Roles of the synergistic reductive O-methyltransferase GilM and of O-methyltransferase GilMT in the gilvocarcin biosynthetic pathway

Article abstract of DOI:10.1021/ja305113d

Two enzymes of the gilvocarcin biosynthetic pathway, GilMT and GilM, with unclear functions were investigated by in vitro studies using purified, recombinant enzymes along with synthetically prepared intermediates. The studies revealed GilMT as a typical

Full text of DOI:10.1021/ja305113d

Communication
pubs.acs.org/JACS
Roles of the Synergistic Reductive O‑Methyltransferase GilM and of
O‑Methyltransferase GilMT in the Gilvocarcin Biosynthetic Pathway
Nidhi Tibrewal,† Theresa E. Downey,† Steven G. Van Lanen,† Ehesan Ul Sharif,‡ George A. O’Doherty,‡
and Jurgen Rohr*,†
̈
†Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington,
Kentucky 40536-0596, United States
‡Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115,
United States
S
* Supporting Information
Scheme 1. Key Oxidative Rearrangement and Follow-up
Sequence of Events Involving GilMT and GilM Reactions en
route to Defuco-gilvocarcin M (7)
ABSTRACT: Two enzymes of the gilvocarcin biosyn-
thetic pathway, GilMT and GilM, with unclear functions
were investigated by in vitro studies using purified,
recombinant enzymes along with synthetically prepared
intermediates. The studies revealed GilMT as a typical S-
adenosylmethionine (SAM) dependent O-methyltransfer-
ase, but GilM was identified as a pivotal enzyme in the
pathway that exhibits dual functionality in that it catalyzes
a reduction of a quinone intermediate to a hydroquinone,
which goes hand-in-hand with a stabilizing O-methylation
and a hemiacetal formation. GilM mediates its reductive
catalysis through the aid of GilR that provides FADH2 for
the GilM reaction, through which FAD is regenerated for
the next catalytic cycle. This unusual synergy eventually
completes the biosynthesis of the polyketide-derived
defuco-gilvocarcin chromphore.
ilvocarcin V (GV, 1), produced by Streptomyces
Ggriseoflavus and various other Streptomyces strains, is the
prototype of the rare group of polycyclic aromatic polyketides
with benzo[d]naphtha[1,2-b]pyran-6-one chromophore, re-
ferred to as the gilvocarcin group. This group shows significant
antitumor activities and remarkably low toxicity.1,2 Gilvocarcin
V exhibits its light-induced anticancer activity by mediating
cross-linking between DNA and histone H3.3,4 The vinyl group
enhances the antitumor activity through formation of a
photoinduced [2 + 2] cycloadduct with thymine residues of
double stranded DNA,5 since the minor congeners gilvocarcin
M (2) and gilvocarcin E, which lack the vinyl group, are
considerably less active than GV.6 For the histone H3
interaction, the D-fucofuranose moiety is considered essential.
intermediate into the unique defuco-gilvocarcin chromophore
typical for this class of anticancer drugs.8 The role of many of
the post-PKS gene products have been assigned based on gene
inactivation, cross-feeding and labeling studies,1,7−12 and most
of the genes of the three known biosynthetic clusters13 of this
group have been assigned to certain enzymatic activities with
gilM (encoding an enzyme of unknown function) and gilMT
(encoding a methyltransferase) being notable exceptions. Thus,
the functions of their gene products, GilM and GilMT,
remained obscure. Both the enzymes appear to be crucial for
the biosynthesis of gilvocarcin and were proposed to be
involved in O-methylation reactions. However, mutant strains
created by deletion of these individual genes did not
accumulate any isolable metabolites, and thus provided no
clue regarding their potential substrates and hence structure of
key biosynthetic intermediates.
The aromatic skeleton of the gilvocarcins is produced by a
type II polyketide synthase (PKS),1,7 which initially yields an
angucycline biosynthetic intermediate (e.g., 3 in Scheme 1).
Intriguing oxidative post-PKS modifications then convert this
Received: May 25, 2012
Published: July 16, 2012
© 2012 American Chemical Society
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Journal of the American Chemical Society
Communication
However, a recent combinatorial biosynthetic enzymology
approach led to the enzymatic total synthesis of defuco-
gilvocarcin M (7) from acetyl-CoA and malonyl-CoA, a model
compound with the completed gilvocarcin chromophore.
Moreover, systematic variations of the enzyme mixtures
revealed a hypothetical pathway suggesting that 2-aryl-5-
hydroxy-1,4-naphthoquinone-3-carboxylic acid (4) is likely the
product of the critical oxidative C−C bond cleavage and
thereby a key intermediate of gilvocarcin V. The results also
suggested that only three more enzymes, namely GilM, GilMT
and GilR, were necessary to finish the biosynthesis of 7. To
confirm this hypothesis and fully enlighten the “biosynthetic
black box”, a potential model substrate, 2-aryl-5-hydroxy-1,4-
naphthoquinone (15) was synthesized and subsequently used
to interrogate the function of GilM and GilMT (Scheme 2).
a
Scheme 2
Figure 1. HPLC traces of the enzymatic reactions with 2-aryl-1,4-
naphthaquinone (15. (A) Standard 2-aryl-1,4-naphthaquinone (15);
(B) standard defuco-gilvocarcin M (7); (C) 15 + GilM; (D) 15 +
GilMT; (E) 15 + GilMT+ SAM; (F) 15 + GilMT + GilM + SAM; (G)
15 + GilMT + GilM + GilR + SAM.
product was isolated and characterized using NMR and HRMS.
The analysis revealed the product as monomethylated
derivative 5. Nuclear Overhauser enhancement (NOE) studies
further confirmed that the hydroxyl group of the phenyl ring of
substrate 15 was methylated.
a
When compound 15 was incubated with both GilM and
GilMT along with SAM, a new product along with 5
accumulated (Figure 1, trace F). The new compound was
identified as defuco-pregilvocarcin M (6) by NMR experiments
(1H NMR, HSQC). Adding GilR led to the accumulation of
defuco-gilvocarcin M (7) (Figure 1, trace G), further
confirming the structure of the GilM/MT product as 6. In
total the results suggested that GilMT is a typical SAM-
dependent O-methyltransferase, while GilM was responsible for
the reduction of the quinone moiety necessary for the observed
second O-methylation. BLAST (basic local alignment search
tool) analysis17,18 revealed GilM to have low similarity (34−
37% sequence identity) to nucleotidyl-S-transferases such as
thiopurine-S-methyltransferases from Rhodococcus equi or
Mycobacterium avium as well as to benzoquinone methyl-
transferases from Mycobacterium tuberculosis (33% sequence
identity). The translated amino acid sequence of GilM contains
VLDLGCGLG as residues 49−57, which appears to be a SAM
binding motif (generally hh(D/E)hGXGXG, where h repre-
sents a hydrophobic residue). Thus, it remained unclear at this
point whether GilMT or GilM catalyzes the second O-
methylation. To solve this ambiguity, product 5 from GilMT
reaction was used as substrate for GilM in the presence of SAM.
The reaction accumulated 6, suggesting that GilM not only
mediates reduction of the quinone but also catalyzes a second
O-methylation. Surprisingly, the GilM reaction seemed to
convert 5 to 6 even in absence of SAM, which prompted us to
further investigate GilM for any bound SAM cofactor. The
enzyme was boiled for 5 min and centrifuged (12000× g, 5
min). The supernatant when analyzed by HPLC showed UV-
absorption at 260, typical of adenosine spectrum. A comparison
with commercially available SAM verified that GilM copurifies
with SAM, thereby solving the mystery of the methyl source
(Supporting Information, Figure 1).
Reagents and Conditions: (a) NBS, dibenzoyl peroxide, CCl4, reflux,
1h, 70% ; (b) NaHCO3, H2O, Me2CO, reflux, 4 h, 86%; (c) PCC,
silica gel, CH2Cl2, 1 h, quant.; (d) 1M BBr3 in CH2Cl2, 2h; (e) 1:1
HCl/AcOH, reflux, 10h, 60%; (f) 1,3-propanediol, TsOH (cat.),
toluene, reflux, 12 h, 75%; (g) chloromethyl methyl ether, iPr2EtN,
CH2Cl2, 40 °C, 24 h, 95%; (h) n-BuLi, n-Bu3SnCl, hexane, 0 °C, 88%;
(i) Pd2(dba)3·CHCl3, PPh3, CuI, THF, 75 °C, 12 h, 75%; (j) 2.4 M
HCl in CH3CN, 74%.
Synthesis of compound 15 began with commercially available
dimethyl anisole (8).14 Bromination with N-bromo-succimide
(NBS) and benzoyl peroxide provided mono bromo benzyl
derivative. A sequential hydroxylation followed by oxidation
with pyridinium chlorochromate (PCC) afforded 9 in 60%
yield from 8.14 Removal of the methoxy group was achieved in
60% yield by treatment with BBr3 at r.t., followed by acidic
hydrolysis. Protection of the aldehyde as a cyclic acetal and the
phenolic OH as methoxylmethyl ether provided 11. An ortho-
metalation with tributyltinchloride yielded the required
stannane 12 in 88% yield.15 2-Bromonaphthoquinone (13)
was prepared from juglone using the reported protocol.16
A
facile Stille coupling between 12 and 13 provided 14. 14 was
then exposed to harsh acidic conditions (2.4 M HCl in
CH3CN) for 4 min to provide fully unprotected 2-aryl-1,4-
naphthaquinone 15 in 74% yield.15
With a potential substrate in hand, both gilM and gilMT
genes were cloned into pET28a and expressed in E. coli to yield
soluble proteins that were purified to near homogeneity.
Synthetic 15 when incubated with purified GilM produced a
complex mixture (Figure 1, trace C). In contrast, HPLC-MS
analysis of 15 with GilMT and S-adenosylmethionine (SAM)
revealed a decrease in the amount of substrate and formation of
a new product (Figure 1, trace E), while in the absence of SAM,
no product was formed (Figure 1, trace D). The GilMT
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Journal of the American Chemical Society
Communication
To further analyze the reaction sequence catalyzed by GilM,
compound 5 was incubated with GilM and the reaction was
studied at different time points (Figure 2). With stoichiometric
Figure 3. HPLC traces of the enzymatic reactions with 5. (A)
Standard 5; (B) standard defuco-gilvocarcin M (7); (C) 5 + GilM;
(D) 5 + GilM + GilR; (E) 5 + GilM + GilR + SAM.
fold increase in the formation of 7 in the GilM-GilR reaction
versus using GilM alone confirmed that GilM works synergisti-
cally with GilR.
Figure 2. HPLC traces of the enzymatic reactions with 5. (A)
Standard 5; (B) 5 + GilM + SAM + 5 min; (C) 5 + GilM + SAM + 10
min; (D) 5 + GilM + SAM + 15 min; (E) intermediate (16) isolated
from HPLC (mixture of closed and open form); (F) 16 + GilM.
Overall, these results described here allowed us to fully prove
the post-PKS reaction sequence of the gilvocarcin biosynthetic
pathway as shown in Scheme 1. The fact that the easy to
synthesize compound 15 is an intermediate of the gilvocarcin
pathway opens up the future possibilities of generating
gilvocarcin analogues through chemo-enzymatic synthesis or
mutasynthesis,21,22 thus coupling the power of chemical
synthesis with metabolic engineering.
enzyme quantities, the substrate was 80% converted into the
product after 15 min (Figure 2, trace D). When the reaction
was analyzed by reversed phase HPLC-MS in 3−5 min
intervals, a new peak (16) appeared with shorter retention time
than the overall product 6 (Figure 2). Although NMR analysis
of 16 was impossible due to its instability, LC-MS analysis
suggested it to be demethyl-defuco-pregilvocarcin M (m/z 323
[M − H]−). To prove that 16 was an intermediate en route to
6 and not a shunt product, 16 was incubated with GilM, and, as
anticipated, was rapidly converted to 6 (Figure 2, trace F).
Overall, the results showed that GilM catalyzes a sequence of
reactions: (i) quinone reduction, (ii) hemiacetal formation and
(iii) O-methylation, to construct the tetracyclic core of the
gilvocarcins.
The only remaining question was the regeneration of GilM
after reduction of the quinone. Interestingly, the respective
GilM-activity in the biosynthetic pathways of other structurally
related compounds, chrysomycin A and ravidomycin V, is
encoded on the same gene as the oxidoreductase GilR-
activity,13 while in gilvocarcin pathway, gilM and gilR are two
separate genes.1 This prompted us to propose that GilM could
be working in conjunction with GilR, an FAD dependent
oxidoreductase that catalyzes the very last step in gilvocarcin
biosynthesis by converting pregilvocarcin to gilvocarcin (2), but
also was shown to convert sugar free defuco-pregilvocarcins.9,19
We hypothesized that GilM utilizes the reduced flavin
generated in the GilR reaction to reduce the quinone thereby
regenerating oxidized flavin for the next catalytic cycle. To
validate this hypothesis, compound 5 was incubated with GilM
and GilR (Figure 3). The assistance of GilR for the reducing
capabilities of GilM was established by measuring the amounts
of the products formed in the reactions. Catalytic amounts of
GilM alone accumulated mostly starting material along with
minor production of 6 (Figure 3, trace C). In the absence of
SAM, GilM and GilR accumulated a new peak (Figure 3, trace
D) that corresponds to demethyl-defuco-gilvocarcin M20 (17,
m/z 321 [M − H]−). Adding SAM to the GilM-GilR reaction
mixture led to the accumulation of 7 (Figure 3, trace E). A 10-
ASSOCIATED CONTENT
* Supporting Information
Synthetic procedures, kinetic studies of GilM and GilMT,
enzymatic total synthesis of 6, and NMR spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
jrohr2@email.uky.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH grants CA 102102 and CA
091901 to J.R. We thank Ms. Manjula Sunkara as well as Drs. J.
Goodman and Andrew Morris for the mass spectra.
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