Assembly of Stephacidin and Notoamide Anticancer Agents
A R T I C L E S
confirming its role as the elusive deoxybrevianamide E synthase,
while only 22 is utilized by NotC (C-7 alkylation) in the
biosynthesis of the stephacidin and notoamides (Figure 2). The
mechanism of the reverse prenyltransferase was also probed by
site-directed mutagenesis to understand the reaction of this group
of enzymes.17,20 Future structural studies of NotF and the
comparison to FgaPT220 are expected to contribute further
information about the regio- and stereospecificity of the reverse
and normal prenyltransfer reactions. We expect this analysis
will likely illuminate the lack of facial selectivity previously
observed for the reverse prenylation step29 and facilitate
expansion of the enzyme substrate range and efficiency. More
importantly, the combined studies of NotF and NotC provide
direct evidence to establish a biosynthetic scheme for this family
of bioactive prenylated fungal alkaloids. Finally, the high in
Vitro catalytic efficiencies of recombinant NotF and NotC
suggest their potential value as biocatalysts for chemoenzymatic
production of bioactive fungal alkaloid analogs in drug
development.
Identification and characterization of the notoamide gene
cluster also provides the initial basis to understand the formation
of three pairs of antipodal natural products derived from a
marine-derived and a terrestrial Aspergillus sp.30,31 In the
marine-derived fungal strain, (-)-notoamide B (10), (-)-
versicolamide B (38), and (+)-stephacidin A (14) are produced,7,30
while their antipodal counterparts, 39, 40, and 41, respectively,
are isolated from the terrestrial A. Versicolor NRRL 25660 strain
(Supplementary Figure 12).31 Based on the putative notoamide
biosynthetic pathway, we propose that formation of 14 and 41
might be controlled by the IMDA reaction. Subsequently, 10
and 39 are possibly derived from 14 and 41, respectively, in
these two distinct fungal strains. It remains unclear whether
generation of (-)- and (+)-versicolamide B (38 and 40) occurs
in the pathway through stephacidin A due to their opposite
stereogenic centers at C-6 (Supplementary Figure 12).30 Instead,
13 might be converted into 11, subsequently producing 38 in
the following IMDA reaction in the marine-derived fungus.30
The detailed biochemical characterization of biosynthetic en-
zymes from both fungal strains is in progress and will shed
more light on the biosynthesis of these unique antipodal natural
products.
be further optimized for the efficient production of a target
metabolite using traditional mutation and selection methods, as
well as new tools from systems biology and synthetic biology.35
Identification of the notoamide gene cluster provides such an
opportunity to produce bioactive fungal alkaloids and analogs
thereof through pathway engineering and heterologous expression.
Methods
Materials and Strains. Authentic deoxybrevianamide E (15),
doubly 13C-labeled brevianamide F (17), and keto-premalbranchea-
mide (23) were synthesized following previously published
procedures.4,36 Standard methods for DNA isolation and manipula-
tion were performed as described by Sambrook et al.37 Genomic
DNA from Aspergillus MF297-2 was isolated with a MasterPure
Yeast DNA Purification kit (Epicenter Biotechnologies) as described
in the manual. The GenBank accession numbers for notC, notF,
and the complete assembled not gene cluster are GU564534,
GU564535, and HM622670, respectively. E. coli DH5R was used
for cloning and plasmid harvesting while E. coli BL21 CodonPlus-
(DE3)-RIPL was used for protein overexpression.
Expression and Purification of NotC and NotF. Details about
the preparation of notC and notF cDNAs and of notF mutant DNAs
are included in the Supporting Information and Supplementary
Table 1. The expressed enzymes were purified with a single Ni-
NTA column (Supporting Information). As determined by SDS-
PAGE analysis, the purity of proteins was more than 90%. The
native status of proteins was determined by gel filtration (Supple-
mentary Figure 1).
Determination of Enzyme Activities. Compounds 22 and 24
were chemically synthesized to examine NotC activity (Supporting
Information). The 100-µL reaction mixture contained 0.5 µg of
NotF, its mutants, or NotC; 5 mM MgCl2; 0.1 mM 17 (NotF or its
mutants) or 22 (NotC); and 0.15 mM DMAPP in the reaction buffer
(50 mM Tris-Cl, pH 7.5, 10% glycerol, and 3 mM ꢀ-mercapto-
ethanol). The reaction was initiated by adding enzyme after
prewarming the other components at room temperature for 1 min.
After mixing well and briefly centrifuging, the reactions were further
incubated at room temperature for 45-60 min and stopped with
10 µL of 1.5 M trichloroacetic acid. The mixtures were mixed and
centrifuged at 13 000g for 5 min. An aliquot of the 100-µL solution
was subjected to HPLC coupled with an XBridge C18 column (5
µm, 4.6 mm × 250 mm), at a wavelength of 222 nm. Solvent B
(acetonitrile in 0.1% TFA) was increased from 30% to 40% for 5
min and then increased to 80% over 20 min for the detection of
products. LC-MS2 analysis was performed by using a ThermoFinni-
gan LTQ linear ion-trap instrument equipped with an electrospray
source and a Surveyor HPLC system at room temperature.
Separations were performed with an XBridge C18 (3.5 µm, 2.1
mm × 150 mm) column at a flow rate of 200 µL/min with solvent
A (water with 0.1% formic acid) and solvent B (acetonitrile with
0.1% formic acid). Solvent B was kept at 2% in solvent A for 4
min and then was gradually increased to 90% over 16 min. After
being washed with 90% solvent B for 2 min, the column was further
re-equilibrated with 2% solvent B for 10 min. The spectra were
recorded in positive ion mode. Product 25 was further characterized
Identification of biocatalysts from fungal alkaloid biosynthetic
pathways may also enable production of natural products and
their analogs through heterologous expression and metabolic
engineering.32-34 It is estimated that >99% of microorganisms
in the environment fail to grow in the laboratory, and the
potential to find pharmaceutically important natural products
from fungal sources remains vastly underexplored. Introducing
natural product gene clusters into more technically and industri-
ally amenable microorganisms such as E. coli and yeast
represents an attractive way to obtain suitable quantities of
natural products and to identity novel leads in drug discovery
and development programs.34 Moreover, a microorganism can
1
with H and 13C NMR analysis (Supporting Information).
Kinetics Analysis. The 100-µL reaction mixture contained 0.25
µg of NotF or 0.11 µg of NotC and 5 mM MgCl2 in the reaction
buffer. Details about the experiment procedures were included in
the Supporting Information. All experiments were performed in
duplicate. The data were fit to the Michaelis-Menten equation in
Prism 4.0 (GraphPad Software).
(29) Stocking, E. M.; Sanz-Cervera, J. F.; Williams, R. M. Angew. Chem.,
Int. Ed. 1999, 38 (6), 786–9.
(30) Tsukamoto, S.; Kawabata, T.; Kato, H.; Greshock, T. J.; Hirota, H.;
Ohta, T.; Williams, R. M. Org. Lett. 2009, 11 (6), 1297–300.
(31) Greshock, T. J.; Grubbs, A. W.; Jiao, P.; Wicklow, D. T.; Gloer, J. B.;
Williams, R. M. Angew. Chem., Int. Ed. 2008, 47 (19), 3573–7.
(32) Chemler, J. A.; Koffas, M. A. Curr. Opin. Biotechnol. 2008, 19 (6),
597–605.
(35) Lee, S. Y.; Kim, H. U.; Park, J. H.; Park, J. M.; Kim, T. Y. Drug
DiscoVery Today 2009, 14 (1-2), 78–88.
(36) Miller, K. A.; Welch, T. R.; Greshock, T. J.; Ding, Y.; Sherman, D. H.;
Williams, R. M. J. Org. Chem. 2008, 73 (8), 3116–9.
(37) Sambrook, J.; Russel, D. W. Molecular Cloning - A Laboratory
Manual, 3rd ed.; Cold Spring Harbor Laboratory Press: New York,
2001.
(33) Wenzel, S. C.; Muller, R. Curr. Opin. Biotechnol. 2005, 16 (6), 594–
606.
(34) Zhang, H.; Wang, Y.; Pfeifer, B. A. Mol. Pharm. 2008, 5 (2), 212–
25.
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Ding, Yousong
