Tandem enzymatic oxygenations in biosynthesis of epoxyquinone pharmacophore of manumycin-type metabolites

Article abstract of DOI:10.1016/j.chembiol.2013.05.006

Many natural products contain epoxyquinone pharmacophore with unknown biosynthetic mechanisms. Recent genetic analysis of the asukamycin biosynthetic gene cluster proposed enzyme candidates related to epoxyquinone formation for manumycin-type metabolites.

Full text of DOI:10.1016/j.chembiol.2013.05.006

Chemistry & Biology
Article
Tandem Enzymatic Oxygenations
in Biosynthesis of Epoxyquinone Pharmacophore
of Manumycin-type Metabolites
Zhe Rui,1 Moriah Sandy,1 Brian Jung,1 and Wenjun Zhang1,
*
1Department of Chemical and Biomolecular Engineering and Energy Biosciences Institute, University of California, Berkeley, CA 94720, USA
*Correspondence: wjzhang@berkeley.edu
http://dx.doi.org/10.1016/j.chembiol.2013.05.006
SUMMARY
The manumycins are a unique group of epoxyquinone natural
products. Since their discovery in 1963, more than 30 manu-
Many natural products contain epoxyquinone phar- mycin-related natural products have been reported (Sattler
et al., 1998). They share a common structural skeleton with a
macophore with unknown biosynthetic mechanisms.
2-amino-4-hydroxyl-5,6-epoxycyclohex-2-enone (mC7N) core
and a variety of acyl side chains on the amino nitrogen (upper
Recent genetic analysis of the asukamycin biosyn-
thetic gene cluster proposed enzyme candidates
related to epoxyquinone formation for manumycin-
type metabolites. Our biochemical studies reveal
that 3-amino-4-hydroxyl benzoic acid (3,4-AHBA)
precursor is activated and loaded on aryl carrier pro-
tein (AsuC12) by ATP-dependent adenylase (AsuA2).
side chain) and at the C4 position (lower side chain). The lower
chain is further terminated by a 2-amino-3-hydroxycyclopent-
2-enone (C5N) moiety to give the manumycin-type framework
(Figure 1). In addition to antibacterial, anticoccidial, and anti-
fungal activities (Sattler et al., 1998), manumycin compounds
displayed strong inhibitory activities against farnesyltransferase
AsuE1 and AsuE3, both single-component flavin-
dependent monooxygenases, catalyze the exquisite
(Hara et al., 1993; Sugita et al., 2007), interleukin-1b converting
enzyme (Tanaka et al., 1996), IkappaB kinase (Bernier et al.,
regio- and enantiospecific postpolyketide synthase 2006), and acetylcholinesterase (Zheng et al., 2007), and thus
are drug candidates to treat cancer, atherosclerosis, inflamma-
tion, and Alzheimer disease. We have begun to focus on the
(PKS) assembly oxygenations. AsuE1 installs a hy-
droxyl group on the 3,4-AHB ring to form a 4-hydrox-
biosynthesis of asukamycins, a group of manumycin-type me-
tabolites produced by Streptomyces nodosus subsp. asukaensis
(Figure 1; Omura et al., 1976). The biosynthetic gene cluster of
yquinone moiety, which is epoxidized by AsuE3 to
yield the epoxyquinone functionality. Despite being
a single-component monooxygenase, AsuE1 activity
is elicited by AsuE2, a pathway-specific flavin reduc-
tase. We further demonstrate that the epoxyquinone
moiety is critical for anti-MRSA activity by analyzing
asukamycin was recently identified and studied using the
blocked mutants of asukamycin producers (Rui et al., 2010).
Although tentative gene functions were assigned based on the
in vivo mutagenesis results, the enzymology of the distinct struc-
the bioactivity of various manumycin-type metabo-
lites produced through mutasynthesis.
tural features embedded in asukamycins, particularly the enzy-
matic mechanism underlying the formation of epoxyquinone
moiety (mC7N), remained unresolved. Herein, we report the
characterization of enzymes required for the biosynthesis of
epoxyquinone moiety in asukamycins.
INTRODUCTION
Feeding experiments indicated that mC7N arose from
The epoxyquinone family of natural products is widely distrib- 3-amino-4-hydroxyl benzoic acid (3,4-AHBA), the same biosyn-
uted in nature and has been attracting much attention because thetic precursor for grixazone produced by S. griseus (Suzuki
of its unique structures and promising biologic activities et al., 2006). 3,4-AHBA was previously demonstrated as a
(Miyashita and Imanishi, 2005). Examples of family members condensation product of two primary metabolites, L-aspartate-
include epoxyquinol A and B (anti-angiogenic and antitumor; 4-semialdehyde (ASA) and dihydroxyacetone phosphate
Kamiyama et al., 2008), preussomerins (Ras farnesyl transferase (DHAP), by the catalytic functions of GriI and GriH in S. griseus
inhibitor; Singh et al., 1994), panepophenanthrin (ubiquitin- (Suzuki et al., 2006). Enzymes highly homologous to GriI and
activating enzyme inhibitor; Sekizawa et al., 2002), EI-1941-1 GriH, AsuA1 and AsuA3, respectively, were encoded in the asu
and EI-1941-2 (interleukin-1b converting enzyme inhibitors; Koi- gene cluster. The subsequent gene disruptions and chemical
zumi et al., 2003), aranorosin (anti-apoptotic Bcl-2 inhibitor; complementations with 3,4-AHBA strongly indicated that
Nakashima et al., 2008), and integrasone (HIV-1 integrase inhib- 3,4-AHBA was an en route biosynthetic intermediate to the
itor; Herath et al., 2004; Figure S1 available online). Despite mC7N moiety (Rui et al., 2010). We propose that 3,4-AHBA is first
extensive efforts on the regio- and enantio-synthesis of epoxy- activated by adenylation, presumably catalyzed by an ATP-
quinone natural products, little is known regarding the biogen- dependent adenylase homolog AsuA2, followed by transferring
esis of the epoxyquinone moiety, the warhead crucial for various of 3,4-AHBA moiety to an acyl carrier protein (ACP) for the down-
biologic functions exhibited by epoxyquinone metabolites.
stream polyketide chain extensions to form the lower chain. Two
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Chemistry & Biology
Biosynthesis of Epoxyquinone Pharmacophore
strated the activation of 3,4-AHBA, with the kinetic parameters
(Km = 21.20 mM, kcat = 5.52 minꢀ1) comparable to those of typical
adenylases in secondary metabolite biosynthetic pathways
(Figures 2A and 2B; Sandy et al., 2012). In addition to 3,4-
AHBA, various tested aryl acids were also strongly activated
by AsuA2, including 4-aminobenzoic acid (4-ABA), 3,4-ACBA,
4-hydroxybenzoic acid (4-HBA), and 3-aminobenzoic acid
(3-ABA) (Figure 2A). Further analysis of the activation pattern
suggested that AsuA2 tolerated functional group substitutions
at the meta- and para- positions rather than the ortho- position
(Figure S3). Aryl-CoA products were not detected upon addition
of CoA to the enzymatic reactions of AsuA2, confirming that
AsuA2 functioned as an adenylation enzyme without forming a
CoA thioester intermediate.
To identify the cognate loading partner of AsuA2, two ACP
candidates, AsuC11 and AsuC12, were supplemented to the
AsuA2 assay, respectively, and the ACPs were monitored for
mass changes with liquid chromatography-mass spectrometry
(LC-MS; Figures 2C and S4). An anticipated increase of
135 Da, corresponding to the formation of 3,4-AHB-S-ACP
from holo-ACP, was observed in the reaction of AsuC12 but
not of AsuC11, demonstrating that AsuC12 served as the spe-
cific ACP for 3,4-AHBA loading (Figure 2D). Other aryl acids
could also be efficiently loaded onto holo-AsuC12 after activa-
tion (Figure S4), suggesting that the loading ACP (AsuC12) was
promiscuous toward aryl substrates.
Figure 1. Chemical Structures of Manumycin-Type Compounds
Asukamycin is A1; protoasukamycin is C1; 4-hydroxyprotoasukamcyin is D1.
See also Figure S1.
AsuE1 Is a Flavin-Dependent Protoasukamycin
Hydroxylase
typical ACP coding genes, asuC11 and asuC12, were identified AsuE1 exhibits moderate sequence similarity to para-hydroxy-
in the asu gene cluster, yet the specificity of them has not been benzoate hydroxylase (PHBH; identity/similarity = 22%/38%;
determined. The key reactions to convert 3,4-AHBA moiety to Cole et al., 2005; Entsch et al., 2005), which catalyzes hydroxyl-
mC7N, which include the de-aromatization and oxidation reac- ation reactions of aromatic substrates utilizing FAD as a
tions, are proposed to take place on the biosynthetic intermedi- cofactor, NADPH as a reducing agent, and molecular oxygen
ate protoasukamycin (C1; Figure 1) after the installation of the to provide the oxygen atom of the incorporated hydroxyl group.
upper and lower chains on 3,4-AHBA. Mutant analysis further The DasuE1 mutant of S. nodosus accumulated protoasukamy-
suggested that at least three oxygenases (AsuE1, AsuE2, and cin (C1), suggesting the substrate of AsuE1 to be C1 (Rui et al.,
AsuE3) encoded in the asu gene cluster were related to the 2010). AsuE1 was purified from Escherichia coli as a colorless
formation of mC7N via insertion of hydroxyl and epoxide oxygens protein without a detectable amount of bound flavin. The incuba-
at the C4 and C5–C6 positions (Rui et al., 2010). With both tion of AsuE1 with NADPH, FAD, and C1 led to the formation of a
in vitro and in vivo analysis, we provide experimental validation compound detected with LC-MS, with the retention time and
and probe the flexibility of the mC7N biosynthetic pathway, mass (m/z = 531.2491 [M + H]+, ppm = 0.2) matching those of
including a cascade of reactions for the priming and postas- the authentic 4-hydroxyprotoasukamycin (D1). Control experi-
sembly modifications of 3,4-AHBA, to yield the epoxyquinone ments showed that the formation of product depended on the
pharmacophore.
presence of nicotinamide reducing agent, flavin cofactor, and
the enzyme. AsuE1 displayed no significant preference toward
flavin cofactors, because FAD, FMN, and riboflavin could be
utilized efficiently in the catalysis (Figure 3A). AsuE1 thus belongs
to the family of single-component monooxygenases: a single
RESULTS AND DISCUSSION
Priming of 3,4-AHBA Starter Unit
Previous studies suggested that 3,4-AHBA is not only the pre- enzyme catalyzes both the reduction of flavin by NADPH, and
cursor of epoxyquinone core, but also serves as a starter unit the hydroxylation using molecular oxygen and reduced flavin
for the lower polyketide chain assembly of manumycin metabo- cofactor (van Berkel et al., 2006). This is distinct from the two-
lites (Hu et al., 1997). We propose that 3,4-AHBA is activated by component monooxygenase system which requires a separate
adenylation catalyzed by AsuA2 and then tethered to a specific flavin reductase component to provide the reduced flavin for
ACP for the PKS extension possibly without the formation of the monooxgenase enzyme (Ballou et al., 2005). This finding is
3,4-AHB-CoA thioester intermediate (Ferreras et al., 2008; Pfen- unanticipated due to the presence of AsuE2, a flavin reductase
nig et al., 1999; Sandy et al., 2012). The ability of AsuA2 to revers- homolog encoded in the asu gene cluster, and that the disruption
ibly adenylate various benzoic acid derivatives was tested using of asuE2 significantly decreased the yield of asukamycin and
the classical ATP-[32P]PPi exchange assay. AsuA2 demon- resulted in the accumulation of C1.
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Chemistry & Biology
Biosynthesis of Epoxyquinone Pharmacophore
Figure 2. Characterization of AsuA2,
AsuC11, and AsuC12
(A) AsuA2 activity toward various aromatic sub-
strates in ATP-[32P]PPi exchange assays. A rela-
tive activity of 100% for 3,4-AHBA-dependent
exchange corresponds to 82k CPM. All chemical
structures are shown in Figure S3.
(B) Determination of AsuA2 kinetic parameters by
ATP-[32P]PPi exchange assays. Error bars repre-
sent SDs from at least three independently per-
formed experiments.
(C) Detection of holo-AsuC12 without AsuA2 and
3,4-AHB-S-AsuC12 in the AsuA2 reaction by
HRMS with deconvolution.
(D) Schematic of 3,4-AHBA activation and loading.
See also Figures S3 and S4.
AsuE2 Is a Flavin-Reductase
Enhancing Activity of AsuE1
AsuE2 shows high sequence similarity to
flavin reductases such as HapC (iden-
tity/similarity = 33%/52%; Kim et al.,
2008) and TftC (identity/similarity = 29%/
41%; Webb et al., 2010) involved in the
hydroxylation of 4-hydroxyphenylacetate
and 2,4,5-trichlorophenol, respectively.
Sequence alignment of flavin reductases
revealed that AsuE2 possesses the
highly conservative Tyr-178 and Val-39
The kinetic parameters of AsuE1 toward C1 were determined to accommodate the dimethyl benzene moiety of the flavin
by monitoring D1 formation using LC-MS. The enzyme demon- (Webb et al., 2010) and His-151 and Ser-60 to stabilize NAD(P)
strated good binding affinity toward C1 (Km = 21.31 mM), albeit H (Kim et al., 2008). Moreover, DasuE2 mutant accumulated a
with relatively slow turnover rate (kcat = 0.183 minꢀ1) for sec- significant amount of C1 (Rui et al., 2010), suggesting that
ondary metabolism (Figure 3B). AsuE1 also converted the AsuE2 might assist the catalysis of AsuE1 by providing a
protoasukamycin derivatives (C3–C5) into the corresponding reduced form of flavin in S. nodosus. AsuE2 contained a trace
4-hydroxyprotoasukamycin derivatives (D3–D5). However, amount of FMN at a molar ratio of ꢁ1:2,000 (FMN:AsuE2) upon
AsuE1 demonstrated only trace activities to install a hydroxyl purification from E. coli (Figure S7). The activity of AsuE2 as a
group on compounds such as 2-amino-4-methyl-phenol and flavin reductase was confirmed by colorimetric assays moni-
ferulic acid (Figure S5) and no activity on 3,4-AHB-S-ACP. toring the consumption of NAD(P)H. With a FMN concentration
These results indicated the stringent substrate requirement of of 10 mM, the enzyme showed a Km of 171.2 mM for NADH with
AsuE1 toward a triene lower chain, which is conserved in most an apparent kcat of 2.34 sꢀ1, and a Km of 1,689 mM for NADPH
manumycin-type metabolites.
The specificity of AsuE1 toward NAD(P)H was probed using utilization of NADH by AsuE2 (Figure 4A).
continuous colorimetric assays. AsuE1 displayed preference to- In vitro AsuE1 assays were then performed by replacing the
ward NADH over NADPH, with kcat/Km for NADH (9.0 sꢀ1mMꢀ1
exogenous flavin with 1 mM of AsuE2. The time-dependent con-
with an apparent kcat of 5.31 sꢀ1, indicating the preferential
)
50-fold higher than that for NADPH (0.18 sꢀ1mMꢀ1; Figure 3B). version of C1 to D1 was observed in the AsuE1/E2 assay without
Notably, the consumption rates of nicotinamide substrates the addition of free flavin (Figure 4B). To probe the necessity of
were independent of the concentration of C1 (Figure S6), AsuE2, a control experiment was further conducted with
different from the catalytic mechanism of PHBH where substrate 0.5 nM of exogenous FMN, the amount equivalent to the bound
PHB must bind before initiation of reduction of the flavin (Cole FMN in 1 mM of AsuE2. No D1 was detected in the AsuE1 assay
et al., 2005; Entsch et al., 2005). Without aromatic substrates, with 0.5 nM of free FMN, indicating that the observed hydroxyl-
the reduced flavin would react with oxygen and eliminate ation activity in AsuE1/E2 reaction was due to the concerted
H2O2, a destructive oxidant that can be damaging to the cell. functions of both enzymes. It is notable that there was no pro-
This futile cycle also wastefully consumes nicotinamide reducing tein-protein interaction between AsuE1 and E2 in pull-down
agents. Due to the low overall catalytic efficiency of AsuE1, we assays, suggesting a possible transient interaction between
next performed the detailed biochemical characterization of the two enzymes instead of forming a stable complex.
the flavin reductase homolog AsuE2 and probed the possible Our biochemical analysis of AsuE1 and E2 demonstrated that
interplays between AsuE1 and E2 in the hydroxylation of although the activity of AsuE1 was not affected by the AsuE2 in
protoasukamycin.
the presence of abundant flavin, AsuE2 elicited the activity of
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Chemistry & Biology
Biosynthesis of Epoxyquinone Pharmacophore
Figure 3. Characterization of AsuE1
(A) Extracted ion chromatograms showing the
conversion of C1 to D1 catalyzed by AsuE1. The
calculated mass with 10 ppm mass error tolerance
was used.
(B) Determination of AsuE1 kinetic parameters.
Error bars represent SDs from at least three inde-
pendently performed experiments.
See also Figures S5 and S6.
of AsuE3. The addition of AsuE2 to the
enzymatic reactions of AsuE3 under
various conditions did not change the
apparent reaction rates of asukamycin
formation, different from a recent obser-
vation that a flavin reductase Fre stimu-
lated the activity of epoxidase Lsd18
in polyether lasalocid biosynthesis (Min-
ami et al., 2012). AsuE3 was therefore
confirmed to be
a single-component
epoxidase catalyzing two half-reactions
in the single protein: (1) reductive half-
reaction using NADH and oxidized FMN
to give reduced FMN, and (2) oxidative
half-reaction using the reduced FMN
and molecular oxygen to afford 4a-hydro-
peroxyflavin followed by epoxidation of
AsuE1 with only a trace amount of flavin in vitro. These results the substrate. A similar epoxidation mechanism may be applied
suggested the importance of AsuE2 in vivo where the cellular to the biosynthesis of a potent antibiotic methylenomycin A,
concentration of free flavin is typically low, consistent with the because an AsuE3 homolog MmyO was identified in its putative
observation that the DasuE2 mutant accumulated the unmodi- biosynthetic gene cluster (Hornemann and Hopwood, 1978;
fied C1 more than asukamycin (A1; Rui et al., 2010). AsuE1/E2 Redenbach et al., 1998).
thus represents an atypical enzyme pair with a divergent evolu-
AsuE3 failed to oxidize C1 in vitro, demonstrating that the pro-
tionary vestige and shows an indistinct boundary between the toasukamycin 4-hydroxylation catalyzed by AsuE1 was a pre-
single- and two-component monooxygenases.
requisite for epoxidation. Therefore, although both one-step
and two-step monooxygenase mechanisms had been proposed
for epoxyquinone formation (Hu and Floss, 2004; Thiericke et al.,
1989), our biochemical characterization of AsuE1/E3 unequivo-
AsuE3 Is a Flavin-Dependent
4-Hydroxyprotoasukamycin Epoxidase
AsuE3 displays high sequence similarity (identity/similarity = cally supported the two-step mechanism for the formation of
46%/63%) to LimB, which catalyzes the FAD- and NADH- mC7N in manumycin-type metabolites. The higher specific activ-
dependent epoxidation of limonene to form limonene- ity of AsuE3 toward D1 (Km = 7.20 mM, kcat = 0.549 minꢀ1
)
1,2-epoxide in Rhodococcus erythropolis (van der Werf et al., compared to the activity of AsuE1 toward C1 (Km = 21.31 mM,
1999). The DasuE3 mutant of S. nodosus accumulated D1 (Rui
cat = 0.183 minꢀ1) further suggesting that the first hydroxylation
k
et al., 2010), suggesting the identity of AsuE3 substrate. AsuE3 reaction promoted by AsuE1 was likely a rate-limiting step in the
was purified from E. coli as a colorless protein without detectable oxygenation cascade (Figures 5B and 6). AsuE3 also converted
amount of bound flavin. To determine the cofactor requirements the 4-hydroxyprotoasukamycin derivatives (D2–D5) to the asu-
and detailed reaction mechanism of AsuE3, the enzymatic reac- kamycin derivatives (A2–A5; Figure S8), but had no activity
tion with D1, NADH, FMN, and AsuE3 was first examined. LC- toward compounds such as 4-isopropyl-6-methylcyclohex-
MS analysis revealed the formation of a compound with a reten- 2-enone, demonstrating a similar substrate tolerance of AsuE3
tion time and mass (m/z = 547.2437 [M + H]+, ppm = 0.4) identical to AsuE1.
to that of the authentic A1. Omission of NADH, FMN, or AsuE3
led to a complete loss of activity. Substitution of FMN with Alternative Starter Unit Feedings Based on Substrate
FAD or riboflavin resulted in a more than 3-fold decrease of the Promiscuity of AsuA2
reaction rate, indicating a stringency of AsuE3 toward flavin Based on the characterized substrate promiscuity of AsuA2, we
cofactors that differs from AsuE1 (Figure 5A). Similar to AsuE1 performed precursor feeding experiments to generate manumy-
and E2, AsuE3 preferred NADH (kcat/Km = 4.3 sꢀ1mMꢀ1) over cin-type products and to probe the flexibility of the overall
NADPH (kcat/Km = 0.24 sꢀ1mMꢀ1; Figure 5B). We next tested biosynthetic pathway. This would also provide insights into the
the possible effect of the flavin reductase AsuE2 to the activity substrate specificity of AsuE1/E3 toward various substitutions
882 Chemistry & Biology 20, 879–887, July 25, 2013 ª2013 Elsevier Ltd All rights reserved

Chemistry & Biology
Biosynthesis of Epoxyquinone Pharmacophore
Figure 4. Characterization of AsuE2
(A) Determination of AsuE2 kinetic parameters.
Error bars represent SDs from at least three inde-
pendently performed experiments.
(B) Extracted ion chromatograms showing the
conversion of C1 to D1 catalyzed by AsuE1/E2.
The calculated mass with 10 ppm mass error
tolerance was used. Assays contain 100 mM
NADH, 100 mM protoasukamycin, 1 mM AsuE1,
and 1 mM AsuE2. The control group lacks AsuE2
but contains 0.5 nM exogenous FMN.
See also Figure S7.
N-acetylated asukamycin derivative (A8)
with an epoxyquinone moiety (Figure 1;
Rui et al., 2010), and the replacement
of the upper chain cyclohexyl group of
asukamycin by different alicyclic and
aliphatic moieties did not affect the for-
mation of expoxyquinone (Hu and Floss,
ꢀ
2006; Pospı´sil et al., 2011).
The manumycin family of natural prod-
ucts was previously demonstrated to
have potent antibiotic activities against
Staphylococcus aureus (Omura et al., 1976). Recently, methi-
cillin-resistant S. aureus (MRSA) has emerged as a major patho-
genic threat that is challenging to combat due to the resistance
to nearly all available antibiotics (Fischbach and Walsh, 2009).
To evaluate the antibiotic activity of asukamycins and their deriv-
atives toward MRSA, agar diffusion assays were performed us-
ing S. aureus USA300 (SF8300), a wound-isolated MRSA strain
that was implicated in numerous outbreaks (Diep et al., 2008).
A1 successfully inhibited the growth of MRSA, therefore estab-
lishing the potential of asukamycins for developing a new gener-
ation of anti-MRSA drugs. All generated metabolites (C6–C16)
exhibited no activity toward MRSA, highlighting the importance
of the epoxyquinone pharmacophore for antibiotic activities (Fig-
ure S11; Kohno et al., 1996; Sattler et al., 1998).
on the aromatic moiety in mC7N formation. 3-ABA, 4-ABA,
4-HBA and 3,4-ACBA were supplemented to the overnight
culture of DasuA3 mutant (Rui et al., 2010) that did not produce
asukamycins due to the deficiency in 3,4-AHBA formation.
High-performance liquid chromatography (HPLC) and LC-MS
revealed the successful incorporation of all compounds to the
asukamycin biosynthetic machinery (Figures 7 and S9). Similar
to a series of asukamycin-type compounds produced by wild-
type, feeding of 3-ABA yielded a suite of major compounds con-
taining the intact upper polyketide chains (C6–C10). 4-HBA lacks
an amino group and was recruited to form C13 without the
attachment of upper polyketide chains. Likewise, 4-ABA was
incorporated to form C15 with an unmodified amino group as a
minor product. The major product in the 4-ABA feeding experi-
ment was C16, which was identified to be an N-acetylated
C15 by HRMS/MS analysis (Figure S9J). The acetyl group
presumably resulted from the action of a nonspecific arylamine
N-acetyltransferase, commonly found in cell to detoxify aryl-
amine or arylhydroxylamine metabolites (Vineis et al., 1994).
Notably, 3,4-ACBA was processed by the asu PKS to afford
C14 as the major product, which was fully characterized by
MS and NMR analysis to be a halogenated asukamycin deriva-
tive containing a free amino group (Figures S9H and S10). The
production of C14 suggested that neither the amide synthetase
AsuC2 encoded by the asu gene cluster nor a putative cellular
arylamine N-acetyltransferase could modify the amino group of
3,4-ACB, possibly due to the steric hindrance of the chlorine
atom. All of the asukamycin derivatives contained a non-oxygen-
ated aromatic core structure, indicating the stringent substrate
requirement of AsuE1 toward the regiospecific substitutions on
the aromatic ring. It is notable that the catalytic functions of
AsuE1/E3 were not affected by the structures of upper polyke-
tide chain, since the DasuC2 mutant with a deficiency for
the upper chain attachment produced a significant amount of
SIGNIFICANCE
We characterized the essential enzymes involved in the prim-
ing and postassembly modifications of 3,4-AHBA to yield the
epoxyquinone pharmacophore in manumycin-type natural
products. Our work demonstrated a two-step monooxyge-
nase mechanism for the formation of epoxyquinone func-
tionality, a warhead prevalent in natural products with a
broad range of biologic activities. Based on the flexibility of
asukamycin biosynthetic machinery, we further generated
a group of manumycin-type metabolites through mutasyn-
thesis and provided evidence for the critical role of the epox-
yquinone moiety in the anti-MRSA activity of asukamycins.
EXPERIMENTAL PROCEDURES
Bacterial Strains, Culture Media, Plasmids, and Instrumentation
S. nodosus subsp. asukaensis ATCC 29757 was obtained from the American
Type Culture Collection. E. coli BL21 Gold (DE3) and BL21 (DE3) plysS cells
were purchased from Invitrogen. Bioassays with S. aureus USA300 (SF8300)
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Chemistry & Biology
Biosynthesis of Epoxyquinone Pharmacophore
Figure 5. Characterization of AsuE3
(A) Extracted ion chromatograms showing the
conversion of D1 to A1 catalyzed by AsuE3. The
calculated mass with 10 ppm mass error tolerance
was used.
(B) Determination of AsuE3 kinetic parameters.
Error bars represent SDs from at least three inde-
pendently performed experiments.
See also Figures S6 and S8.
chromosomal copy of the phosphopantetheinyl
transferase Sfp was used for AsuC11 and C12
expression to ensure their posttranslational modifi-
cation to the pantetheinylated forms (Pfeifer et al.,
2001). For expressions of AsuA2, AsuC11, and
AsuC12, the cells were grown at 37ꢂC in 1 l of LB
medium supplemented with 50 mg/ml kanamycin,
and then induced at 16ꢂC and optical density 600
of 0.4–0.6 with 0.1 mM of isopropyl-b-D-thiogalac-
topyranoside (IPTG) for 16 hr. For expression of
AsuE1-E3, the cells were grown in 1 l of LB medium
containing 2.5 mM betaine and 1 M sorbitol with
100 mg/ml of ampicillin and 25 mg/ml of chloram-
phenicol, induced, and harvested as described
above. The cells were resuspended in 35 ml of lysis
buffer (20 mM HEPES, pH 7.5, 0.5 M NaCl, and
5 mM imidazole), and lysed by sonication on ice.
The soluble proteins were collected by centrifuga-
were performed in Professor Binh Diep’s lab in the Department of Medicine,
San Francisco General Hospital. The media and chemicals were purchased
from Difco, Sigma-Aldrich, and EMD Chemicals. Oligonucleotide primers
were synthesized by Elim Biopharm, and PCR was performed with Phusion
High-Fidelity PCR Master Mix (NEB). Cloning was performed using the Xa/LIC
Cloning Kit (Novagen) or the restriction enzymes from NEB. Recombinant
plasmid DNA was purified with a QIAprep kit (QIAGEN). DNA sequencing
was performed at the UC Berkeley DNA Sequencing Facility. SDS-PAGE
gels and nickel-nitrilotriacetic acid agarose (Ni-NTA) superflow resin were pur-
chased from Biorad and QIAGEN, respectively. Protein samples were concen-
trated using the 10 kDa or 30 kDa cutoff Amicon Ultra-15 Centrifugal Filter
Units (Millipore). DNA and protein concentrations were determined by Nano-
drop 1000 spectrophotometer (Thermo Scientific). HPLC analysis was per-
formed on an Agilent 1260 Infinity system with Inertsil ODS-4 C18 column
(5 mm, 4.6 3 250 mm). LC-MS analysis was carried out on an Agilent Technol-
ogies 6520 Accurate-Mass Q-TOF LC-MS instrument equipped with an Agilent
Eclipse Plus C18 column (3.5 mm, 4.6 3 100 mm). 1H-nuclear magnetic reso-
nance (NMR), 13C-NMR, heteronuclear single quantum correlation, and heter-
onuclear multiple bond correlation spectra were recorded in d6-DMSO on a
Bruker Biospin 900 MHz spectrometer with a cryoprobe.
tion at 12,000 3 g and 4ꢂC for 1 hr, and mixed with 1 ml of Ni-NTA agarose
resin on a nutator at 4ꢂC for 1 hr. The proteins were then loaded onto a gravity
flow column, washed with washing buffer (20 mM HEPES, pH 7.5, and 20 mM
imidazole), and eluted with eluting buffer (20 mM HEPES, pH 7.5, and 150 mM
imidazole). Purified proteins were concentrated into 20 mM of HEPES (pH 7.5)
with 10% glycerol using Amicon Centrifugal Filter Units, flash-frozen in liquid
nitrogen, and stored at ꢀ80ꢂC. The approximate protein yields were
0.9 mg/l for AsuA2, 17 mg/l for AsuC11, 6.7 mg/l for AsuC12, 1.5 mg/l for
AsuE1, 1.8 mg/l for AsuE2, and 1.8 mg/l for AsuE3.
ATP-[32P]PPi Exchange Assay
The assays were performed in 100 ml of reaction mixture that contains 50 mM
Tris buffer (pH 7.5), 2 mM MgCl2, 5 mM ATP, 1 mM Na4PPi (containing 250–400
kcpm of Na4[32P]PPi), 1 mM TCEP, 5 mM aromatic substrate, and 1 mM AsuA2
at 25ꢂC. Reactions were initiated by adding AsuA2 and quenched with 500 ml
of a charcoal suspension (100 mM Na4PPi, 350 mM HClO4, and 16 g/l char-
coal) after 60 min. The pellets were collected by centrifugation and washed
twice with 500 ml of washing solution (100 mM Na4PPi and 350 mM HClO4).
AsuA2 kinetic analysis was carried out in 500 ml of reaction mixture as
described above except for a varying concentration of 3,4-AHBA (5, 10, 20,
50, 100, or 250 mM). Reactions were initiated by adding AsuA2 and quenched
with 500 ml of a charcoal suspension at 0, 5, 10, 15, and 20 min. The rate of sub-
strate consumption was calculated as the incorporation rate of the charcoal-
bound [32P]-ATP measured on a Beckman LS 6500 scintillation counter. The
resulting data were fitted to the Michaelis-Menten equation in GraphPad Prism
Cloning, Overexpression, and Purification of AsuA2, AsuC11,
AsuC12, and AsuE1-E3
The corresponding proteins (AsuA2, 51 kDa; AsuC11 and AsuC12, 10 kDa;
AsuE1, 43 kDa; AsuE2, 19 kDa; AsuE3, 41 kDa) were overproduced in E. coli
cells and purified using Ni-NTA affinity chromatography (Figure S2). First,
asuA2, asuC11, asuC12, and asuE1-E3 were PCR-amplified from cosmid
pART1361 with the primers shown in Table S1. For AsuA2, gel-purified PCR
products were ligated to pET-30 Xa/LIC to yield pZR01 following the standard
protocol. For AsuC11 and AsuC12, PCR products were digested with NdeI/
EcoRI and inserted into the NdeI/EcoRI cut pET-24b to give pZR02 and
pZR03, respectively. For AsuE1-E3, PCR products were digested with BglII/
HindIII and inserted into BamHI/HindIII cut pRSETB to afford pART1321/
1301/1311, respectively. All plasmid constructs were confirmed by DNA
sequencing. Then, pZR01 was expressed in E. coli BL21 Gold (DE3); pZR02
and pZR03 were expressed in E. coli BAP1; pART1321/1301/1311 were
expressed in E. coli BL21 (DE3) plysS. E. coli BAP1 strain that contains a
to obtain estimates for kcat and Km
.
LC-MS Identification of ACP-Bound Biosynthetic Intermediate
Assays were performed in 50 ml of 50 mM Tris (pH 7.5), 2 mM MgCl2, 1 mM
TCEP, 5 mM ATP, 1 mM aromatic substrate, 1 mM AsuA2, and 1 mM AsuC12
or AsuC11. Reactions were incubated for 1 hr and analyzed with nanocapillary
LC-MS using a chip column (Agilent Zorbax 300SB-C18 5 mm; separation:
43 mm 3 75 mm; enrichment: 4 mm 40 nl) in line with a QTOF (Agilent 6510
Q-TOF LC-MS). A linear gradient of 2%–95% CH3CN over 9 min in H2O with
0.1% formic acid at a flow rate of 0.6 ml/min was used for analysis. The pro-
tein mass was determined by deconvolution using MassHunter Qualitative
Analysis (Agilent).
884 Chemistry & Biology 20, 879–887, July 25, 2013 ª2013 Elsevier Ltd All rights reserved

Chemistry & Biology
Biosynthesis of Epoxyquinone Pharmacophore
Figure 6. Schematic of the Epoxyquinone
Moiety Formation in Asukamycin
See also Figure S2 and Table S1.
To obtain the kinetic parameters for NADH/
NADPH, 100 ml of AsuE1 or AsuE3 assays were
performed in 50 mM phosphate buffer (pH 7.0),
20 mM flavin (FAD for AsuE1 and FMN for AsuE3),
1 mM either AsuE1 or AsuE3, and variable con-
centrations of NADH/NADPH. The decrease in
absorbance at 340 nm was monitored after the
addition of enzyme in a Greiner UV-Star 96-well
plate on a Tecan Infinite 200 Pro plate reader.
The acquired turnover rates were fit to the
Michaelis-Menten equation using GraphPad Prism
to obtain kcat and Km
.
AsuE1 and AsuE3 Enzymatic Assays
To obtain the kinetic parameters of AsuE1 for protoasukamycin, 1 ml of
AsuE1 assay was performed in 50 mM phosphate buffer (pH 7.0), 20 mM
FAD, 1 mM NADH, 1 mM AsuE1, and variable concentrations of protoasukamy-
cin. Aliquots of 200 ml were quenched at 3, 5, 7, 9, and 11 min using an equal
volume of ethyl acetate. The extraction method and LC-MS analysis were the
same as mentioned before. The quantification of 4-hydroxyprotoasukamycin
was conducted by integrating the extracted 4-hydroxyprotoasukamycin
peak. The acquired turnover rates were fit to the Michaelis-Menten equation
using GraphPad Prism to obtain kcat and Km. The determination of kinetic
parameters of AsuE3 for 4-hydroxyprotoasukamycin was similar to that of
AsuE1, except that FMN was used as the flavin cofactor and the production
of asukamycin was recorded for calculation.
The authentic protoasukamycin and 4-hydroxyprotoasukamycin were
obtained from the fermentation cultures of DasuE1 and DasuE3 mutants,
respectively, as described previously (Rui et al., 2010). Typical conditions
were: a 50 ml AsuE1 or AsuE3 reaction mixture contains 50 mM phosphate
buffer (pH 7.0), 1 mM NAD(P)H, 20 mM flavin, 200 mM of either protoasukamycin
or 4-hydroxyprotoasukamycin, and 10 mM of either AsuE1 or AsuE3, respec-
tively. The reaction was performed at 30ꢂC and quenched with an equal volume
of ethyl acetate. The organic layer was concentrated in vacuo and resuspended
into 50 ml of methanol. Two microliters of the solution was injected into LC-MS
and a linear gradient of 55%–95% CH3CN (vol/vol) over 15 min in H2O with
0.1% (vol/vol) formic acid at a flow rate of 0.5 ml/min was used for analysis.
Figure 7. HPLC Analysis of Asukamycin Derivatives in DasuA3 Supplemented with 3-ABA, 4-ABA, 4-HBA, and 3,4-ACBA
R0 = -H in C12 and C15; R0 = -COCH3 in C11 and C16; R = R1–R5 in C6–C10; l = 325 nm.
DasuA3, 3,4-AHBA deficient mutant.
See also Figures S9–S11.
Chemistry & Biology 20, 879–887, July 25, 2013 ª2013 Elsevier Ltd All rights reserved 885

Chemistry & Biology
Biosynthesis of Epoxyquinone Pharmacophore
LC-MS Analysis of Flavin Bound to AsuE2
Received: February 20, 2013
Ten nanomoles of AsuE2 was denatured by addition of methanol to release any
bound cofactor. After centrifugation, the supernatant was analyzed with
LC-MS using a linear gradient from 5% to 95% acetonitrile containing 0.1%
formic acid at a flow rate of 0.5 ml/min. The released flavin was detected in
the negative ion mode; 200 pmol of authentic FAD and FMN were used as
standard.
Revised: April 23, 2013
Accepted: May 9, 2013
Published: July 25, 2013
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