Genome mining in Streptomyces. Discovery of an unprecedented P450-catalyzed oxidative rearrangement that is the final step in the biosynthesis of pentalenolactone

Article abstract of DOI:10.1021/ja111279h

The penM and pntM genes from the pentalenolactone biosynthetic gene clusters of Streptomyces exfoliatus UC5319 and Streptomyces arenae TUe469 were predicted to encode orthologous cytochrome P450s, CYP161C3 and CYP161C2, responsible for the final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1). Synthetic genes optimized for expression in Escherichia coli were used to obtain recombinant PenM and PntM, each carrying an N-terminal His₆-tag. Both proteins showed typical reduced-CO UV maxima at 450 nm, and each bound the predicted substrate, pentalenolactone F (4), with K_D values of 153 ± 14 and 126 ± 11 μM for PenM and PntM, respectively, as determined by UV shift titrations. PenM and PntM both catalyzed the oxidative rearrangement of 4 to 1 when incubated in the presence of NADPH, spinach ferredoxin, ferredoxin reductase, and O₂. The steady-state kinetic parameters were k_cat = 10.5 ± 1.7 min^-1 and K_m = 340 ± 100 μM 4 for PenM and k_cat = 8.8 ± 0.9 min^-1 and K_m = 430 ± 100 μM 4 for PntM. The in vivo function of both gene products was confirmed by the finding that the corresponding deletion mutants S. exfoliatus/ΔpenM ZD22 and S. arenae/ΔpntM ZD23 no longer produced pentalenolactone but accumulated the precursor pentalenolactone F. Complementation of each deletion mutant with either penM or pntM restored production of antibiotic 1. Pentalenolactone was also produced by an engineered strain of Streptomyces avermitilis that had been complemented with pntE, pntD, and either pntM or penM, as well as the S. avermitilis electron-transport genes for ferredoxin and ferrodoxin reductase, fdxD and fprD.

Full text of DOI:10.1021/ja111279h

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pubs.acs.org/JACS
Genome Mining in Streptomyces. Discovery of an Unprecedented
P450-Catalyzed Oxidative Rearrangement That Is the Final Step in the
Biosynthesis of Pentalenolactone
Dongqing Zhu,†,§ Myung-Ji Seo,†,§ Haruo Ikeda,‡ and David E. Cane*,†
†Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, United States
‡Laboratory of Microbial Engineering, Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara, Minami-ku,
Kanagawa 252-0373, Japan
S Supporting Information
b
have previously identified the responsible pentalenene synthase
ABSTRACT: The penM and pntM genes from the penta-
from Streptomyces exfoliatus UC5319 and reported the crystal
structure of the recombinant enzyme.3b,3c Experiments using
chirally deuterated and tritiated samples of FPP established the
detailed stereochemistry of the cyclization reaction itself, while
feeding of labeled pentalenene to cultures of S. exfoliatus and
analysis of the derived pentalenolactone confirmed the role of 1
as the parent hydrocarbon of the pentalenolactone family of
metabolites.3a Very recently, we isolated and sequenced two
closely related 13-kb gene clusters from two different pentale-
lenolactone biosynthetic gene clusters of Streptomyces
::
exfoliatus UC5319 and Streptomyces arenae TU469 were
predicted to encode orthologous cytochrome P450s, CYP-
161C3 and CYP161C2, responsible for the final step in the
biosynthesis of the sesquiterpenoid antibiotic pentalenolac-
tone (1). Synthetic genes optimized for expression in
Escherichia coli were used to obtain recombinant PenM
and PntM, each carrying an N-terminal His6-tag. Both
proteins showed typical reduced-CO UV maxima at 450
nm, and each bound the predicted substrate, pentalenolac-
tone F (4), with KD values of 153 ( 14 and 126 ( 11 μM for
PenM and PntM, respectively, as determined by UV shift
titrations. PenM and PntM both catalyzed the oxidative
rearrangement of 4 to 1 when incubated in the presence of
NADPH, spinach ferredoxin, ferredoxin reductase, and O2.
The steady-state kinetic parameters were kcat = 10.5 ( 1.7
min-1 and Km = 340 ( 100 μM 4 for PenM and kcat = 8.8 (
0.9 min-1 and Km = 430 ( 100 μM 4 for PntM. The in vivo
function of both gene products was confirmed by the finding
that the corresponding deletion mutants S. exfoliatus/
ΔpenM ZD22 and S. arenae/ΔpntM ZD23 no longer
produced pentalenolactone but accumulated the precursor
pentalenolactone F. Complementation of each deletion
mutant with either penM or pntM restored production of
antibiotic 1. Pentalenolactone was also produced by an
engineered strain of Streptomyces avermitilis that had been
complemented with pntE, pntD, and either pntM or penM, as
well as the S. avermitilis electron-transport genes for ferre-
doxin and ferrodoxin reductase, fdxD and fprD.
nolactone producers, S. exfoliatus UC5319 and Streptomyces
::
arenae TU469, which are responsible for the biosynthesis of this
sesquiterpenoid antibiotic and have been designated as the pen
and pnt clusters (Supporting Information (SI), Figure S1).4 Each
biosynthetic gene cluster encodes 11 open reading frames
(ORFs), with an average >90% mutual sequence similarity
between each pair of orthologous gene products. We also
identified and characterized the closely related ptl cluster from
Streptomyces avermitilis, which we showed to be responsible for
the biosynthesis of the recently discovered neopentalenolactone
branch of the pentalenolactone family of metabolites.5 We have
already assigned the biochemical function of eight of the key
ORFs from each cluster, several of which are closely related to
those of the ptl cluster, including five oxidative enzymes, four
oxygenases (PenI/PntI, PenH/PntH, PenE/PntE, PenD/
PntD), and the dehydrogenase (PenF/PntF) that together are
responsible for the multistep oxidative conversion of pentalenene
(3) to pentalenolactone F (4) (Scheme 1 and SI, Scheme S1).4a,5
The only step in the biosynthesis that remains to be elucidated
is the mechanism of the net oxidative rearrangement of penta-
lenolactone F (4) to pentalenolactone (1) itself. In fact, the only
structural genes within the complete pen and pnt biosynthetic
gene clusters whose functions have yet to be assigned are the
orthologous genes penM and pntM. Each of these genes encodes
a predicted cytochrome P450 of 398 amino acids, PenM and
PntM, respectively, with 81% mutual sequence identity and
87% similarity.6
entalenolactone (1) is a widely occurring sesquiterpenoid
P
antibiotic that has been isolated from more than 30 species
of Streptomyces.1 Pentalenolactone exerts its antibiotic action
against both Gram-positive and Gram-negative bacteria as well as
fungi and protozoa by reaction of the electrophilic epoxylactone
moiety with the active-site cysteine of glyceraldehyde-3-phos-
phate dehydrogenase, resulting in irreversible inactivation of this
target glycolytic enzyme.2 The committed step in the biosyn-
thesis of pentalenolactone is the cyclization of farnesyl diphosphate
(2, FPP) to the triquinane sesquiterpene pentalenene (3).3 We
To confirm the predicted biochemical function of both PenM
and PntM, synthetic genes corresponding to penM and pntM
with codons optimized for expression in Escherichia coli were
each inserted into the T7-based expression vector pET-28a(þ),
Received: December 14, 2010
Published: February 1, 2011
r
2011 American Chemical Society
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|

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Scheme 1
Figure 2. Construction of ΔpenM deletion mutant S. exfoliatus ZD22.
N2), GC-MS analysis indicated complete consumption of sub-
strate 4 (absence of 4-Me, m/z 292) and formation of the
pentalenolactone methyl ester (1-Me, m/z 290) (SI, Figure
S4).7 The structure of the enzymatically generated 1-Me was
established by direct comparison by capillary GC-MS with an
authentic sample. The product 1-Me was accompanied by ∼25%
of a second, m/z 290 component of unknown structure whose
GC-MS properties did not match those of authentic samples of
any of the known isomers of pentalenolactone in either retention
time (tR) or mass spectrum. The steady-state kinetic parameters
of both P450-catalyzed reactions were established by a series of
10 min incubations with PenM or PntM over a range of
concentrations of 4 from 72 to 860 μM, followed by calibrated
GC-MS analysis of the resulting 1-Me, giving kcat = 10.5 ( 1.7
min-1 and Km = 340 ( 100 μM 4 for PenM and kcat = 8.8 ( 0.9
min-1 and Km = 430 ( 100 μM 4 for PntM (SI, Figure S5).
We also investigated the in vivo role of the PenM and PntM
P450s by using Streptomyces gene replacement to generate the
corresponding in-frame deletion mutants, S. exfoliatus/ΔpenM
ZD22 and S. arenae/ΔpntM ZD23 (Figure 2 and SI, Figure S6).8
Thus, PCR was used to amplify 1860- and 1752-bp segments of
DNA flanking the penM gene harbored in a 7-kb segment of the
pen cluster.4a The two fragments were religated and inserted into
the vector pDQ44 to generate plasmid pDQ60, in which an
1170-bp internal segment of penM had been replaced by a 6-bp
scar flanked by 14 bp from the original 50-end of penM and 13 bp
from the 30-terminus of penM. Conjugation of plasmid DQ60
into wild-type S. exfoliatus UC5319 and two successive rounds of
homologous recombination gave the targeted penM deletion
mutant ZD22, whose integrity was confirmed by PCR. In an
analogous manner, PCR-targeted gene replacement was used to
replace a 1563-bp segment of pntM with an 81-bp scar (Figure
S6). Two successive rounds of homologous recombination then
gave the targeted in-frame deletion mutant, S. arenae/ΔpntM
ZD23, using PCR to screen the exconjugants and to confirm the
Figure 1. Characterization of PenM, CYP161C3. (A) Reduced CO
difference spectra. Solid line, free, Fe3þ-PenM; dotted line, Fe2þ-PenM
after Na2S2O4 reduction; broken line, reduced CO complex. (B)
Titration with pentalenolactone F (4), UV difference spectra.
and the corresponding plasmids pET28a-penM and pET28a-
pntM were individually transformed into the expression host
E. coli BL21(DE3). After induction with IPTG, the resulting
recombinant PenM and PntM proteins, each carrying an N-ter-
minal His6-tag, were purified to >95% homogeneity by immobi-
lized metal ion affinity chromatography on Ni2þ-NTA resin in
yields of 21 and 27 mg/L of culture, respectively (SI, Figure S2).
The purified His6-tag-PenM had an MD m/z 46 428 by ESI-MS
(predicted 46 428 for P-Met protein), while His6-tag-PntM had
an MD m/z 46 196 (predicted 46 195 for P-Met protein). High-
resolution gel filtration chromatography established that both
PenM and PntM are monomers.
The resting Fe3þ forms of both PenM and PntM each
exhibited characteristic heme Soret bands at 420 nm (Figure 1
and SI, Figure S3). Upon treatment of each protein with sodium
dithionite and exposure to CO, the reduced Fe2þ-CO differ-
ence spectra each showed a typical absorption maximum at 450
nm. Comparison of the P450 content with the concentration of
each protein gave a calculated ratio of 1.05-1.10 P450 per
subunit of protein. Titration of both PenM and PntM with
pentalenolactone F (4) gave rise to the characteristic Type I
P450 substrate-binding spectra, with a hypsochromic shift of the
low-spin 420 nm absorption to a high-spin band at 390 nm.
Analysis of the concentration dependence of the UV difference
spectra gave calculated KD values for 4 of 153 ( 14 and 126 (
11 μM for PenM and PntM, respectively.
To establish directly the biochemical function of both PenM
and PntM, the individual purified recombinant proteins were
incubated with pentalenolactone F (4) in the presence of spinach
ferredoxin, spinach ferredoxin-NADPþ reductase, and a 25-fold
molar excess of NADPH (Scheme 1). After 4 h incubation at
30 °C, followed by quenching with HCl and treatment of the
organic extracts with trimethylsilyl-diazomethane (TMS-CH-
desired deletion.
::
Both wild-type S. exfoliatus UC5319 and S. arenae TU469 as
well as the corresponding deletion mutants S. exfoliatus/ΔpenM
ZD22 and S. arenae/ΔpntM ZD23 were cultivated in the appro-
priate liquid production media, and the resulting culture broths
were assayed by GC-MS after methylation of the chloroform
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COMMUNICATION
Scheme 2
The P450-catalyzed oxidative rearrangement of pentalenolac-
tone F (4) to pentalenolactone (1) has no biochemical pre-
cedent. Indeed, the vast majority of P450-catalyzed oxidations
are simple oxygenations, such as hydroxylation of C-H bonds,
epoxidation of alkenes, and heteroatom oxidation, or oxidative
C-C bond cleavages and C-C bond-forming reactions, such as
phenolic couplings.11 Wagner-Meerwein-type carbocation re-
arrangements and elimination reactions are, of course, very
common in nonoxidative terpene synthase-catalyzed transforma-
tions. Generation of the intermediate C-1 cation 6 from 4
presumably involves direct transfer of the H-1 si hydride3a to a
reactive ferryl iron-oxo species, or alternatively hydrogen atom
abstraction of H-1 si followed by rapid electron transfer to the
paired hydroxyl radical-iron species (Scheme 2). The transient,
initially generated, highly oxidizing ferryl iron-oxo species, P450
compound I, has been implicated in the vast majority of P450-
catalyzed reactions.11,12 P450-generated caged radical pair
intermediates normally recombine to give the hydroxylated pro-
duct with a rebound rate of >1010-1011 s-1, with competing
formation of cationic species representing at most only a very
minor reaction channel.11b,13 The neopentyl cation intermediate
6 produced by oxidation of pentalenolactone F will undergo
successive syn migration of the C-12 methyl group and coupled
anti deprotonation with loss of H-3 re to give pentalenolactone
(1).3a,14 Although we cannot definitively rule out an alternative
radical rearrangement-hydrogen atom abstraction pathway for
the formation of 1, enzyme-catalyzed radical rearrangements are
relatively rare, with the exception of P450-catalyzed reactions of
artificial radical clock substrates such as cyclopropylmethylene
derivatives11b,13 or the rearrangements catalyzed by corrinoid- or
S-adenosyl methionine-dependent enzymes.15
The previously isolated (1R)-hydroxy shunt metabolite pentale-
nolactone H (7)16 is excluded as an intermediate or side product of
the PenM- and PntM-catalyzed reactions since the configuration of
the hydroxyl group of 7 and the retention of H-1 si are incompatible
with the demonstrated loss of H-1 si from pentalenolactone F in the
formation of 1.3a On the other hand, the isomeric products pen-
talenolactones A (8), B (9), and P (10), each of which has
previously been isolated as a trace component in the culture extracts
of pentalenolactone-producing Streptomyces,9,16 are presumably
formed by competing deprotonation of 6 or the derived carboca-
tionic intermediates of the dominant oxidative rearrangement
pathway. Indeed, the formation of pentalenolactone P (10) suggests
that the 1,2-methyl migration may involve a corner-protonated
cyclopropane as an intermediate or transition state. None of these
three very minor components could be detected, however, in the
small-scale in vitro incubations of 4 with either PenM or PntM.
PenM and PntM have 84% and 89% sequence identity,
respectively, to the SBI_10607 protein (CYP161C1) of
Figure 3. ΔpenM and ΔpntM deletion mutants. (A) Accumulation of
pentalenolactone F (4) by S. exfoliatus ZD22, S. arenae ZD23, and
S. avermitilis SUKA16 ΔptlEΔptlD::ermEp-pntE-pntD. (B) Complementa-
tion by penM or pntM restores production of pentalenolactone (1).
extracts with TMS-CHN2 (Figure 3 and SI, Figure S7). While
both wild-type strains produced, as expected, pentalenolactone
methyl ester (1-Me, m/z 290), identified by direct comparison
with an authentic sample, 1-Me could not be detected in the
methylated extracts of either P450- deletion mutant, ZD22 or
ZD23. Instead, both S. exfoliatus/ΔpenM ZD22 and S. arenae/
ΔpntM ZD23 accumulated enhanced proportions of pentaleno-
lactone F, analyzed as the corresponding methyl ester 4-Me (tR =
12.82 min, m/z 292), identical by direct GC-MS comparison
with authentic 4-Me. Interestingly, the proportion of pentaleno-
lactone D (5)9 also decreased in cultures of both deletion
mutants. Complementation of either S. exfoliatus/ΔpenM ZD22
or S. arenae/ΔpntM ZD23 with pntM under control of the
constitutive ermE promoter restored production of pentaleno-
lactone (1), as established by GC-MS analysis and detection of
enhanced levels of 1-Me in the methylated extracts of each of
the corresponding exconjugants (Figure 3 and SI, Figure S8).
Similarly, conjugation of penM under control of ermEp into the
corresponding ZD22 and ZD23 deletion mutants also restored
production of pentalenolactone (1). Interestingly, all of the
complemented strains also produced an additional unidentified
m/z 290 component (tR = 13.44 min) that also could be detected
in extracts of both wild-type strains but was absent from both the
penM and pntM deletion mutants and was identical to the m/z
290 side product of the in vitro incubation of recombinant PenM
and PntM with pentalenolactone F. The structure of this addi-
tional product is under investigation.
We previously described the construction of an engineered
strain, S. avermitilis SUKA16 ΔptlEΔptlD::ermEp-pntE-pntD,
that produces pentalenolactone F (4), along with the shunt
metabolite 9,10-epi-pentalenolactone F (epi-4) (Figure 3A).4a
Complementation of this strain with either penM or pntM as well
as the S. avermitilis ferredoxin and ferredoxin reductase genes,
fdxD and fprD, all under control of the ermE promoter, led in
both cases to production of pentalenolactone (1), as determined
by GC-MS analysis of the methylated extracts (Figure 3B). In the
absence of enhanced levels of the P450 electron transport
proteins fdxD and fprD, the production of pentalenolactone
(1) was significantly reduced (SI, Figure S9).10
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Streptomyces bingchenggensis.17 In fact, SBI_10607 is itself
found within a predicted 11-ORF pentalenolactone biosyn-
thetic gene cluster4a and, therefore, is expected to catalyze
the identical oxidative conversion of 4 to 1. Neither PenM
nor PntM has more than 50% identity to any other P450, as
revealed by a BLAST search of the nonredundant protein
database. The vast majority of the low similarity matches are
to proteins that are either predicted hydroxylases or of
altogether unknown function. For example, PntM has only
40% identity and 56% positive matches to the predicted 397-
amino acid PimD protein (UniProt ID Q9EW92) that is
believed to be responsible for an unspecified late-stage hy-
droxylation or epoxidation in the biosynthesis of the anti-
fungal polyene antibiotic pimaricin.18 We anticipate that
further mining of Streptomyces genomes will continue to
unearth unusual and intriguing biochemical nuggets.
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’ AUTHOR INFORMATION
Corresponding Author
David_Cane@brown.edu
Author Contributions
§These authors contributed equally to this work.
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This work was supported by NIH grant GM30301 (D.E.C.)
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