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Journal of Bacteriology, January 1999, p. 298-304, Vol. 181, No. 1
Department of
Microbiology1 and
College of
Pharmacy,2 Ohio State University, Columbus, Ohio
43210
Received 27 July 1998/Accepted 16 October 1998
DoxA is a cytochrome P-450 monooxygenase involved in the late
stages of daunorubicin and doxorubicin biosynthesis that has a broad
substrate specificity for anthracycline glycone substrates. Recombinant
DoxA was purified to homogeneity from Streptomyces lividans
transformed with a plasmid containing the Streptomyces sp.
strain C5 doxA gene under the control of the strong
SnpR-activated snpA promoter. The purified enzyme was a
monomeric, soluble protein with an apparent Mr
of 47,000. Purified DoxA catalyzed the 13-hydroxylation of
13-deoxydaunorubicin, the 13-oxidation of 13-dihydrocarminomycin and
13-dihydrodaunorubicin, and the 14-hydroxylation of daunorubicin. The
pH optimum for heme activation was pH 7.5, and the temperature optimum
was 30°C. The kcat/Km
values for the oxidation of anthracycline substrates by purified DoxA,
incubated with appropriate electron-donating components, were as
follows: for 13-deoxydaunorubicin, 22,000 M Streptomyces sp. strain
C5 produces the anthracycline antibiotics Using both in vivo and in vitro reactions, we have shown previously
that DoxA, a cytochrome P-450, catalyzes three oxidation steps in the
biosynthesis of doxorubicin: (i) the oxidation of 13-deoxycarminomycin
and 13-deoxydaunorubicin to 13-hydroxycarminomycin and
13-dihydrodaunorubicin, respectively; (ii) the oxidation of 13-dihydrocarminomycin and 13-dihydrodaunorubicin to carminomycin and
daunorubicin, respectively; and (iii) the 14-hydroxylation of
daunorubicin to doxorubicin (9, 24) (Fig.
1). The 13-oxidation of the 13-dihydro
anthracyclines was shown to be NADPH and oxygen dependent, indicating
that the oxidation step had to proceed through an additional
hydroxylation event (9, 24). For that step, we hypothesized
that a 13-dihydroxy intermediate was formed which resolved to the
13-keto form (i.e., daunorubicin or carminomycin) via spontaneous
dehydration (9, 24).
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Purification, Properties, and Characterization of
Recombinant Streptomyces sp. Strain C5 DoxA, a Cytochrome
P-450 Catalyzing Multiple Steps in Doxorubicin Biosynthesis
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
1 · s1; for 13-dihydrodaunorubicin, 14,000 M1 · s1; for 13-dihydrocarminomycin,
280 M1 · s1; and for daunorubicin,
130 M1 · s1. Our results indicate
that the conversion of daunorubicin to doxorubicin by this enzyme is
not a favored reaction and that the main anthracycline flux through the
late steps of the daunorubicin biosynthetic pathway catalyzed by DoxA
is likely directed through the 4-O-methyl series of anthracyclines.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
-rhodomycinone,
daunorubicin (daunomycin), and baumycins (24). Most
daunorubicin-producing strains, including wild-type Streptomyces
peucetius, accumulate these three compounds as well as other
intermediates of baumycin biosynthesis (24). Arcamone et al.
(2), however, isolated a mutant of S. peucetius,
which they named S. peucetius subsp. caesius,
that produced doxorubicin (14-hydroxydaunorubicin), which added a
functionality. There has since been great interest in elucidating the
mechanism of doxorubicin biosynthesis and its relationship to
daunorubicin biosynthesis (9, 10, 14, 18, 24).
View larger version (23K):
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FIG. 1.
Summary of reactions catalyzed by purified DoxA and
their respective kcat/Km
values expressed in M 1 · s1. DOC,
13-deoxycarminomycin; DHC, 13-dihydrocarminomycin; CAR, carminomycin;
DOD, 13-deoxydaunorubicin; DHD, 13-dihydrodaunorubicin; DAU,
daunorubicin; DOX, doxorubicin; ND, not done (but previously shown to
occur [9]). DauK (anthracycline
4-O-methyltransferase) catalyzes the methylation of
4-hydroxyanthracyclines to the 4-methoxy analogues.
Our previous experiments did not delineate the preferred substrates for DoxA, and because they were run with either whole cells or crude extracts, other enzymes present in those cells or extracts (particularly nonspecific 13-keto dehydrogenases) had profound effects on the results obtained, making interpretations about flux and substrate specificities difficult and tentative (9, 10).
Here we report the purification from Streptomyces lividans TK24(pANT195) of recombinant Streptomyces sp. strain C5 DoxA, and we describe its physical properties and kinetic characteristics. The kinetic data obtained in this work indicate that DoxA appears to have a strong preference for 4-methoxy anthracycline intermediates over their 4-hydroxy analogues as well as a preference for hydroxylation of the C-13 position over the C-14 position.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
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Bacterial strains, media, and growth conditions.
S.
lividans TK24(pANT195), which contains the Streptomyces
sp. strain C5 doxA gene expressed from the strong
SnpR-activated snpA promoter (8) in a
high-copy-number plasmid, has been described previously (9,
10). Primary seed cultures of S. lividans TK24(pANT195) were incubated for 48 h by rotary shaking (250 rpm; 30°C) in 50 ml of yeast extract-malt extract (YEME) medium,
supplemented with 20% (wt/vol) sucrose and 40 µg of
thiostrepton · ml1, in 250-ml shake flasks
containing a coiled spring (9). These cultures were used to
inoculate 450 ml of the same medium (in 2-liter flasks), which were
incubated for 48 h before being used to inoculate a 10-liter
(working volume) stirred tank fermentor (MicroFerm; New Brunswick
Scientific, New Brunswick, N.J.) containing the same medium.
Fermentation conditions were as follows: temperature, 30°C; aeration,
constant at 10 liters/min; agitation, 250 rpm; and no pH control. After
100 to 120 h, the mycelium in the fermentation broth was harvested
by continuous centrifugation at 16,000 × g at a flow
rate of approximately 100 ml · min1 with a
Contifuge 17 RS continuous centrifuge (Heraeus Instruments, Hanau,
Germany). Mycelial pellets were stored frozen at 
70°C and thawed as required.
Substrates and inhibitors. Authentic 13-dihydrocarminomycin and rhodomycin D were obtained from the National Cancer Institute, Bethesda, Md. Authentic daunorubicin and doxorubicin were obtained from Calbiochem (La Jolla, Calif.) and Sigma Chemical Co. (St. Louis, Mo.), respectively. Authentic 13-dihydrocarminomycin and carminomycin were donated by Rhône-Poulenc, and 13-dihydrodaunorubicin was a gift from Adria Labs (Dublin, Ohio; now Pharmacia-Upjohn). 13-Deoxydaunorubicin was biosynthesized from rhodomycin D in vitro with recombinant S. lividans(pANT144), containing recombinant Streptomyces sp. strain C5 dauK (encoding anthracycline 4-O-methyltransferase [9, 11]) and dauP (encoding rhodomycin D methylesterase [9, 11]), as described by Dickens et al. (9). 13-Deoxydaunorubicin was then purified by methods previously described and quantified against standards previously generated in our laboratory (9).
The mammalian cytochrome P-450 inhibitors 4-methylpyrazole, quinidine, troleandomycin, and sulfaphenazole were obtained from Sigma. All other chemicals were reagent grade or better.DoxA monooxygenase assay. DoxA activity was assayed by the method described previously by Dickens et al. (9). Each assay mixture contained, in a final volume of 500 µl, the following components: cell extract from recombinant S. lividans TK24(pANT195) containing Streptomyces sp. strain C5 DoxA (ca. 0.165 to 3 mg of protein); NADP+ (Sigma Chemical Co.), 0.5 µmol; NADPH (Sigma), 0.5 µmol; glucose-6-phosphate (Sigma), 5 µmol; glucose-6-phosphate dehydrogenase (Sigma), 0.42 U; spinach ferredoxin (Sigma), 22 µg; spinach ferredoxin: NADP+ reductase (Sigma), 0.05 U; 10 mM sodium phosphate buffer (pH 7.5); and anthracycline substrate, 0.25 to 5 µl of stock solutions of various concentrations, ranging from 70 µM to 1.9 mM, dissolved in nanopure water. The NADP+, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase constituted an NADPH-regenerating system. The assay mixtures were incubated at 30°C in 16-mm wells of a cell culture plate (Corning Costar, Cambridge, Mass.), with rotary shaking at 250 rpm for periods ranging from 15 s to 1 h. The enzymatic reactions were stopped by the addition of 125 µl of 100 mM Tris-HCl buffer (pH 10.0), and the reaction products were extracted three times each with 500 µl of chloroform-methanol (9:1). The organic extracts were combined, filtered through 0.2-µm-pore-size nylon Acrodisc 13 filters (Gelman Sciences, Ann Arbor, Mich.), and air dried. The reaction products were resuspended in 10 µl of methanol and analyzed qualitatively and quantitatively by thin-layer chromatography and high-pressure liquid chromatography (HPLC) analysis, respectively, as described in detail previously (9).
Spectral analysis, protein concentration determination, and
cytochrome P-450 assay.
Visible and UV absorption spectra of
protein samples were obtained with a UV 160-U spectrophotometer
(Shimadzu Instruments, Kyoto, Japan). All samples were analyzed in 10 mM phosphate buffer (pH 7.5) containing 20% (vol/vol) glycerol, except
that samples for the determination of protein concentration were
analyzed in nanopure water. Protein content was quantified by the
dye-binding assay developed by Bradford (3). The cytochrome
P-450 content of protein samples was assayed spectrophotometrically as
described previously by Dickens and Strohl (10) and
quantified by the method described by Omura and Sato (21).
Reference and experimental samples to be quantified for P-450 content
were reduced with a few grains of sodium dithionite, followed by
bubbling with CO for 1 min prior to analysis. Spectra were obtained
with a Shimadzu UV 160-U spectrophotometer scanning between 400 and 600 nm, and reduced-plus-CO minus reduced difference spectra were obtained by digital subtraction. The difference spectra revealed a strong peak
at 450 nm, which was absent in control extracts of S. lividans TK24(pANT849) (recombinant strain containing vector alone
[8-10]). An extinction coefficient of 91 cm1 M1 was employed for the quantitation of
the cytochrome P-450 heme in protein solutions, using the absorbance
difference between 450 and 490 nm (21).
Protein purification. All enzyme purification procedures were carried out at 4°C or performed on ice when applicable. In preliminary experiments, we determined that DoxA represented the only protein in S. lividans(pANT195) possessing a reduced-plus-CO minus reduced spectral peak at 450 nm (9, 10), so purification of DoxA was routinely followed by spectral analysis rather than by HPLC analysis of activity. DoxA activity (as measured by oxidation of 13-dihydrodaunorubicin to daunorubicin) was confirmed at each purification step in fractions containing dithionite-reduced-plus-CO minus reduced maximum absorbance at 450 nm.
Frozen mycelium (150 to 200 g [wet weight]) of S. lividans TK24(pANT195) was thawed and resuspended in 10 mM sodium phosphate buffer (pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride, washed through a centrifugation step at 16,000 × g for 45 min, and resuspended in 10 mM sodium phosphate buffer (pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride and 10% (vol/vol) glycerol (buffer A). The mycelial suspension (approximately 400 ml) was disrupted by using a French pressure cell (American Instrument Co., Urbana, Ill.) at 16,000 lb/in2. The crude mycelial extracts were clarified of unbroken mycelia and insoluble material by centrifugation at 16,000 × g for 45 min, and the proteins in the clarified supernatant were fractionated by precipitation with ammonium sulfate. The 30 to 60% (wt/vol) saturation fraction, found to contain most of the DoxA, was pelleted by centrifugation at 16,000 × g (45 min), resuspended in a minimum amount of 10 mM sodium phosphate buffer (pH 7.5) containing 20% glycerol (vol/vol) (buffer B), and dialyzed overnight against 4 liters of the same buffer. The dialyzed protein solution was loaded onto a Q-Sepharose Fast Flow (Pharmacia, Piscataway, N.J.) column (33 by 2.5 cm) at a flow rate of 2 ml · min1 by using
an EP-1 Econo pump (Bio-Rad Laboratories, Hercules, Calif.) with buffer
B and eluted with a linear gradient of 1.0 to 0 M NaCl. Fractions
containing the peak P-450 content were collected, pooled, concentrated
by precipitation with 80% (wt/vol) ammonium sulfate, and dialyzed as
described above against buffer B. The dialyzed protein solution was
then loaded onto three 5-ml Econo-Pac Hi-S (Bio-Rad) columns assembled
in series and eluted with buffer B at a flow rate of 1 ml · min1. Fractions containing DoxA were collected, pooled,
and concentrated by centrifugation for 1 h at 3,000 × g through Centricon-10 concentrators (Amicon, Beverly, Mass.). The
concentrated protein solution was dialyzed overnight against 2 liters
of 100 mM sodium phosphate buffer (pH 7.5) containing 20% (vol/vol)
glycerol (buffer C), and the dialyzed protein was loaded onto a phenyl
Sepharose CL-4B (Pharmacia) column (9 by 2.5 cm) at room temperature to
prevent precipitation of the salts. The column was washed with 2 column volumes of 100 mM sodium phosphate buffer (pH 7.5) containing 1.0 M
NaCl and 20% glycerol (vol/vol) (buffer D), followed by a 60-ml
linear, negative gradient of 1.0 to 0 M NaCl (all run at 1 ml · min1). DoxA was then eluted from the column by a step
gradient down from buffer C to 10 mM sodium phosphate buffer (pH 7.5)
containing 20% glycerol (buffer B), concentrated to 2 ml with
Centricon-10 concentrators as described above, and dialyzed overnight
against buffer B. Approximately 400 µl of the dialyzed protein
solution was added to a Superose 6 HR 10/30 fast protein liquid
chromatography (FPLC) column (Pharmacia) and fractionated with buffer B
at a flow rate of 0.1 ml · min1; 200-µl
fractions were collected. The peak 450-nm fraction contained pure DoxA,
as revealed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and activity assays. SDS-PAGE was performed
as described by Laemmli (16), and proteins in the gels were
detected by silver staining as described by Morrissey (19).
Protein characterization.
The approximate molecular weight
of denatured DoxA was determined by SDS-PAGE by comparison to protein
standards of known molecular weight (Gibco BRL, Gaithersburg, Md.),
using a Mini-Protean II gel apparatus (Bio-Rad) running a constant
current of 15 mA per gel. The apparent molecular weight of recombinant,
native DoxA was determined by gel filtration chromatography in buffer B
with a Superose 6 HR 10/30 column connected to a GP-250 Programmer Plus
FPLC system (Pharmacia, Uppsala, Sweden) calibrated with protein
molecular weight standards (100 µl each; 2 mg · ml1) obtained from Boehringer Mannheim (Indianapolis,
Ind.). The ratio of the elution volume of peak recombinant DoxA to the
void volume of the Superose 6 column was used to interpolate the
Mr of recombinant DoxA from the linear plot of
the logarithm of the native Mr as a function of
the ratio of elution volume to void volume.
Enzyme kinetics. For enzyme kinetics determinations, DoxA monooxygenase activity was assayed as described above, except that the reactions were reduced in volume to 25 or 50 µl, depending on the substrate used, and carried out in capped 1.5-ml microcentrifuge tubes to avoid evaporation. All components in the kinetic assay mixture were scaled down directly from the 500-µl-volume assay mixture. The reactions were run for periods of 15 s to 1 h and stopped by the addition of 125 µl of 100 mM Tris-HCl buffer (pH 10.0), the reaction products were extracted three times each with 125 µl of chloroform-methanol (9:1), and the organic extracts were combined and filtered as described above. The organic phase was collected and filtered, dried in a Speed-Vac ISS-100 vacuum evaporator (Savant, Farmingdale, N.Y.), and then resuspended in 10 µl of methanol for quantitative analysis via HPLC. DoxA activity was quantified by peak integration of substrate and product as described previously (9).
The assays were optimized with respect to pH and temperature, and the activity of DoxA for each anthracycline substrate was tested for linearity, both with respect to time and protein concentration, by the HPLC quantitation assay. After optimal pH and temperature values were determined and linear ranges were found, a range-finding experiment was run to determine approximate Km values for each substrate. For final determinations, four or five substrate concentrations were used that spanned the concentration range of 0.5 to 10 times the Km. Kinetic data were analyzed, and the kinetic constants Km and Vmax, as well as all statistical analyses, were obtained and calculated by using nonlinear least-squares regression algorithms from Microsoft Excel 7.0. The kinetic constants kcat and kcat/Km were derived. For each concentration of anthracycline substrate tested, four identical assays were run, with each reaction stopped at the respective time points noted above. The errors of the kinetic rates at each substrate concentration were calculated from these data.Inhibition of DoxA activity. Known mammalian cytochrome P-450 inhibitors 4-methylpyrazole, quinidine, troleandomycin, and sulfaphenazole (all from Sigma) were prepared by procedures described by Shafiee et al. (23). Additionally, the effects on DoxA activity of 1 and 5 mM rhodomycin D (non-DoxA-metabolized precursor) and doxorubicin (product) were tested. Both troleandomycin and sulfaphenazole were first dissolved in acetone before being added to an aqueous solution. The acetone was removed by bubbling nitrogen gas through the solution until the necessary volume of each stock solution was reached. The inhibition assays were run at 50-µl volumes by using the DoxA kinetic monooxygenase assay for 13-dihydrodaunorubicin oxidation to daunorubicin. Inhibition reactions contained 10 mM 13-dihydrodaunorubicin and 1 or 5 mM inhibitor and were terminated as described above after 15 min. All inhibition reactions were performed in duplicate.
For determination of the kinetics of inhibition of DoxA activity by doxorubicin, the kinetic reactions were run as described above with 13-dihydrodaunorubicin as the substrate. Four concentrations of doxorubicin and four concentrations of substrate were tested in duplicate, and the Ki for doxorubicin inhibition of 13-dihydrodaunorubicin oxidation activity by DoxA was calculated.| |
RESULTS AND DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
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Purification of DoxA. The Streptomyces sp. strain C5 doxA gene was overexpressed in S. lividans TK24 under the control of the SnpR-activated snpA promoter in pANT195 (9, 10), resulting in an observable overproduction of the protein in crude extracts of the recombinant strain (Fig. 2).
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-lactoglobulin, 18; carbonic anhydrase, 29; ovalbumin, 43;
bovine serum albumin, 68; phosphorylase B, 97; and myosin (heavy
chain), 200. Lane 2, S. lividans TK24(pANT195) crude
extract. Lane 3, the 30 to 60% ammonium sulfate precipitation
concentrate. Lane 4, pooled Q-Sepharose Fast Flow (anion exchange)
fractions. Lane 5, pooled Hi-S (cation exchange) fractions. Lane 6, pooled phenyl Sepharose CL-4B (hydrophobic interaction) fractions. Lane
7, the fraction containing purified DoxA from Superose 6 (HPLC gel
filtration). Purified DoxA was obtained after Superose 6 gel filtration
(denoted by the arrow). The approximate Mr as
determined from the SDS-PAGE gel is 47,000.
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Stability of purified DoxA. Oxidation of 13-dihydrodaunorubicin by DoxA was found to occur optimally at pH 7.5 and at a temperature of 30°C (data not shown). This optimal pH, slightly higher than normal physiological conditions, may be necessary to ensure that the proper ionic nature of the zwitterionic anthracycline substrates is maintained for optimal recognition. A similar hypothesis was offered by Connors (4) for explaining this same pH requirement for optimal anthracycline-4-O-methyltransferase (DauK) activity. The presence of 20% (vol/vol) glycerol was shown to be absolutely required for DoxA activity and stability. Purified DoxA showed no appreciable loss of activity even after 7 days at 4°C in the presence of 20% (vol/vol) glycerol (data not shown). Moreover, at room temperature, no discernible loss of activity was observed after 1 h in the presence of 20% glycerol, whereas all activity was lost after 30 min at room temperature in the absence of glycerol (data not shown).
Phenylmethylsulfonyl fluoride (serine protease inhibitor) had no effect on DoxA activity. Dithiothreitol, on the other hand, was strongly inhibitory. Dithiothreitol inhibition was reversible after desalting. High ionic strength buffers, such as buffer C, which contains 100 mM sodium phosphate, also strongly inhibited DoxA activity, but this effect was partially reversible after exchange into low ionic strength buffers, such as 20 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) or buffer B (10 mM sodium phosphate [pH 7.5]).Determination of the apparent Mr of recombinant DoxA. DoxA eluted from a calibrated Superose 6 HR 10/30 FPLC gel filtration at a voided volume/elution volume ratio of 1.79, corresponding to a native protein with an Mr of approximately 49,000 (Fig. 3). Denatured DoxA analyzed by SDS-PAGE was determined to have an apparent Mr of approximately 47,000, indicating that the protein exists in a monomeric form. These results compare favorably with the predicted native Mr of 46,096, obtained by gene sequence analysis (10). Almost all other known streptomycete cytochrome P-450s are also monomers of relatively similar size (20), including P-450Soy from Streptomyces griseus (Mr, ca. 42,000) (25) and P-450ChoP from Streptomyces sp. strain SA-COO (Mr, ca. 47,500) (13).
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Kinetics of recombinant Streptomyces sp. strain C5 DoxA. Cytochrome P-450 enzymes require electron transport from NADPH via ferredoxin and ferredoxin:NADP+ oxidoreductase (or a similar system). The genes encoding native Streptomyces strain sp. C5 ferredoxin and ferredoxin:NADP+ reductase have not yet been found (9, 10, 18, 24) and therefore could not be used for the assay of DoxA. Instead, an electron-donating system of spinach ferredoxin and spinach ferredoxin:NADP+ reductase, which has been shown to function adequately for other bacterial cytochrome P-450 activities (1, 23), was used. To ensure that electron shuttling efficiency was not limiting DoxA activity, a relatively high concentration (1 mM) of NADPH was present in all assays and an efficient NADPH regenerating system was used.
Streptomyces sp. strain C5 DoxA oxidizes 13-deoxycarminomycin and 13-deoxydaunorubicin to their respective 13-dihydro forms, with subsequent oxidation of the 13-dihydro moieties to carminomycin and daunorubicin, respectively (9, 24) (Fig. 1). Thus, DoxA exhibits a relatively broad substrate specificity for anthracyclines lacking the 10-carbomethoxyl moiety. This generalized broad substrate specificity exhibited by DoxA is not uncommon for secondary metabolic enzymes (4-6, 9, 24). Anthracyclines possessing functionality at C-10 (e.g., rhodomycin D, 10-carboxy-13-deoxycarminomycin, aklavin, and aclacinomycin A) are not substrates for DoxA, probably due to steric inhibition at the functional end of the molecule (9, 24). Additionally, anthracyclinones (aglycones) are not substrates for DoxA (data not shown), likely indicating a requirement for the sugar moiety (9, 24). The kinetic constants obtained for DoxA on four anthracycline substrates are shown in Table 2. The Km values for all four substrates are relatively similar, ranging from 0.9 µM for daunorubicin to 4.6 µM for 13-dihydrodaunorubicin. These data suggest that DoxA binds its substrates with relatively similar affinities, irrespective of whether the 4-hydroxyl group is methylated or of the oxidation state at C-13 (methylene, hydroxyl, or keto). 14-Hydroxylation of carminomycin by purified DoxA was not observed, however, even after a 24-h incubation with a high concentration of enzyme. This suggests a stringent specificity for the methoxyl moiety at C-4, perhaps required for proper positioning of the anthracycline side chain, for 14-hydroxylase activity, as we previously suggested (9).
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1 · mg1 for
daunorubicin to 3.9 nmol · min1 · mg1 for 13-dihydrodaunorubicin, a 520-fold difference.
The kcat values were highly substrate dependent.
The most rapid turnover was observed when 13-dihydrodaunorubicin was
the substrate (6.3 × 101 s1), whereas
turnover with daunorubicin as substrate was extremely slow (1.2 × 104 s1). Moreover, the
kcat/Km values indicate
that 13-deoxydaunorubicin (22,000 M1 · s1) and 13-dihydrodaunorubicin (14,000 M1 · s1) are by far the preferred
substrates, whereas daunorubicin (130 M1 · s1) and 13-dihydrocarminomycin (280 M1
· s1) are far less preferred. All of these kinetic data
reflect activity relative to the total purified DoxA in the assays;
nevertheless, the data are affected by the percentage of DoxA in the
active (i.e., P-450) form. Based on the ratio of nanomoles of P-450
heme/nanomoles of purified DoxA, we estimate that the DoxA used for the
kinetic data was approximately 20% in the P-450 (i.e., active) form.
In the case of mycinamycin biosynthesis, in which two adjacent carbons
are oxidized by the same P-450-like enzyme, both reactions appear to be
important steps in the biosynthetic pathway in vivo (15).
The data presented herein, on the other hand, strongly suggest that
14-hydroxylation of daunorubicin by DoxA is, under wild-type
conditions, physiologically irrelevant to the producing organisms. The
primary anthracycline biosynthesis products in most
daunorubicin-producing strains are actually baumycins, higher glycoside
derivatives of daunorubicin (24). In fact, cultures of
Streptomyces sp. strain C5 blocked mutants to which
radiolabeled daunorubicin was exogenously added converted over 90% of
the daunorubicin to baumycins, with no measurable doxorubicin formed
(9, 24). Only when doxA was strongly
overexpressed in Streptomyces sp. strain C5 did we observe
even 10% of exogenously added daunorubicin being converted to
doxorubicin (9, 24). From those studies, we concluded that
under normal conditions, 14-hydroxylation of daunorubicin could not
favorably compete with baumycin biosynthesis from daunorubicin (9,
24), which is supported by the current pure enzyme data. A caveat
to this conclusion, however, is the finding by Lomovskaya et al.
(18) that DnrV (originally identified as OrfA in
Streptomyces sp. strain C5 [10]; the
Streptomyces sp. C5 version has recently been renamed DauV
[24]), encoded by the gene directly upstream of DoxA,
has a dramatic effect on DoxA activity. This may, in the producing
organisms, alter significantly the physiological conditions of these
reactions. On the other hand, we did not observe that the presence of
dauV had any stimulatory effect on DoxA activity in S. lividans TK24 (10). This suggests that additional
factors present in daunorubicin-producing strains, but absent from
S. lividans TK24, may be required to exacerbate the effect
of DnrV (or DauV of Streptomyces sp. C5) on DoxA.
Dickens and Strohl (10) reported that daunorubicin can be
reduced back to 13-dihydrodaunorubicin by a putative ketoreductase of
low substrate specificity in S. lividans TK24, the
heterologous host used in that study. Similarly, Lomovskaya et al.
(18) have shown that S. peucetius DnrU (the
corresponding gene product in Streptomyces sp. strain C5,
originally labeled Orf1 [10], has recently been
renamed DauU [24]) possesses 13-ketoreductase activity. Thus, the high Vmax and
kcat values for 13-dihydrodaunorubicin likely
reveal that DoxA in Streptomyces sp. strain C5 is necessary to compensate for the 13-keto-reductase activity in situ.
Where tested, the catalytic efficiency was dramatically affected by the
presence of the methoxy moiety at C-4. The maximal rate of
13-dihydrodaunorubicin (4-methoxy) catalysis is approximately 100-fold
higher than that obtained with its 4-hydroxy derivative, 13-dihydrocarminomycin; turnover of the 4-methoxy substrate was 2,423-fold faster than that obtained with the 4-hydroxy analog. Thus,
while DoxA binds these two molecules with relatively similar affinities
(see above), 13-oxidation of the molecule possessing the 4-methoxy
substitution is greatly favored. Based on the kinetic trends obtained
in this study, it seems reasonable to suggest that 13-deoxycarminomycin
catalysis would proceed at a rate similar to that of
13-dihydrocarminomycin and much slower than that of its 4-methoxy
analogue, 13-deoxydaunorubicin. Unfortunately, we cannot confirm this
due to the inability to synthesize and stabilize large-enough
quantities of 13-deoxycarminomycin for this study (data not shown).
The late steps in daunorubicin biosynthesis, including the (known)
calculated kcat/Km values
for DoxA, are shown in Fig. 1. Since
kcat/Km defines substrate
specificity, the data indicate that oxidation of the 4-methoxy
anthracyclines may occur preferentially within the producing organism.
This would suggest that flux through the daunorubicin (i.e., 4-methoxy)
series is preferred over flux through the carminomycin (i.e.,
4-hydroxy) series. We recently showed that DauK, anthracycline
4-O-methyltransferase (known as DnrK in S. peucetius [24]), can methylate rhodomycin D,
10-carboxy-13-deoxycarminomycin, 13-deoxycarminomycin,
13-dihydrocarminomycin, and carminomycin (4-6, 9).
Although comparative kinetics for these methylation reactions are not
known, our current data suggest that the most efficient biosynthesis of
daunorubicin would require methylation prior to the formation of
carminomycin. We previously had hypothesized that
4-O-methylation of carminomycin might be the penultimate step in daunorubicin biosynthesis (4-6), but the current
data suggest that this is probably not the case.
Based on these observations, it appears that the primary function of
DoxA is to catalyze the enzymatic conversion of 13-deoxydaunorubicin to
daunorubicin via 13-dihydrodaunorubicin.
Inhibition of DoxA. Known inhibitors of mammalian cytochrome P-450 monooxygenases (1, 7), quinidine, sulfaphenazole, and troleandomycin, inhibited DoxA oxidation of 13-dihydrodaunorubicin by 11, 20, and 35%, respectively, at 1 mM; at 5 mM, all inhibited DoxA activity at >95%. 4-Methylpyrazole did not inhibit DoxA activity at either 1 or 5 mM. Mechanistically, quinidine and sulfaphenazole exhibit reversible heme complexation, with sulfaphenazole acting competitively. Troleandomycin, on the other hand, forms metabolic intermediate heme complexes and thus is considered quasi-irreversible (7). Although these inhibitors function by heme complexation, it is unclear why 4-methylpyrazole had no inhibitory effect on DoxA, especially since the molecule is much smaller than either sulfaphenazole or troleandomycin and thus has less steric hindrance to the heme group. According to Ortiz de Montellano (22), inhibitors that bind not only to the heme group but also to lipophilic regions within the protein are inherently more effective. It is likely that both troleandomycin and sulfaphenazole bind to hydrophobic portions of DoxA, assisting in their inhibition capability, which is noticeably lacking in 4-methylpyrazole.
Rhodomycin D, a precursor anthracycline possessing a 10-carbomethoxyl moiety (9, 24), did not inhibit DoxA activity at either 1 or 5 mM. Although we have no supporting substrate-binding data, it is likely that the large 10-carbomethoxy group present at the reactive end of the anthracycline molecule sterically prevents it from accessing the heme group of the P-450, making it neither a substrate nor an inhibitor. Doxorubicin completely inhibited DoxA activity at both 1 and 5 mM in preliminary trials. Subsequently, doxorubicin inhibition of DoxA activity was determined to be competitive with respect to 13-dihydrodaunorubicin oxidation, with a Ki of 21 µM. This inhibition constant (equivalent to 11.4 mg of doxorubicin/liter) may be physiologically significant, since high concentrations of doxorubicin may accumulate in the proximity of the enzyme. S. peucetius has been shown to possess an active export mechanism for doxorubicin (12), one of at least three separate mechanisms involved in self-resistance (12, 14, 17, 24). Similarly, Streptomyces sp. strain C5 possesses the same resistance genes, although their functionality has not been proven (24). According to our data, active export of doxorubicin would be important not only as a resistance mechanism but also from a metabolic flux standpoint. If doxorubicin were allowed to accumulate, it might interfere with the biosynthesis of daunorubicin, its immediate precursor, from 10-deoxydaunorubicin. We speculate, therefore, that it is important that doxorubicin be exported from the cell in order for the organism to continue to generate more daunorubicin and doxorubicin.| |
ACKNOWLEDGMENTS |
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We thank C. R. Hutchinson for allowing us to preview his manuscript prior to publication. We also thank Jill Johnson, Synthetic Chemistry Laboratory, National Cancer Institute, for authentic samples of carminomycin and rhodomycin D and Rhône-Poulenc and Adria (now Pharmacia-Upjohn) for other authentic anthracycline substrates and standards.
This work was supported by the National Science Foundation under grant no. MCB-94-05730. M.L.D. was supported in part by a presidential fellowship from Ohio State University.
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FOOTNOTES |
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* Corresponding author. Present address: Merck Research Laboratories, P.O. Box 2000, R80Y-215, Rahway, NJ 07065. Phone: (732) 594-8433. Fax: (732) 594-1399. E-mail: william_strohl{at}merck.com.
Present address: Merck Research Laboratories, P.O. Box 2000, RY810, Rahway, NJ 07065.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
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Journal of Bacteriology, January 1999, p. 298-304, Vol. 181, No. 1
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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