most significantly, the P450 monooxygenase is shown to catalyze successive hydroxylation, bicyclic ring-
opening, and two epoxidations that generate the sesquiterpenoid core skeleton of 1. We also characterized a truncated polyketide
synthase with a ketoreductase function that controls the configuration at C-5 of hydroxylated intermediates.
INTRODUCTION
■
Cytochrome P450 enzymes (P450s) are heme-containing
monooxygenases that catalyze a variety of oxidative trans-
formations in primary and secondary metabolism.1 P450
enzymes are widely distributed in natural product biosynthetic
pathways from bacteria, fungi, and plants. These enzymes play a
key role in the generation of structural complexity, including
hydroxylation by activation of C−H bonds,2 epoxidation,2b C−
C bond cleavage,3 and functional group rearrangement/
migration.4 In addition, P450s from secondary metabolism
have also been shown to catalyze less common reactions,
Figure 1. Examples of natural products of which multifunctional P450s
including recent examples of heterocycle formation1b and
play important roles in the biosynthesis. Functional groups introduced
cationic terpene cyclization.5 An increasing number of oxidative
by P450s are shown in red.6a,d,7,8
enzymes have been shown to catalyze multistep oxidation at
distinct carbon atoms of substrates, resulting in drastic
structural transformations (Figure 1).5,6 For example, during
novel multifunctional P450s from nature is of both fundamental
mechanistic significance and practical importance.
Fumagillin (1) from Aspergillus fumigatus is a meroterpenoid
that has numerous biological activities. Compound 1 and its
derivatives have been intensely studied for their potential use in
lovastatin biosynthesis, LovA catalyzes consecutive oxidations
at opposite sides of the decalin ring to yield the precursor
monacolin J,7 while TamI catalyzes successive hydroxylation
and epoxidation reactions to afford the bicyclic ketal system in
tirandamycin.6d Considering the central roles of such P450s in
biosynthetic pathways, as well as the potential applications of
these enzymes as C−H activation biocatalysts, discovery of
Received: January 26, 2014
Published: February 26, 2014
© 2014 American Chemical Society
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the treatment of amebiasis,9 microsporidiosis,10 rheumatoid
arthritis,11 and for their antiangiogenic properties by the
irreversible inhibition of human type 2 methionine amino-
peptidase (MetAP2).12,13 Structurally, 1 consists of a highly
oxygenated, rearranged sesquiterpene esterified to a polyketide-
derived tetraenoic diacid. The terpenoid portion, fumagillol (2)
is derived by cleavage and multiple oxidation of the bicyclic
sesquiterpene hydrocarbon intermediate β-trans-bergamotene
(3)14 (Figure 2). The transformation of 3 to 2 involves a
Figure 2. Multiple oxidative-tailoring steps in fumagillin (1)
biosynthesis.
dramatic skeletal rearrangement in which the strained cyclo-
butane bridge is opened to yield the 1,8-bisepoxide-containing
cyclohexanediol with six contiguous stereocenters. Therefore,
given the unusual C−C cleavage and the requisite pair of C−H
activation reactions starting from 3, it appeared likely that a
P450 might be centrally involved in the biosynthesis of 2 and 1.
We recently discovered the biosynthetic gene cluster for 1 in
A. fumigatus.15 This fma cluster contains the first reported
membrane bound class I terpene cyclase (Fma-TC) that
catalyzes the formation of 3 from farnesyl pyrophosphate
(FPP). The fma cluster also encodes a polyketide synthase
(Fma-PKS) that synthesizes a dodecapentaenoate precursor
that is trans-esterified to 2 by the acyltransferase Fma-AT to
yield the intermediate prefumagillin 4.15 It was proposed that
the polyene portion of 4 would be oxidatively cleaved to yield
1. Following our discovery, Wiemann et al. reported that the
fma gene cluster is embedded within a supercluster on
chromosome 8 of A. fumigatus that also encodes the
biosynthetic pathways for fumitremorgin and pseurotin.16 The
fma gene cluster (Figure 3A) contains four oxygenases that are
most likely responsible for the oxidative tailoring steps that
transform 3 to 1, including Af470 (Antibiotic Biosynthesis
Monoxygenase superfamily monooxygenase), Af480 (nonheme
iron-dependent dioxygenase), Af510 (cytochrome P450 mono-
oxygenase) and Af440 (flavin-binding monooxygenase/meth-
yltransferase). An additional redox enzyme in the fma gene
cluster is the partial PKS (Af490), in which only the
dehydratase (DH) and ketoreductase (KR) domains are
present. The mechanistic role of each of these enzymes,
especially that of the P450 (encoded by Af510), which is
hypothesized to play a central role in the conversion of 3 to 2,
has remained unknown.
Figure 3. Genetic verification of Af510, Af480, Af470, and Af490. (A)
The fma gene cluster. (B) HPLC analysis of metabolites extracted
from isogenic control and ΔAf510, Af480, and Af470 strain showing
loss of 1 and accumulation of 5 from ΔAf480 and 4 from ΔAf470
strain, respectively. (C) GC-FID analysis of the ΔAf510 strain showing
the accumulation of 3.
spectacular range of catalytic prowess, including hydroxylation
of 3, oxidative cleavage of the bridging cyclobutane ring, and
two epoxidation reactions. In addition to the on-pathway
intermediates, we also demonstrate the range of off-pathway
compounds that can be obtained using P450 starting from 3.
Another notable finding includes the functional assignment of
Af490 as a stereoselective ketoreductase in the biosynthesis of
2.
RESULTS
■
Gene Inactivation Studies and Identification of
Intermediates. We first set out to determine the roles and
timing of the several redox enzymes in the fma gene cluster.
We individually deleted five genes, Af510, Af490, Af480, Af470,
and Af440 in A. fumigatus CEA17 akuBKU80 strain (pyrG89,
ΔakuBKU80), which is deficient in nonhomologous end joining
(Supporting Information).17 In comparison to the isogenic
control strain, the metabolic profile of ΔAf510, ΔAf480 and
ΔAf470 showed complete abolishment of the formation of 1
(Figure 3B), while ΔAf440 retained production of 1. The
ΔAf490 strain showed decreased titers of 1 (86% decreasing in
the culture of 4 days using CYA medium). The nonessential
role of Af440 is in accordance with that reported by Wiemann
et al., in which Af440 (which corresponds to psoF) was shown
to be involved in pseurotin biosynthesis.16 However in contrast
to the Wiemann report, in the analysis of the ΔAf470 profile,
we found the accumulation of 4 with m/z 471 [M+Na]+
(Figure 3B), therefore suggesting a role for Af470 in the
oxidative cleavage of the terminal alkene of the dodecapentae-
noate side chain into the carboxylic acid present in 1. On the
other hand, deletion of Af480 led to the accumulation of a
Here we describe the complete characterization of the
biosynthetic pathway of fumagillin (1). Using a combination of
genetic knockout, chemical complementation, heterologous
reconstitution in Saccharomyces cerevisiae, and in vitro
biochemical assays, we have identified the role of each of the
required pathway enzymes and biosynthetic intermediates. We
show that Af510 indeed encodes a multifunctional P450 with a
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polyene-containing compound 5, with maximum UV absorb-
ance at 333 nm and m/z 451 [M+Na]+ (Figure 3B).
Compound 5 was isolated from a 6-day liquid culture of A.
fumigatus ΔAf480 mutant strain and structurally characterized.
1
On the basis of the H, 13C and 2D NMR analyses, 5 was
elucidated as 6-demethoxyfumagillin, in contrast to an aldehyde
shunt product reported by Wiemann et al.16 The structure of 5
revealed that the nonheme iron-dependent oxygenase encoded
by Af480 catalyzes hydroxylation of C6 in the biosynthesis of 1.
The only remaining unassigned oxygenase in the gene cluster
was therefore the P450 gene, Af510, which shows the strongest
sequence homology to the multifunctional P450 OrdA18 in
aflatoxin biosynthesis (47% protein identity). Wiemann et al.
reported that deletion of Af510 in A. fumigatus led to no
detectable accumulation of any intermediates. While the same
phenotype was observed here using LC−MS assay conditions, a
more careful extraction with hexane and analysis of the
hydrocarbon extract by GC-FID revealed the presence of β-
trans-bergamotene (3) (Figure 3C). In the isogenic control, no
accumulation of 3 was observed. This result suggested that the
initial oxidation of 3 is catalyzed by the enzyme encoded by
Af510.
Verification of Activities of Enzymes Encoded by
Af470 and Af480. The enzyme encoded by Af470 (Fma-
ABM) was initially revealed to harbor a DUF4188 conserved
domain of unknown function by NCBI Conserved Domain
Search. A further homology modeling search with Phyre219
showed, however, that Fma-ABM appears to be related to the
cofactor-independent ABM superfamily monoxygenases with a
ferrodoxin-like fold.20 To further test the role of Fma-ABM, we
cloned the intron-less Af470 (for reconstruction of the gene
from mRNA, see Supporting Information) into a yeast 2 μm
expression vector which was transformed into S. cerevisiae strain
BJ5464-NpgA. Upon supplementing 4 to the yeast culture
expressing Af470, the biotransformation into 1 was detected
(Figure 4A). On the basis of protein structure prediction
(Supporting Information, Figure S7A), Fma-ABM was
predicted to be a membrane-bound protein. Upon purification
of the microsomal fractions of the yeast strain expressing Fma-
ABM and incubation with 4, the same conversion to 1 was
observed (Figure 4B). These results confirmed the role of Fma-
ABM in the oxidative cleavage of 4 to 1.
To confirm the role of the enzyme encoded by Af480 (Fma-
C6H), the 6xHis-tagged enzyme was solubly expressed from E.
coli BL21 (DE3) and purified to homogeneity (Supporting
Information, Figure S5). Bioinformatics analysis showed that
Fma-C6H shares a similar sequence with the members of the
phytanoyl-CoA dioxygenase (PhyH) superfamily, thereby
requiring Fe (II) and α-ketoglutarate (α-KG) for its activity.
Incubation of recombinant Fma-C6H with α-ketoglutarate,
sodium ascorbate, and substrate 5, however, did not lead to the
formation of any hydroxylated product. We therefore reasoned
that Fma-C6H may function upstream in the pathway prior to
attachment of the polyene portion. To obtain such a substrate,
the hydrolysis of 5 under basic condition was performed to
afford 6-demethoxyfumagillol (6) (Supporting Information).
When recombinant Fma-C6H and 6 were incubated under the
same condition as above, complete conversion of 6 to 6-
hydroxylfumagillol (7) was obtained (Figure 4C). Furthermore,
coincubation of Fma-C6H with the recombinant methyltrans-
ferase (Fma-MT) encoded by Af390−400 in the presence of
the necessary cofactors (α-ketoglutarate, sodium ascorbate, and
SAM) and 6 resulted in the formation of the expected 2 (Figure
Figure 4. Verification of the function of Fma-ABM, Fma-C6H, and
Fma-MT. (A) LC−MS detection of 1 upon expression of Fma-ABM
in S. cerevisiae BJ5464-NpgA with supplement of 4. S. cerevisiae control
is untransformed BJ5464-NpgA. (B) LC−MS analysis of in vitro assays
of yeast microsomes containing Fma-ABM toward 4. (C) LC−MS
analysis of in vitro assays of Fma-C6H with 6; and combined Fma-
C6H and Fma-MT with 6, respectively.
4C). Collectively, these studies confirmed the function of Af480
and revealed that 6 (or a related compound) may be a key
intermediate in the biosynthetic pathway of 1. To test this
hypothesis, feeding of 6 to A. fumigatus blocked in
bergamotene formation (deletion of Fma-TC, ΔAf520)
restored biosynthesis of 1 as well as the production of 5 (see
compilation of chemical complementation traces in Figure 7).
With this information in hand, we then turned to investigation
of the possible routes of oxidative modification of 3 into 6.
Yeast Reconstitution of Fma-P450 Activity and
Characterization of Products. The transformation of 3
into 6 requires at least three oxidation steps to introduce the
one C5 hydroxyl group and the two epoxide functionalities. In
addition, the cleavage of the C−C bond between C5 and C8
must also take place. Several proposals for this mechanistically
unresolved transformation have been advanced, including
desaturation of the C5−C6 bond followed by concomitant
cleavage of the C5−C8 bond.21 In light of the functional
assignment of Af440, Af470, and Af480 described above, it
appeared likely that the P450 encoded by Af510 (Fma-P450) is
a multifunctional P450 that is responsible for most, if not all, of
the oxidative steps between 3 and an advanced intermediate
such as 6.
We previously showed that expression of Fma-TC alone in S.
cerevisiae can lead to accumulation of β-trans-bergamotene
(3).15 To investigate the activity of Fma-P450 toward 3, we
cloned the intron-less Af510 into a yeast 2 μm expression
vector for microsomal expression. To equip Fma-P450 with the
optimal redox partner, we also cloned the A. fumigatus
cytochrome P450 oxidoreductase (AfCPR) for coexpression.22
All three genes (encoding Fma-TC, Fma-P450, and AfCPR)
were placed under the ADH2 promoter and transformed into
BJ5464-NpgA. After four days of culturing followed by
extraction with hexanes/EtOAc (1:1), we observed a series of
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sesquiterpene compounds (6, 8−19) that are derived from 3
(Figure5A). Of these compounds, 10, 11, 14, and 17 were the
opposite stereochemistry of the C5 hydroxyl groups was
readily established through the coupling patterns of H-5 in H
1
NMR (6 and 9: br.s; 10 and 11: br.t, J = 9.4 and 10.5 Hz,
respectively). To identify which of these compounds are on-
pathway intermediates in the biosynthesis of 1, each compound
was supplied to the ΔAf520 blocked mutant at ∼40 μg/mL. As
expected, only 6 and 9 restored production of 1, even though
each strain also produced the fumagillin analogues 5 and 20,
respectively (Figure 7A and Supporting Information, Figure
S11), through the direct esterification with the polyketide side
chain (more on this below). In contrast, neither 10 nor 11
restored biosynthesis of 1 (Figure 7A and Supporting
Information, Figure S11), thereby confirming that they are
shunt products in S. cerevisiae, and no further epimerization of
the C5 hydroxyl can take place in A. fumigatus. This result also
provides insight into the intrinsic stereoselectivity of the Fma-
AT which prefers the natural R-OH group at C5.
Less Oxidized Products. Compound 8 contains one
additional oxygen atom compared to 3, and was found to be
5R-hydroxyl-β-trans-bergamotene (Supporting Information,
Table S5/Figures S21−S26). 8 is therefore the product of
direct oxidation of C5 of 3 by Fma-P450. Compounds 14 and
15 are dihydroxylated versions of 3. NMR characterization
revealed the structures of 14 and 15 to be 5,9-dihydroxyl-β-
trans-bergamotene and 5,10-dihydroxyl-trans-bergamotene, re-
spectively (Supporting Information, Tables S11−S12/Figures
S48−S57). All three compounds retained the trans-bergamo-
tene scaffold found in 3, and therefore represent possible early
intermediates in the pathway prior to the C5−C8 bond
cleavage step. Interestingly, only the feeding of 8 to the ΔAf520
blocked mutant restored production of 1, while addition of
either 14 or 15 failed to do so (Figure 7A and Supporting
Information, Figure S11). Therefore 14 and 15 are shunt
products of Fma-P450 activity in yeast. In particular, the high
levels of 14 in the yeast culture extract hints that C9 is the site
of the second P450 oxidation and may be subject to rapid
hydroxylation (see discussion below). To prove the multifunc-
tional nature of Fma-P450, 8 was added to the ΔAf510 mutant
(Figure 7B). No restoration of 1 was observed, as would be
expected for any additional role of Fma-P450 in the pathway.
Finally, addition of purified 8 to the yeast culture expressing
Fma-P450 and AfCPR similarly led to the formation of more
oxidized products such as 11 and 14 (Figure 5C).
Ketone-Containing Product 12. Among the products
isolated, 5-keto-isocordycol (12) contains a ketone functionality
at C5 and an allylic alcohol resulting from prototopic ring-
opening/rearrangement of the C8−C9 epoxide (Supporting
Information, Table S9/Figure S40−S41). Although this
compound is only present in minor quantities, the unexpected
cyclohexanone shunt product suggests that the C5 hydroxyl (in
8, for example) may serve as the source of electrons in the
oxidative cleavage of the cyclobutane ring, thereby transforming
the bicyclic trans-bergamotene scaffold into an 2,5,7-trisub-
stituted cyclohexanone. The corresponding C5-reduced de-
rivative, epi-isocordycol (13), was also found in the extract with
the 5S-hydroxyl (Supporting Information, Table S10/Figures
S42−S47). Both 12 and 13 will be revisited in the following
sections.
Figure 5. Products of Fma-P450 when reconstituted in S. cerevisiae.
(A) LC−MS metabolic profiles of coexpression of Fma-TC (encoded
by Af520), Fma-P450 (encoded by Af510), and AfCPR. Compounds
13, 15, 16, 18, and 19 are present at trace quantities and are not
shown. (B) LC−MS analysis of coexpression of Fma-P450 and AfCPR
in S. cerevisiae BJ5464-NpgA with supplement of 3; (C) same as trace
B but with supplement of 8. (D) Elucidated structures of compounds
8−19.
major products (0.6−1.1 mg/L); 8 and 12 were minor
products (0.3−0.5 mg/L); and the remaining (6, 9, 13, 15,
16, 18, and 19) were present at less amounts (<0.3 mg/L). The
same set of metabolites was also observed when 3 was directly
supplied to the yeast culture expressing only Fma-P450 and
AfCPR (Figure 5B). In the absence of 3 or Fma-P450, none of
these 13 compounds was observable in the extract. To elucidate
the structures of these products, large scale (12 L) fermentation
of the triply transformed yeast strain was performed, followed
by isolation and characterization of each compound (Figure 5D
and Supporting Information).
Highly Oxidized Products. Compounds 6 and 9 were
identified as 6-demethoxyfumagillol23 and cordycol,24 respec-
tively, based on MS and NMR comparison to standards. 9 is the
monoepoxide relative of 6 and may therefore be an immediate
precursor of 6. Formation of these compounds does indicate
that Fma-P450 alone is sufficient to transform 3 into 6.
However, both compounds are found in very minute quantities
(Figure 5A). Surprisingly, the 5-epimeric forms of 6 and 9,
which are 5-epi-demethoxyfumagillol (11) and 5-epi-cordycol
(10), respectively, were present as major products. The
Other Shunt Products. The other four compounds 16−19
were all found to be structurally related, but clearly off-pathway
products. Compound 16 is particularly interesting with its
trisubstituted sesquifenchyl core (Supporting Information,
Table S13/Figures S58−S62). The 5-hydroxy-β-cis-bergamo-
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tene (17), 5,10-dihydroxyl-β-cis-bergamotene (18), and 19 all
have the unexpected β-cis-bergamotene scaffold and are
presumably formed by isomerization of reactive intermediates
derived from 8 (Supporting Information, Tables S14−S16/
Figures S63−S80). 17 is a major product from the yeast
expression of Fma-P450, while 18 and 19 are further oxidized
products of 17. The feeding of 17 to the ΔAf520 blocked
mutant did not restore production of 1 (Figure 7A). Possible
mechanisms of formation of 16−19 are shown in Figure 10 and
will be discussed below.
Biochemical Assay of Fma-P450 and Identification of
C5-Ketones as Biosynthetic Intermediates. The array of
products recovered from S. cerevisiae clearly showcased the
multifunctional capability of Fma-P450 (hydroxylation, epox-
idation, rearrangement) in the oxidation of β-trans-bergamo-
tene (3). The large number of shunt products, however, may be
a result of the weak expression of fungal membrane-bound
P450 in yeast. As a result of the low concentration of the P450,
reactive intermediates may be readily intercepted or hydrolyzed
to yield off pathway products such as 14 and 17. Therefore, to
analyze the function of Fma-P450 under more controlled
conditions, we prepared microsomal fractions from the yeast
strain that overexpressed Fma-P450 and AfCPR for in vitro
assay.
transformation of 3 most likely involves a C5 ketone
intermediate that is stereoselectively reduced into the 5R
isomer.
Several ketoreductases (KRs) such as the 3-KR in sterol
biosynthesis and 3-KR for long-chain fatty acid synthase have
been reported in S. cerevisiae,25 hence it is possible that an
endogenous yeast KR could catalyze the reduction of the C-5
ketone to form the 5S-hydroxy moiety observed in compounds
10, 11, and 13. To test this hypothesis, we prepared both
soluble and microsomal extracts of the untransformed host
BJ5464-NpgA. We first tested if the isolated ketone 12 could be
selectively reduced to 13. As shown in Supporting Information,
Figure S9, complete reduction of 12 to 13 was observed. The
R-isomer of 13 may be present at a very low concentrations as
suggested by ion-monitoring, but the amount was insufficient
for purification and characterization. To further test whether
this endogenous yeast KR activity is responsible for formation
of the other 5S-containing products shown in Figure 5D, we
chemically prepared the three ketone substrates, 5-keto-
cordycol (21), 5-keto-demethoxyfumagillol (22), and 5-keto-
fumagillol (23) (Figure 7D, spectroscopic data in Supporting
Information, Tables S17−S19 and Figures S81−S86). These
compounds were synthesized from 10, 11, and 2 by PCC
oxidation, respectively. When either 21 or 22 was added to the
control yeast extract (soluble or microsomal), we observed the
corresponding ketoreduction to 10 or 11 (Supporting
Information, Figure S10), respectively, thereby validating the
involvement of endogenous yeast KRs in the formation of
compounds with the 5S hydroxyl functional group.
When 3 was incubated with 10 mg/mL of microsomal
fractions and NADPH overnight, we detected 11 as essentially
the single product in the extract (Figure 6A). Other major
With the ketones 21−23 in hand, we performed chemical
complementation experiments by feeding each of these
compounds individually to the ΔAf520-blocked mutant. In
each case, complete restoration of biosynthesis of 1 was
observed (Figure 7A). Therefore, the C5-ketone moiety in each
of the compounds can be processed correctly. Furthermore,
chemical complementation of ΔAf510 mutant by 22 restored
fumagillin production, indicating that Fma-P450 is not involved
in the biosynthetic pathway beyond 22 and is not essential for
reduction of the C5 ketone. Lastly, complementation of 23 to
the ΔAf480 mutant efficiently restored fumagillin production,
hinting that reduction of C5 may occur as the last step in the
formation of 2.
Identification of Fma-KR as the Stereospecific 5-
Ketoreductase. We hypothesized that a KR encoded in the
Fma gene cluster might be responsible for the stereospecific 5R
reduction of the ketone during the biosynthesis of 1. Although
no standalone KR is found in the Fma cluster shown in Figure
3A, Af490 encodes a partial PKS in which only the DH-KR
domains are present. Upon initial discovery of the fma cluster,
the pseudo/partial PKS was assumed to be an inactive enzyme
that had no likely role in the biosynthetic pathway. Although
deletion of Af490 did not completely abolish the production of
1, we did notice an 86% decrease in the titer of fumagillin (1)
(Figure 3B). The incomplete abolishment of the biosynthesis of
1 may be due to the presence of endogenous KRs in A.
fumigatus that have overlapping functions to that encoded by
the pathway enzyme Af490 (Fma-KR). To investigate the role
of Fma-KR, the 110 kDa protein was therefore expressed as a
6xHis tag fusion and purified from S. cerevisiae (Supporting
Information, Figure S6). Upon individual incubation of each of
the ketone derivatives 21, 22, or 23 with recombinant Fma-KR,
we observed complete conversion to the corresponding 5R-
Figure 6. LC−MS analysis of in vitro assays of yeast microsomes
containing Af510 and Af CPR toward (A) 3 and (B) 9.
compounds observed in vivo, such as 10, 14, and 17, etc. are
only present at very low levels when searched for using selective
ion monitoring. Similarly, when the assay is repeated using 8 as
the substrate, near complete conversion of 8 to 11 was
observed with nearly no side products (Supporting Informa-
tion, Figure S8). To analyze one of the individual steps
catalyzed by the multifunctional P450, we assayed the
epoxidation of 9 to 6 using the same microsomal extract. As
shown in Figure 6B, we could observe incomplete conversion
of 9 to 6 only in the presence of Fma-P450, thereby confirming
the ability of this enzyme to catalyze the specific formation of
epoxide.
Collectively, these in vivo and vitro results confirmed the
multifunctional role of Fma-P450, including C5 hydroxylation,
coupled or sequential 4e-oxidative cleavage, and rearrangement
to yield epoxycyclohexanone, and the tandem epoxidation. At
the same time, it is also evident that Fma-P450 alone is
insufficient to generate the correct stereoisomer of 5R-
demethoxyfumagillol 6, instead the 5S diastereomer 11 being
formed in yeast or using yeast microsomes. Therefore, given
that a ketone intermediate 12 could be isolated, the
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Figure 8. Fma-KR as the R-specific ketoreductase. (A−C) LC−MS
analysis of in vitro assays of Fma-KR with 21−23. (D) LC−MS
analysis of coexpression of Fma-KR, Fma-P450, and AfCPR in S.
cerevisiae BJ5464-NpgA with supplement of 3. (E) LC−MS analysis of
in vitro assay of Fma-KR, and yeast microsomes containing Af510 and
Af CPR plus 8.
TC.16 The initial oxidation of 3 by Fma-P450 involves C−H
hydroxylation at the bridgehead C5 position to yield 8.
Subsequently, a four electron oxidation initiated at C-9 coupled
to cleavage of the cyclobutane C5−C8 bond of the
bicyclo[3.1.1] core yields the on-pathway intermediate
epoxyketone 21. An additional epoxidation reaction also
catalyzed by Fma-P450 then furnishes the characteristic
bisepoxide ketone 22. A possible mechanism for the Fma-
P450-catalyzed conversion of 3 to 22 is shown in Figure 10 and
will be elaborated further in the Discussion section.
The diepoxyketone 22 is then subjected to successive C-6
hydroxylation and O-methylation by Fma-C6H and Fma-MT,
respectively, to yield 23, which is then stereoselectively reduced
by Fma-KR to 5R-hydroxy-seco-sesquiterpene 2. Acylation
catalyzed by the Fma-AT with the dodecapentaenoyl group
on Fma-PKS to yield 4 has been previously demonstrated.15
Finally, oxidative cleavage of 4 by the oxygenase (Fma-ABM)
encoded by Af470 arrives at 1.
We have also shown that Fma-KR can stereospecifically
reduce each of the three potential ketone intermediates 21−23
into the corresponding 5R-hydroxyl compounds, thereby
implying that this ketoreduction step may occur at different
stages during the biosynthesis of 2. In the proposed pathway,
however, we have assigned the enzyme to catalyze reduction of
the most advanced intermediate 23. We have shown that
compounds 6 and 9, which can be produced by reduction of 22
and 21, respectively, can each be acylated efficiently by Fma-AT
to yield the analogues 5 and 20. For example, feeding 6 to the
ΔAf520 strain resulted in the production of both 5 and 1
(Figure 7A), with a relative ratio around 4:1. This indicated that
C-6 hydroxylation by Fma-C6H may be slower than the C-5
acylation catalyzed by Fma-AT. The formation of 5 and 20 also
demonstrates that Fma-AT has significant tolerance toward the
substitution pattern of the terpene substrate, and can intercept
Figure 7. LC−MS analysis of metabolites produced as a result of
chemical complementation experiments. Compounds are supple-
mented at ∼40 μg/mL to (A) ΔAf520; (B) ΔAf510; (C) ΔAf480.
(D) Structures of 21−23.
hydroxy-containing compounds, 9, 6, or 2, respectively (Figure
8A−C).
Lastly, to examine the product profile of Fma-P450 in the
presence of Fma-KR, we repeated the biotransformation of 3 in
S. cerevisiae by coexpression of Fma-P450, Fma-KR, and AfCPR.
As shown in Figure 8D, the product profile is altered
considerably to reflect the presence of the dedicated C5
reductase. The 5R-hydroxy isomers 6 and 9 are now clearly
major products of the assay, while the 5S-hydroxy diaster-
eomers 10 and 11 have essentially disappeared. The same
results were obtained in the in vitro assay when Fma-P450-
containing yeast microsomes were coincubated with purified
Fma-KR. Whereas 11 was formed as the single product in the
previous assay shown in Figure 6A, inclusion Fma-KR led to the
production of the natural diastereomer 6 (Figure 8E).21b,26
The Complete Fumagillin Biosynthetic Pathway. On
the basis of the new enzymes characterized and the new
compounds isolated and identified in this work, we can now
deduce a complete biosynthetic pathway for fumagillin (1), as
shown in Figure 9. The pathway begins with the conversion of
FPP to β-trans-bergamotene (3) by a membrane-bound Fma-
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Figure 9. The fumagillin (1) biosynthetic pathway.
these 5R-reduced products in vivo. In contrast, we have
established that Fma-C6H is highly specific toward the
unacylated intermediate 6. Therefore, 5 and 20 cannot be
oxidized to 1. However, no trace of 5 or 20 is recovered from A.
fumigatus, thereby suggesting that neither 6 nor 9 is an
intermediate in vivo. Hence, we proposed that reduction of the
C5 ketone takes place after the 6-methoxyl function has been
installed in 23, thereby preventing premature C5 acylation of
the terpenoid by Fma-AT.
The multifunctional Fma-P450 is the central player in the
consecutive series of chemically and mechanistically distinct
catalytic processes. This enzyme initiates the oxidative
transformation on 3 by performing C-5 hydroxylation, followed
by oxidative ring-opening coupled to epoxidation and a second
tandem epoxidation to generate the key diepoxyketone
intermediate 22 that serves as the substrate for later
hydroxylation, methylation, ketoreduction, and acylation
catalyzed by other pathway enzymes from the fma cluster.
When Fma-P450 was reconstituted in S. cerevesaie, we observed
numerous oxidized products derived from the bioconversion of
3. On the basis of the structures of these compounds, we can
propose a detailed mechanism for Fma-P450. Either radical or
cationic rearrangement mechanisms can be proposed for this
enzyme and are shown in Figure 10 and Supporting
Information, Figure S12. Although most of the P450-catalyzed
C−H bond activation reaction has been shown to involve caged
radical intermediates rather than carbocations,2b,4b,31 several
recently discovered P450-catalyzed reactions almost certainly
proceed through carbocationic mechanisms.5,32 It is possible
that the various products produced by Af510 result from a
partitioning between radical pair and cationic intermediates.
The first C−O bond installation occurs by C−H bond
cleavage and hydroxylation at the C-5 of hydrocarbon 3 to form
5-hydroxybergamotene 8. Next, the hydrogen atom at C-9 is
abstracted by the key ferryl-oxo intermediate (FeIVO,
porphyrin π cation radical) to generate the hydroxycyclobu-
tylcarbinyl radical, A. This reactive intermediate can either
undergo an oxygen rebound to generate 14 or be converted to
a hydroxycyclobutylcarbinyl cation (B) after one-electron
oxidation by the oxidative ion (IV)-hydroxo intermediate.33
Both reactions result in a ferric heme center and resume the
catalytic cycle of Fma-P450 in the presence of AfCPR.
Carbocation B can be subjected to attack by the “super-
DISCUSSION
■
In this study, we have identified all the enzymes in the
fumagillin pathway that transform the bicyclo[3.1.1] sesqui-
terpene 3 into the richly decorated pharmacophore 2, which
acts by binding to type 1 and 2 MetAP.27 The highly oxidized
cyclohexane portion of 2, which is also present in the
metabolite ovalicin from Pseudeurotium ovalis,28 has been the
subject of intensive synthetic efforts (see review in Yamagushi
et al. and references therein).29 Considering the number of
oxidative steps required to modify 3 into 2, it is remarkable that
only two oxygenases are responsible. Most impressively, Fma-
P450 encoded by Af510 alone catalyzes the eight electron
oxidation of 3 into the intermediate 22 (Figure 9). The same
set of reactions must therefore also be involved in the
conversion of 3 into ovalicin, which is structurally identical to
23 except for the additional hydroxylation at C7 (Figure 8).
The biosynthesis of ovalicin has previously been studied using
labeled samples of mevalonate and of 3.14,30 From these
investigations, several mechanisms and intermediates were
ruled out, including the involvement 8,9-didehydrobergamo-
tene that had been suggested by Birch (Figure 9).21a The
pathway deduced from our genetic and biochemical experi-
ments is fully consistent with these earlier findings.
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Figure 10. Proposed mechanism of Fma-P450. Radical mechanism from A to 21 was shown in Supporting Information, Figure S12.
nucleophile” iron(III)-peroxo intermediate generated from the
following P450 cycle.1a The later reaction produces inter-
mediate C and induces the subsequent ring-opening rearrange-
ment through C5−C8 bond cleavage to form the 8,9-epoxide
21 (Figure 10), followed by a 1,2-epoxidation to generate 22.
Nucleophilic addition from water or ferric hydroxo (FeIII−OH)
complex to B also generates 14. This shunt pathway can readily
take place, as indicated by the high level of 14 observed in yeast
(Figure 5A). Formation of the shunt β-cis-bergamotene
derivatives 17−19 can result from ring-opening/closure of
the C-9 radical intermediate (D) to generate the isomeric
radical (or cation) cis-5-hydroxybergamotene intermediate.
Compounds 17−19 can then readily be obtained through
direct nucleophilic attack of water or intervening 1,2-hydride
shift. Lastly, if Fma-P450 epoxidizes the 1,2-exomethylene
double bond of 3 prior to C-5 hydroxylation, the resulting
epoxide intermediate could undergo well precedented cationic
rearrangement of its bicyclo[3.1.1] skeleton to form the tetra-
substituted sesquifenchol derivative 16.34 Overall, the oxidative
cleavage of 8 to 21 is particularly intriguing, and would parallel
the proposed conversion of loganin to secologanin,35 as well as
that involved in the formation of furanocoumarin.36
that catalyzes stereospecific 5-ketoreduction of cyclohexanone
23 to 2 is intriguing given that PKS KR domain typically acts
on β-ketoacyl thioester substrates attached on the acyl carrier
proteins. Interestingly, a BLAST search for similar Fma-KR-like
enzymes indicates that in A. fumigatus the hybrid PKS/NRPS
(PsoA)6b in pseurotin biosynthesis (and the corresponding
orthologues in A. clavatus and M. anisopliae)17 are the closest
match with up to 56% protein identity (65% nucleotide
identity) within the KR domain region. Hence, it appears that
the Fma-KR may be an evolutionary product originating from
classic gene duplication, divergence, and differential loss, but
with the mechanisms acting on the functional domain level
rather than on the entire gene. Although both A. clavatus and
M. anisopliae have a close orthologue of psoA, the Fma-KR gene
appears to be absent, suggesting the duplication has only
occurred in A. fumigatus. Curiously, the pseurotin biosynthetic
gene cluster6b is intertwined with the fma gene cluster and was
shown recently to be coregulated by a single transcriptional
factor (FapR) embedded in this fma-pso supercluster.16 The
possibility that Af490 originated from a duplicated partial psoA
gene but neofunctionalized to participate in the fma pathway
further adds to the complex evolutionary history of this
supercluster. Even more interesting, although we verified the
role of Fma-KR in vitro and in S. cerevisiae, deletion of this gene
in A. fumigatus did not lead to complete abolition of fumagillin
(1) biosynthesis. It remains highly possible that the activity of
the KR domain of PsoA can partially complement the deletion
of Af490, therefore maintaining the ketoreductase activity,
The Af490 (Fma-KR) gene has previously been annotated as
a PKS-like enzyme that harbors conserved motifs characteristic
of the PKS-DH (smart00826) superfamily and PKS-KR
(smart00822) domains as revealed by a NCBI Conserved
Domain-Search. The discovery that the truncated PKS gene
Af490 with only DH-KR domains encodes a functional enzyme
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General Techniques for DNA Manipulation. A. fumigatus
genomic DNA was prepared using CTAB isolation buffer as described
elsewhere.41 Polymerase chain reactions were performed using
Phusion DNA Polymerase (New England Biolabs) or Platinum Pfx
DNA polymerase (Invitrogen). DNA restriction enzymes were used as
recommended by the manufacturer (New England Biolabs). RNA
extraction was performed using a RiboPure Yeast Kit (Ambion), and
ImProm-II Reverse Transcription System for RT-PCR (Invitrogen)
was used to synthesize complementary DNA (cDNA) from total RNA.
PCR products were subcloned to a pCR-Blunt vector (Invitrogen) and
confirmed by DNA sequencing. Primers used to amplify the genes
were synthesized by Integrated DNA Technologies and are listed in
Supporting Information, Table S2. In vivo yeast recombination cloning
was performed by transforming the S. cerevisiae BJ5464-NpgA with
DNA fragments with >35 bp overlaps and includes a 2 μm plasmid
backbone (derived from YEplac195 or YEplac112) using an S.c.
EasyComp Transformation kit (Invitrogen).
albeit at lower efficiency as indicated by the lowered titer of 1 in
the ΔAf490 strain. Alternatively, other endogenous KRs in A.
fumigatus may also possess this activity and catalyze the
requisite reduction.
Fma-ABM encoded by Af470 has been shown to be the
oxygenase responsible for the C10′-C11′ cleavage of the
dodecapentaenoate of 4 and further oxidation of the aldehyde
intermediate to the decatetraenedioic ester 1. Oxidative
cleavage of olefins by a single oxygenase has been implicated
in the biosynthesis of carotenoids,37 as well as in the formation
of microbial secondary metabolites such as rifamycin.38 The
carotenoid oxygenases are nonheme iron-dependent dioxyge-
nases, while the cleavaging enzyme in the rifamycin pathway is
a P450 monooxygenase. Interestingly, Fma-ABM belongs to a
potentially new class of oxygenases that catalyze such reactions.
It contains a DUF4188 conserved domain of still unknown
function but homology modeling using Phyre2 suggests that
the main portion of Fma-ABM is structurally related to
aldoxime dehydratase (Supporting Information, Figure S7B)
which is a heme-containing enzyme from Bacillus sp.,39 as well
as the cofactor-free ABM superfamily of monooxygenases,
which is typified by ActVA-Orf6 in actinorhodin biosynthesis
and SnoaB in nogalonic acid biosynthesis.20 It is uncertain
whether Fma-ABM requires a heme as a prosthetic group as it
is much smaller than aldoxime hydratase (275 versus 373
amino acids). Further biochemical investigation of Fma-ABM
enzyme is warranted as a BLAST search reveals that Fma-ABM
homologues are ubiquitous in fungi.
Chemical Analysis. For production of 1 and other metabolites, A.
fumigatus and mutant strains were cultured in CYA medium (Czapek-
Dox agar supplemented with 5 g/L yeast extract). Total RNA for RT-
PCR was extracted from A. fumigatus grown on Czapek-Dox liquid
medium with 5 g/L yeast extract (CYB) after 4 days of cultivation. For
chemical complementation of ΔAf520, ΔAf510, and ΔAf480 mutants,
purified compounds are supplemented to the CYA medium at 0.2 mg/
mL individually. LC−MS analyses of conversion of 7 to 8 by Af480
and 7 to 2 by Af480 and Af 390−400 were performed on Prevail 3 μm,
2.1 × 100 mm2 C18 reversed-phase column (Alltech) and separated
on a 5−95% (v/v) CH3CN linear gradient in H2O supplemented with
0.05% (v/v) formic acid at a flow rate of 125 μL/min. All LC−MS
analyses except the aforementioned were performed on a Shimadzu
2010 EV LC−MS (Phenomenex Luna, 5 μm, 2.0 × 100 mm2, C18
column) using positive and negative mode electrospray ionization with
a linear gradient of 5−95% MeCN-H2O in 30 min followed by 95%
CONCLUSIONS
■
1
MeCN for 15 min with a flow rate of 0.1 mL/min. H, 13C, and 2D
We have uncovered the roles of three oxygenases, Fma-P450,
Fma-C6H, and Fma-ABM, as well as an O-methyltransferase,
Fma-MT, involved in the biosynthesis of fumagillin (1). Among
these enzymes, Fma-P450, a P450 monooxygenase, catalyzes a
multistep transformation that includes a simple hydroxylation
followed by ring-opening of the bicyclic substrate coupled to
generation of an epoxide, followed by a second epoxidation that
results in the conversion of β-trans-bergamotene (3) to a highly
oxygenated structure, 5-keto-demethoxyfumagillol (22). In the
course of these studies we identified nine compounds as off-
pathway products that shed light on the mechanism of action
and catalytic potential of Fma-P450. The catalytic versatility of
Fma-P450 therefore significantly augments the already
impressive chemical virtuosity of this important class of
enzymes.
NMR spectra were obtained on Bruker AV500 spectrometer with a 5
mm dual cryoprobe at the UCLA Molecular Instrumentation Center.
Overexpression and Purification of His6-tagged Fma-C6H
and Fma-MT. Af 390 and Af400 were obtained as one transcript by
RT-PCR verifying that Af400 was misannotated (for revised
annotation, see Supporting Information). The DNA fragemnt of
Af 390−400 and Af480 from cDNA were inserted into pET21
(Novagen) digested with NdeI and XhoI to yield pKW20174 and
pKW20172, respectively. Primers used for the amplification and
cloning are listed in Supporting Information, Table S2. Recombinant
enzymes were expressed with C-terminal His6-tagged in E. coli BL21
(DE3) and purified by nickel affinity chromatography. The cells were
cultured at 37 °C, 250 rpm in 500 mL of LB medium with 35 μg/mL
carbenicillin. Isopropylthio-β-D-galactoside (IPTG, 0.1 mM) to induce
protein expression was added at OD600 between 0.4 to 0.6 and the cells
were further cultured for 12−16 h at 16 °C. The cells were then
harvested by centrifugation (3500 rpm, 15 min, 4 °C), resuspended in
∼25 mL of lysis buffer (100 mM Tris-HCL, pH 7.4, 0.1 M NaCl, 20
mM imidazole), followed by lysed by sonication on ice. Cell debris was
removed by centrifugation (15 000 rpm, 30 min, 4 °C). The His6-
tagged proteins were purified by using Ni-NTA agarose (Qiagen)
according to manufacturer’s instructions. Purified enzyme was
concentrated and exchanged into buffer A (50 mM Tris-HCl, pH
7.9, 2 mM EDTA, 2 mM DTT) + 10% glycerol with the centriprep
filters (Amicon) and stored at −80 °C for enzyme assays.
We have also characterized the function of Fma-KR as the
ketoreductase that controls the configuration at C-5 of
hydroxylated intermediates that then undergo further acylation
catalyzed by Fma-AT. This study has elucidated all the tailoring
reactions in the biosynthesis of 1 and will provide opportunities
for derivatization of 1 using enzymatic approaches.
MATERIALS AND METHODS
■
Assay for Fma-C6H and Fma-MT Activity. For in vitro synthesis
of 2 and 7, 10 μM Fma-C6H480 was incubated with 20 μM substrate
6, 1 mM sodium ascorbate, 1 mM α-ketoglutarate, and 100 mM NaCl
in 100 mM Tri-HCl (pH 7.4). For in vitro synthesis of 2, the assay was
performed using the same condition as above with additional 1 mM
SAM and 10 μM Fma-MT. The reaction was incubated at 1 h and
extracted twice with ethyl acetate. The organic phases were dried and
dissolved in 20 μL of MeOH and subjected for analysis by LC−MS as
described in Chemical Analysis.
Strains and Culture Conditions. A. fumigatus strain used in this
study was A. fumigatus CEA17 akuBKU80 strain (pyrG89, ΔakuBKU80),
which is deficient in nonhomologous end joining17 and was
maintained on Czapek-Dox agar or glucose minimal agar (GMM).40
Details on AFUA_8G00390 and AFUA_8G00470−510 deletion
mutants are shown in the Supporting Information. E. coli TOP10
(Invitrogen) and XL1-Blue (Stratagene) were used for DNA
manipulation, and BL21 (DE3) was used for protein expression.
Saccharomyces cerevisiae strain BJ5464-NpgA (MATα ura3−52 his3-
Δ200 leu2- Δ1 trp1 pep4::HIS3prb1 Δ1.6R can1 GAL) was used as the
yeast expression host.
Biotransformation in S.c. For biotransformation in S.c. of Fma-
P450 and AfCPR, S. cerevisiae strain BJ5464-NpgA harboring both
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Fma-P450 and AfCPR plasmid were inoculated to 4 mL of Yeast
Synthetic Drop-Out medium without uracil and leucine. The cells
were grown for 72 h with constant shaking at 28 °C. A 15 μL aliquot
of the seed culture was inoculated with 2 mL of YPD (10 g yeast
extract, 20 g peptone, and 950 mL of Milli-Q water) supplemented
with 1% dextrose. 3, 8, and 9 (0.5 mg in 10 μL of DMSO) was added
to the culture after 48 h at 28 °C with shaking and the cells were
cultivated for another 24 h. The cultures were extracted by hexanes-
ethyl acetate (1:1) twice, the organic layers were concentrated in vacuo
and redissolved in 100 μL of MeOH. A 10 μL aliquot of samples was
further analyzed by LC−MS with the method described in Chemical
Analysis. For biotransformation in S.c. of Af470, the culture of S.
cerevisiae strain BJ5464-NpgA harboring Af470 was prepared by a
similar method above and 4 (0.1 mg in 10 μL of DMSO) was added to
the culture after 48 h.
Microsome Assay for Fma-P450 Activity. Details in preparation
of Fma-P450 and AfCPR-containing microsomes for in vitro assay are
shown in the Supporting Information. For in vitro microsomes assay,
10 mg/mL (wet weight) microsomal fractions containing Af510 and
Af CPR, 1 mM substrates, 2 mM NADPH, and NADPH regeneration
system (BD) solution A (5 μL) and B (1 μL), and 100 mM PBS, pH
7.4 were incubated in a 100 μL reaction. The reaction was incubated at
room temperature for overnight and extracted with 100 μL of hexanes-
ethyl acetate (1:1) twice. The organic phase was dried and redissolved
in 20 μL of MeOH for analysis by LC−MS. The amount of protein in
10 mg/mL microsomes was calculated to be 180 μg/mL based on a
modified Bradford assay against a BSA standard curve (protein
samples were predenatured in 0.1 M NaOH).
ASSOCIATED CONTENT
* Supporting Information
Experimental details and spectroscopic data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
yitang@ucla.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
H−C.L. is supported by National Science Council of Taiwan
(102-2917-I-564-008). This work was supported by grants from
the US National Institutes of Health to Y.T. (Grant Nos.
1R01GM085128 and 1DP1GM106413) and to D.E.C. (Grant
No. 5R01GM030301), and from JSPS through the Funding
Program for NEXT (Grant No. LS103) (K.W.) and by Nagase
Science and Technology Foundation Japan (K.W). A.M.C and
S.D. work was supported by Northern Illinois University. NMR
instrumentation was supported by the NSF equipment Grant
CHE-1048804. We thank Prof. Yongquan Li at Zhejiang
University for a standard of 9. We thank Ralph A. Cacho, Dr.
Youcai Hu, and Prof. Neil Garg for helpful discussions.
Expression and Purification of Fma-KR from S. cerevisiae. S.
cerevisiae BJ5464-NpgA was transformed with pHCfmaKR. For 1 L
culture, the cells were grown at YPD (10 g/L yeast extract, 20 g/L
peptone) supplemented with 1% dextrose and incubated at 28 °C with
shaking for 72 h. The cells were harvested by centrifugation (3750 rpm
at 4 °C for 10 min), and the cell pellet was resuspended in 20 mL of
lysis buffer (50 mM NaH2PO4, 150 mM NaCl, 10 mM imidazole, pH
8.0) and lysed by sonication on ice in one minute intervals until
homogeneous. To remove cellular debris, the homogeneous mixture
was centrifuged at 17000 rpm for 1 h at 4 °C. Ni-NTA agarose resin
was added to the supernatant (2 mL) and the solution was stirred at 4
°C overnight. Soluble Fma-KR was purified by gravity-flow column
chromatography with increasing concentrations of imidazole in Buffer
A (50 mM Tris-HCl, 500 mM NaCl, 20 mM−250 mM imidazole, pH
7.9). Purified protein was concentrated and buffer was exchanged into
Buffer B (50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl, pH 8.0)
using an Amicon Ultra-15 Centrifugal Filter Unit and stored in 10%
glycerol. The purified Fma-KR was analyzed by SDS-PAGE
(Supporting Information, Figure S6) and their concentration was
calculated to be 8.7 mg/L, using the Bradford assay with BSA as a
standard.
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