aromatic methylation of the antitumour drug mithramycin

45 Pages·2003·1.48 MB·English

JBC Papers in Press. Published on December 5, 2003 as Manuscript M312351200

MtmMII-mediated C-methylation during biosynthesis of the antitumor
drug mithramycin is essential for biological activity and DNA-drug
interaction

Running title: C-Methylation during mithramycin biosynthesis
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David Rodríguez1,2, Luis M. Quirós2 and José A. Salas1,2* ww
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1 Departamento de Biología Funcional, Universidad de Oviedo, 33006 Oviedo, Spain es
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2 Instituto Universitario de Oncología del Principado de Asturias (I.U.O.P.A), nu
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Universidad de Oviedo, 33006 Oviedo, Spain. , 20
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* Corresponding author. Tel/Fax [phone]. email: [email protected]

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
SUMMARY
The antitumor drug mithramycin consists of a polyketide chromophore glycosylated
with a trisaccharide and a disaccharide. Two post-polyketide methylations take place
during mithramycin biosynthesis. One of these methylations has shown to be very
relevant for biological activity, that is the introduction of a methyl group at aromatic C-
7. We have purified 282-fold the MtmMII methyltransferase involved in this reaction.
The protein is a monomer and results from kinetic studies were consistent with a model
for the enzyme acting via a compulsory order mechanism. The enzyme showed high
substrate specificity, being unable to operate on structurally closely related molecules.
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Structural predictions suggest that the molecule is integrated by two domains, an ow
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essentially all-α amino domain, and an α/β carboxyl domain displaying a variation of a ed
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Rossmann-fold and that contains the cofactor binding site. Although 7-demethyl- http
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mithramycin did not show any biological activity, it was able to reach the nucleus of w
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eukaryotic cells, with the subsequent binding to DNA. Mithramycin and 7- rg
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demethylmithramycin were able to form similar complexes with Mg2+, but their es
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respective DNA-binding isotherms showed to be very different. The dinucleotide nu
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binding model fitted very well the isotherms recorded for both compounds predicting , 20
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that the C-7 methyl group was essential for high affinity binding to specific GC and CG
sequences. Considering previous structural studies, we propose that this effect is
performed by positioning the group in the floor of the minor groove, allowing the
interaction with the third sugar moiety of the trisaccharide, D-mycarose, which is
involved in sequence selectivity.
INTRODUCTION
Polyketides comprise a family of compounds synthesized via linear poly-β-
ketones by repetitive head-to tail additions of acyl units. These compounds may
undergo a variety of further reactions, including cyclizations, methylations,
hydroxylations, glycosylations, oxidations and reductions. The result is an extremely
diverse family of natural products, many of which display useful biological activities
with clinical (antibiotics, antifungal, antitumor drugs, antiparasitic and
immunosuppressive agents) or agricultural (insecticides, herbicides) applications. The
aureolic acid group of antitumor drugs comprise mithramycin (MTM), chromomycins,
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olivomycins, chromocyclomycin, durhamycin and UCH9 (1) (Fig. 1). All these ow
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compounds belong to the large and important family of aromatic polyketides. Their ed
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structures are constituted by a tricyclic chromophore that is glycosylated at two different http
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positions of the aglycon with saccharides of various chain lengths (1). The only w
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exception being chromocyclomycin, which possesses a tetracyclic chromophore. The rg
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polyketide aglycon of these compounds is derived from the condensation of ten acetates es
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in a series of reactions catalysed by a type II polyketide synthase (2). These compounds nu
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are active against Gram-positive bacteria but not against Gram-negative because of a , 20
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permeability barrier. While these compounds are too toxic to be used as antibiotics, they
show good antitumor activity and some of them are in clinical use. They interact with
GC-rich regions of DNA in a non-intercalative way in the presence of Mg2+ ions which
are essential for activity, forming 2:1 antibiotic:Mg2+ complexes and inhibiting
replication and transcription (3).
MTM is produced by Streptomyces argillaceus and some other streptomycetes.
It has been clinically used for the treatment of testicular carcinoma, Paget’s bone
disease and other bone growth disorders and is also used for control of hypercalcemia
in patients with malignant disease (4). The biosynthetic pathway for MTM biosynthesis
has been extensively studied by biochemical and genetical means (2, 5-12). Although
MTM is a tricyclic aromatic polyketide with a side chain, its biosynthesis proceeds
through tetracyclic tetracycline-like intermediates (6). The MTM polyketide skeleton
derives from one acetyl-CoA and nine malonyl-CoA units and, through condensation
reactions, give rise to a decaketide (5). This molecule will be substrate for several
aromatizations and cyclizations to produce 4-demethyl-premithramycinone (the first
biosynthetic intermediate so far isolated) and, after a O-methylation step, it renders
premithramycinone (2,6). This tetracyclic intermediate is then glycosylated through a
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series of biosynthetic intermediates with the addition of D-olivose, D-oliose and D- ow
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mycarose sugar moieties finally making a trisaccharide chain, and generating 9- ed
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demethyl-premithramycin A3 (9-DMPA3). This intermediate is then C-methylated to http
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premithramycin A3 (PMA3) (11) prior to the addition of a disaccharide of D-olivose-D- w
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olivose to originate premithramycin B (9). All these biosynthetic intermediates have rg
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been isolated from mutants blocked at different steps in the biosynthesis and share a es
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common feature, that is the presence of a tetracyclic aglycon. The final tricyclic nu
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structure of MTM is obtained after the oxidative cleavage of the fourth ring of , 20
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premithramycin B, in a reaction catalyzed by a monooxygenase and followed by the
ketoreduction of the 4’-position of the generated aliphatic chain (9,12). This renders the
fully biologically active compound. None of the other biosynthetic intermediates
obtained by insertional inactivation show any biological activity. One outstanding case
is 7-DMTM, a product accumulated by a mutant lacking the mtmMII methyltransferase
gene, whose structure resembles that of MTM, varying only in the absence of the C-7
methyl group (11). Interestingly, 7-DMTM displays no activity over several tumoral
cell lines (13).
Biological methylations have been found to play a crucial role in drug
metabolism and biosynthesis. In secondary meatbolism pathways, S-adenosyl-
methionine (SAM) is commonly utilised as ubiquitous methyl donor, while folate-
dependent methylations are mainly found in primary metabolism in different organisms,
including mammals (14). Substrates of SAM-dependent methyltransferases include
lipids, proteins, polynucleotides, polysaccharides and small molecules as polyketides.
The methylated functional groups include carboxyl, phenol and hydroxyl groups
(oxygen atoms), aliphatic and aromatic amines (nitrogen atoms), thiols and sulfides
(sulfur atoms), olefins and ring carbons (carbon atoms), halides and metal ions (14).
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The structure of MTM contains nine methyl groups. Six of them are part of the sugar ow
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moieties, since each deoxysugar unit contains a C-6 methyl group derived from the ed
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dehydration step affecting C-4 and C-6 during its biosynthesis; another methyl side http
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chain is attached at C-3 of the D-mycarose moiety, the third sugar of the trisaccharide w
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chain. The remaining three methyl groups are part of the aglycon: C-5’ is derived from rg
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the acetate starter unit, meanwhile methyl groups at C-1’ and C-7 are transferred by es
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methyltransferases coded by the mtmMI and mtmMII genes respectively (11). nu
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In this report, we purify the MtmMII methyltransferase and characterise the , 20
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methylation step occurring at the aromatic C-9 of the aglycon of 9-DMPA3. A
conformational model for the protein based on structural predictions is also presented.
Finally, we analyse the relevance of the methylation for the biological activity of the
drug and propose a model to explain drug-DNA interaction and its structural basis.
EXPERIMENTAL PROCEDURES
Reagents
Phenyl Sepharose High Performance, Sephacryl S-200 HR, Superdex 200 and S-
Adenosyl-L-[methyl-3H]-Methionine (specific activity 2,92 TBq/mmol) were purchased
from Amersham Biosciences. DTT and alkaline phosphatase were from Roche
Diagnostics Gmh. Ethylen glycol, Octanol, Olivomycin, SAM, and SAH were
purchased from Sigma-Aldrich. Acrylamide and Bisacrilamide were from Bio-Rad,
EDTA and ammonium sulfate from Merck and Tris from USB. Calf thymus DNA was
purchased from Fluka & Riedel; phosphate buffered saline (PBS) from Oxoid and
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Daunorubicin from Farmitalia Carlo Erba S.A. Dulbecco's modified Eagle's medium ow
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and fetal calf serum were obtained from Gibco. Tripticasein soy broth (TSB) was from ed
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Oxoid. http
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Purification Procedure rg
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For the purification of the methyltransferase coded by the mtmMII gene, es
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S. argillaceus ∆AH, a mithramycin overproducing strain (Fernández Lozano Mª.J., nu
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unpublished results), was grown in 2-liter Erlenmeyer flasks containing 500 ml of TSB , 20
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liquid medium in the presence of 5 µg ml 1 thiostrepton. The cultures were inoculated
with 25 µl of a dense spore suspension and, after incubation for 48 h at 30 °C on an
orbital shaker incubator (200 rpm), the mycelia was collected by centrifugation at
10,000 ×g during 10 min. The mycelial paste was washed twice with buffer A (50 mM
Tris-HCl buffer, pH 8.0, 1 mM EDTA, and 1 mM DTT) and then was disrupted by two
passes through a French Press at a pressure of 1,500 psi. DNA was broken by
ultrasounds (3 pulses of 15 s each with intermittent cooling on ice water) in a MSE
ultrasonic disintegrator at 150 W and 20 kHz. Unbroken cells and debris were removed
by centrifugation at 14,000 ×g for 30 min. Nucleic acids were precipitated with
streptomycin sulfate (1% final concentration) and the supernatant fractionated by
precipitation with ammonium sulfate. Fractions precipitating between 25% and 50%
saturation were recovered and extensively dialysed against buffer A containing 1.3 M
ammonium sulfate. This fraction was applied to a Phenyl sepharose High Performance
column (15 ml volume) at a flow rate of 3 ml min 1; the column was eluted using a
decreasing linear gradient from 1.3 to 0 M (NH ) SO in 10 column volumes, followed
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by 10 column volumes of buffer A and, finally, 5 volumes of buffer A containing 30%
v/v ethylene glycol. Active fractions were concentrated by ammonium sulfate
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precipitation (80% saturation) and, after centrifugation, the precipitates were ow
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resuspended in 3 ml of buffer B (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT ed
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and 150 mM NaCl). The sample was then applied to a Sephacryl S-200 column http
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(2.6 × 90 cm) at a flow rate of 0.3 ml min 1. Active fractions were extensively dialysed ww
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against buffer C (25 mM Tris-HCl, pH 8.0, 1 mM DTT, and 20% glycerol) and then rg
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applied to a adenosine-agarose column (2 ml), previously prepared as described (15). es
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The column was eluted at a flow rate of 0.5 ml.min 1. Elution was carried out in two nu
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steps: first, buffer C including 0.2M NaCl is applied until no protein eluting from the , 20
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column can be detected; second, the enzyme is eluted using buffer C containing 0.6 M
NaCl.

Enzyme Assays
Methylation reactions consisted of the following components (50 µl final
volume): 1 µl of S-adenosyl-L-[methyl-3H]methionine (0.1 µCi/ml), 1µl of the
correspondent acceptor molecule (100 µM final concentration) and a variable volume of
enzyme and buffer (total 50 µl) depending on the protein concentration. After
incubation at 30 °C for 0.5 h, the reactions were stopped by adding one-tenth of their
volume of 0.2 N HCl and extracted twice with 50 µl of ethyl acetate. After phase
separation, the organic phase was recovered, the solvent evaporated and the residue was
suspended in 100 µl of methanol and, after addition of scintillation mixture, the
radioactivity in the samples was determined.

Polyacrylamide Gel Electrophoresis and Protein Analysis
Analysis of the samples during protein purification was performed using SDS-
PAGE (16). Protein estimation was carried out by measurement of absorbance at
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280 nm and by a protein-dye binding assay (17). ow
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Isolation of MTM and biosynthetic intermediates. http
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MTM, 9-DMPA3, 7-demethyl-mithramycin (7-DMTM), premithramycin A1 w
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(PMA1), 4-demethyl-premithramycinone (4-DMPC) and premithramycinone (PMC) rg
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were obtained as previously described (7,11). es
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Steady State Kinetics , 20
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Initial velocity assays were carried out using the radiolabeled assay as described
above. The concentration of one substrate was varied while the concentration of the
second substrate was held at a nonsaturating concentration. After stopping the reaction,
the amount of methylated molecule was estimated. The experimental data reported here
were all carried out at least in duplicate. Data were analysed by linear and nonlinear
regression using the program Statistica for Windows (Statsoft, Inc.).
Measure of Isotopic Exchange Reactions Catalysed by MtmMII
The exchange reactions between [3H]SAM and [3H]PMA3 were assayed by
measuring the formation of [3H]PMA3 from [3H]SAM and 9-DMPA3. Assays were
performed as described under "Steady State Kinetics," except that the concentrations of
unlabeled reactants and products were adjusted to the equilibrium conditions at the
beginning of the experiment. The concentrations for the unchanged pair were 1.12 and
10 µM, and the concentrations of the changed pair were varied from 1 to 75 times the
concentration of the other pair depending on the assay. After addition of enzyme, the
reaction mixture was incubated at 35 °C for 20 min. A small concentration (119 nM) of
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S-Adenosyl-L-[methyl-3H]-Methionine was added subsequently; samples were taken at ow
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different times, processed as described (18) and used to calculate the rate of isotopic ed
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Biological Assays rg
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The biological activities of MTM and 7-DMTM were tested by performing es
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bioassays against Micrococcus luteus ATCC 10240 as previously described (19). nu
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Structural predictions
Secondary structure predictions and fold recognition were performed using the
PSIPRED (20-23) and 3D-PSSM (24) servers.

Fluorescence microscopy
HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum, 100 IU.ml-1 penicillin, and 100 µg.ml-1 streptomycin in a
humidified atmosphere of 5% CO . For fluorescence experiments, cells were
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subcultured on microscope slides by incubation at 37 °C for 2 min with 0.0125% trypsin
in 0.002% EDTA, followed by the addition of complete medium, washing, and
resuspension in fresh medium. After grown, slides were washed with PBS, and MTM or
7-DMTM where added at 1 mM in the same buffer. After 30 min at 37ºC in 5% CO ,
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cells were washed with PBS and intrinsic fluorescence was observed in a Leica DMR-
XA microscope using a filter L5 Blue BP 480/40 BP 527/530.

Determination of physico-chemical parameters
MTM and 7-DMTM hydrophobicities were determined measuring their partition
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coefficients (KD) between water and octanol. 1 ml aliquots of both molecules (30 µM) ow
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dissolved in Tris-HCl buffer 50 mM pH 2.6 at 30ºC were extracted by addition of 1 ml ed
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octanol followed by vigorous shaking. The final concentrations in both solvents were http
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determined spectrofotometrically at 280 nm. w
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For the determination of the pK of the molecules, the compounds were rg
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dissolved at a final concentration of 30µM in a wide-range three-component buffer es
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(acetic acid, MES, Tris), and then extracted using 1 ml of octanol. The final nu
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concentrations were measured spectrofotometrically. Graphic representation of the , 20
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results gave rise to sigmoid plots from which the apparent pK was estimated.
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Absorbance and fluorescence measurements
Absorption and fluorescence spectra were recorded with a Kontron 930 (Uvikon
Instruments) spectrophotometer and a Perkin Elmer LS50B luminescence spectrometer,
respectively. All fluorescence measurements for MTM and 7-DMTM and their
complexes with Mg2+ were carried out at an excitation wavelength of 470 nm to avoid
time dependent decrease of the fluorescence emission produced by photodegradation of