From the Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, United Kingdom
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ABSTRACT |
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Differentiation-inducing factor (DIF)-1 is a
chlorinated alkyl phenone released by developing
Dictyostelium amoebae, which induces them to differentiate
into stalk cells. A biosynthetic pathway for DIF-1 is proposed from
labeling, inhibitor, and enzymological experiments. Cells incorporate
36Cl into DIF-1 during development, showing
that the chlorine atoms originate from chloride ions; peak
incorporation is at the first finger stage. DIF-1 synthesis can be
blocked by cerulenin, a polyketide synthase inhibitor, suggesting that
it is made from a polyketide. This is most likely the C12
polyketide (2,4,6-trihydroxyphenyl)-1-hexan-1-one (THPH). Feeding
experiments confirm that living cells can convert THPH to DIF-1.
Conversion requires both chlorination and methylation of THPH, and
enzymatic activities able to do this exist in cell lysates. The
chlorinating activity, assayed using 36Cl
, is
stimulated by H2O2 and requires both soluble
and particulate components. It is specific for THPH and does not use
this compound after O-methylation. The
methyltransferase is soluble, uses
S-adenosyl-L-methionine as a co-substrate, has
a Km for dichloro-THPH of about 1.1 µM, and strongly prefers this substrate to close
analogues. Both chlorinating and methyltransferase activities
increase in development in parallel with DIF-1 production, and both are
greatly reduced in a mutant strain that makes little DIF-1. It is
proposed that DIF-1 is made by the initial assembly of a
C12 polyketide skeleton, which is then chlorinated and
methylated.
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INTRODUCTION |
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DIF-11 regulates the central cell fate decision during Dictyostelium development. In suitable conditions, isolated cells are induced by DIF-1 to differentiate into stalk cells, whereas without it they become spores (1-6). Likewise, when Dictyostelium aggregates develop on a substratum containing DIF-1, the proportion of stalk precursor cells (prestalk cells) increases, and the proportion of prespores decreases (7, 8). DIF-1 levels rise strongly in development as prestalk cells first differentiate (9, 10), and our current view is that these rising DIF-1 levels induce the most sensitive cells in the aggregate to differentiate into prestalk cells. These cells rapidly produce DIF-1 dechlorinase, which inactivates DIF-1 and prevents a further rise in levels, allowing the majority of cells to differentiate as prespores (11, 12). To understand this process further requires a better knowledge of DIF-1 signaling and, to this end, I have attempted to discover the biosynthetic pathway for DIF-1.
DIF-1 is an unusual signal molecule, a chlorinated alkyl phenone (13),
for which neither the biosynthetic pathway nor any of the biosynthetic
enzymes are known. The methods used for elucidating the biosynthetic
pathways of many natural products are difficult to apply to DIF-1.
Genetic analysis has identified a number of mutants potentially
defective in DIF-1 biosynthesis (4), but efforts to clone the mutated
genes by complementation (14) have so far
failed2; nor have such
mutants been isolated yet by insertional
mutagenesis,3 which would
facilitate subsequent cloning of the mutated genes (15). DIF-1 is
active at 109 M and so is only present at
concentrations orders of magnitude lower than many secondary
metabolites; it took a massive effort to isolate 50 µg for
identification of the structure (13, 16). Standard methods for
determining the biosynthetic origin of the carbon backbone and oxygen
substituents by stable isotopic labeling are therefore difficult.
However, it has been possible to deduce a likely biosynthetic pathway
for DIF-1 from labeling experiments with
36Cl
, from inhibitor studies, and by
searching biochemically for some of the predicted biosynthetic enzymes.
The initial experiments were guided by clues provided by the structure
of DIF-1 itself and by various biosynthetic precedents.
Since DIF-1 does not resemble any known intermediary metabolite, it is probably made by a dedicated biosynthetic pathway. The aromatic ring of DIF-1 could arise either from the shikimate pathway of aromatic amino acid biosynthesis (17) or from a polyketide. Of these alternatives, a polyketide origin for DIF-1 is the more likely, since it automatically explains the four alternating oxysubstitutions of the final molecule (18) and because this is the way that many simple aromatic metabolites are made, including acetylphloroglucinol (19), a homologue of the proposed polyketide precursor of DIF-1.
Polyketides are typically formed by condensing together acetate units, from malonyl CoA, onto a starter such as acetate in a reaction closely related to fatty acid biosynthesis (19, 20). The initial carbonyl groups of the polyketide are either left intact or variously reduced to hydroxyl, alkenyl (after elimination of water), or fully to alkyl, depending on the programming of the particular polyketide synthase. The polyketide can be ring closed, aromatized, and substituted in various ways, allowing a great diversity of products to be made. Polyketide synthases usually combine the activities required for polyketide synthesis into a large complex, often with a single polypeptide combining several activities. The condensing enzymes of polyketide and fatty acid synthases share conserved sequence motifs around the active site (21), and the active site cysteine is normally the target for the covalent inhibitor, cerulenin (22-24). Inhibition by cerulenin therefore provides a diagnostic test for the proposed polyketide origin of DIF-1 (25, 26).
The most likely polyketide precursor of DIF-1 is a hexaketide, which, after complete reduction of two carbonyls and ring closure, would yield (2,4,6-trihydroxyphenyl)-1-hexan-1-one (THPH; see Fig. 8 for structures). THPH has the complete 12-carbon backbone of DIF-1 and only requires chlorination and O-methylation to form DIF-1. Chlorinations can be carried out by chloroperoxidase enzymes, which utilize hydrogen peroxide to oxidize chloride ions and chlorinate a variety of phenolic compounds (27, 28). Methylations are normally carried out by methyltransferases, using S-adenosylmethionine as the methyl donor, and a number of O-methyltransferases have been identified in the biosynthesis of secondary metabolites (29-31). Since THPH and a variety of other potential substrates have been synthesized (32, 33), it was possible to devise biochemical assays for the predicted chlorinating and methylating enzymes, which were duly detected.
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MATERIALS AND METHODS |
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General-- All reagents, unless otherwise stated, were from Sigma or Aldrich. S-Adenosyl-[methyl-3H]methionine and Na36Cl were from Amersham Corp. DIF-1, DIF-3, THPH, (3-chloro-2,4,6-trihydroxyphenyl)-1-hexan-1-one (monochloro-THPH), and (2,6-dihydroxy-4-methoxyphenyl)-1-hexan-1-one (methoxy-THPH) were synthesized essentially as described previously and purified by Flash chromatography on silica, eluting with 7.5% ethyl acetate in hexane, followed by reverse phase HPLC (32, 33). Protein was assayed using the Bio-Rad dye-binding assay with bovine serum albumin as the standard. RNA was extracted, and Northern blots were performed as previously (34).
Cell Growth and Labeling-- Cells were grown and developed at 22 °C. Strain Ax2 was grown in axenic medium with shaking (35), and strain V12M2 was grown on nutrient plates in association with Klebsiella aerogenes (36) and washed free of bacteria in KK2 (20 mM K1K2PO4, 2 mM MgSO4, pH 6.2) before use.
To label with 36Cl
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Cell Lysates-- Lysates were made by freeze/thawing or filter lysis (38) of Ax2 cells (unless otherwise stated) developed to the appropriate stage on 1.8% L28 agar (Oxoid) containing KK2. For the chlorination assay, lysates were in 50 mM K1K2PO4, pH 7.5, 2 mM MgSO4, 10% glycerol, 1 mM dithiothreitol plus 1 × protease inhibitors (1000 × is 5 mg/ml leupeptin, 2.5 mg/ml pepstatin, 150 mg/ml benzaminide); for the methyltransferase assay, lysates were in 50 mM K1K2PO4, pH 7.5, 2 mM EDTA, 10% glycerol, 1 mM dithiothreitol plus 1 × protease inhibitors. Lysates were fractionated into pellet and supernatant by centrifugation in a Beckman TL-100 centrifuge (30,000 × g for 30 min for chlorination; 300,000 × g for 30 min for methyltransferase assays). DIF-1 dechlorinase was assayed as previously (39).
Chlorination Assay--
100 µl of sample in lysis buffer
containing 0.1 µCi of 36Cl (equivalent to 2 mM Cl
), 0.1 mM THPH, and 50 mM H2O2, unless otherwise stated,
was incubated at 25 °C, and the incubation was terminated at the
appropriate times by the addition of 100 µl of stop solution (90/10/2
ethyl acetate/hexane/acetic acid, containing 5 mg/ml butylated
hydroxytoluene and 1 mg/ml 50% tocopherol as antioxidants). After
centrifugation, the upper phase was taken off, and the lower phase was
reextracted with 150 µl of ethyl acetate. The combined organic phases
were dried down in a Savant Speed-Vac and quantitatively loaded onto TLC plates.
Methyltransferase Assay (Dichloro-THPH Methyltransferase)-- A 50-µl sample in lysis buffer was incubated with 50 µM dichloro-THPH and 1 µM AdoMet (including 0.5 µCi of [3H]AdoMet) at 25 °C, and the reaction was terminated by adding 50 µl of stop solution. After centrifugation in a microcentrifuge, 35 µl of the organic phase was loaded directly onto each lane of the TLC plate. Kinetic data was analyzed using the Enzyme Kinetics software of D.G. Gilbert.
Partial Purification of Dichloro-THPH Methylase-- The 35-60% ammonium sulfate cut of a high speed supernatant from a lysate of 1.2 × 1010 slug stage cells was dialyzed into 20 mM MOPS, 2 mM EDTA, 10% glycerol, 1 mM dithiothreitol, pH 7.5 (MEG buffer) and loaded onto a POROS 20HQ strong anion exchange column (10 × 0.46 cm). Proteins were eluted with a gradient of 0-1 M KCl in MEG buffer over 5 min at 5 ml/min, and methyltransferase activity was located. The two steps gave a purification of 11.5-fold over the high speed supernatant.
TLC and HPLC--
TLC was on activated Whatman LK6D silica
plates developed with 60/40/2 ethyl acetate/hexane/acetic acid for the
enzyme assays (RF values were as follows: THPH,
0.51; monochloro-THPH, 0.50; dichloro-THPH, 0.65; DIF-3, 0.58; DIF-1,
0.72) or 90/21/3/3 chloroform/methanol/acetic acid/water for whole cell
extracts. Tritiated compounds were detected by autoradiography after
spraying the plates with 3H-Enhance (NEN Life Science Products) and
36Cl-labeled compounds directly, either on
film (Kodak XAR5) or on phosphor imaging plates (Eastman Kodak Co.)
exposed in a lead box to reduce background. Tritiated compounds were
quantitated by scintillation counting after scraping the labeled bands
from the TLC plate and 36Cl by reference to standard spots
on the TLC plates, using Molecular Dynamics scanners for film and image
plates as appropriate. The standard curve was linear from 0.5-16 cpm
on film and over a much wider range on phosphor imaging plates.
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RESULTS |
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In Vivo Labeling--
In vivo labeling with potential
precursors was used to define the period of peak DIF-1 accumulation
during development and to search for possible biosynthetic
intermediates. Labeling with radioactive acetate or methionine (as a
methyl donor) yielded strong incorporation into a number of lipids (42)
but not detectably into DIF-1. Alternatively, the DIF-1 released into
the medium by developing cells can be labeled with
36Cl (41). Since none of the accompanying
labeled compounds are potential precursors (40), cell-associated
compounds were examined. Developing cells were labeled with
36Cl
and extracted with organic solvents, and
the extracts were separated by TLC. The very low specific activity of
36Cl
necessitated autoradiographic exposures
of a few months on film or a few days on phosphor imaging plates. Fig.
2 shows that only two labeled compounds
are detected in cell extracts. One is DIF-1, as shown by
co-chromatography on two different TLC systems (not shown) and by the
previous demonstration of DIF-1 in slug extracts by HPLC (43); the
other compound (X) is unidentified. This compound runs in
approximately the same place on TLC as dichloro-THPH, a proposed
precursor of DIF-1 (Fig. 8), but further characterization was not
attempted due to the low amounts present.
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Inhibition of DIF-1 Biosynthesis by Cerulenin--
To test whether
the polyketide synthase inhibitor, cerulenin, inhibits DIF-1
biosynthesis, cells were incubated in submerged culture with cAMP to
stimulate their development and with 36Cl to
label the DIF-1 produced. After 16 h, DIF-1 was extracted from the
medium and resolved by TLC. Fig.
3A shows that cerulenin efficiently inhibits DIF-1 synthesis at concentrations similar to those
inhibiting condensing enzymes in other organisms (25, 26, 44, 45).
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Utilization of THPH by Cerulenin-treated Cells-- The likeliest polyketide precursor for DIF-1 is the C12 polyketide THPH, which is DIF-1 less the chlorines and methoxy group (see Fig. 8). It was synthesized, along with some related compounds, and fed to cells inhibited with cerulenin, to test whether it could be converted to DIF-1. Fig. 4 shows that it can be. DIF-1 synthesis is inhibited by cerulenin, as before, but it is fully restored when either THPH or monochloro-THPH is supplied to the cells. THPH is converted to compounds co-eluting with monochloro-THPH and dichloro-THPH, which are therefore likely intermediates in the biosynthetic pathway, and finally to DIF-1 itself. Monochloro-THPH is converted to dichloro-THPH and DIF-1 (the trace of label in the position of monochloro-THPH is most likely due to a trace contamination of THPH in the original substrate). The compounds running below monochloro-THPH are most likely metabolites of DIF-1, which would be made in these conditions (40).
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Detection of a Specific Methyltransferase in Cell Lysates-- Cell lysates were made from slugs, when DIF-1 synthesis is near maximal, and tested for the presence of a methylating enzyme by incubating them with S-adenosyl-[methyl-3H]methionine as methyl donor and a number of potential DIF-1 precursors. After incubation at 25 °C, nonpolar products were extracted from the reaction mixes with ethyl acetate/hexane and analyzed by TLC. Fig. 5 shows that, although lysates make a background of methylated compounds from endogenous substrates, there is a massive incorporation of label into a new product when dichloro-THPH is supplied. TLC and HPLC co-elution with authentic DIF-1 shows that this product is DIF-1 (not shown). Monochloro-THPH can also be methylated to make DIF-3 (which coincides with a background band in Fig. 5), and 2-methoxy DIF-1 is also utilized, albeit poorly (not visible in Fig. 5 but apparent with purified enzyme preparations).
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Detection of a Chlorinating Activity in Cell Lysates--
A
chlorinating activity was detected in slug cell lysates using
36Cl as the radioactive tracer and testing
for substrate-dependent incorporation in much the same way
as for the methyltransferase. Fig. 7
shows that THPH supports the formation of a chlorinated product, whose
production is greatly stimulated by 50 mM
H2O2. Such high concentrations of
H2O2 may be necessary due to the presence of a
strong catalase activity in these lysates (48).
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DIF-1 Biosynthesis in Mutant Strains-- The "DIFless" mutant strain HM44, which arrests in development as a tight mound, has been widely used to examine the effects of DIF-1 on gene expression because it makes very little DIF-1 but remains fully responsive to it (4). DIF-1 biosynthesis by this mutant was therefore investigated. It only accumulates about 2.5% as much DIF-1 as its parental strain, HM27, in submerged culture (Table II), confirming the original observations. Although DIF-1 production is stimulated by THPH, it is still less than 10% of wild type. Both the chlorinating and methylating enzymes are greatly reduced in lysates from HM44, compared with HM27, at the time of DIF-1 synthesis. Similar results were obtained with two other DIFless strains, HM42 and HM43, which have a less severe phenotype than HM44 (not shown). These results are consistent with the methylating and chlorinating enzymes being involved in DIF-1 biosynthesis, since their activities are greatly reduced in the mutants. However, since both activities are affected, it seems likely that the underlying mutations in these strains are regulatory and not in the structural genes of the biosynthetic enzymes.
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DISCUSSION |
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The experiments described in this paper make a cumulative argument that DIF-1 is synthesized by the pathway shown in Fig. 8. In this pathway, a C12 polyketide skeleton is first assembled by a polyketide synthase and then chlorinated and methylated by specific enzymes. The biosynthesis of a minor stalk cell-inducing activity, DIF-2, which has a C4 alkyl side chain instead of the C5 of DIF-1 (49), can also be accounted for by the same pathway, if it is assumed that a propionyl group is incorporated into the polyketide instead of two acetyls, resulting in one fewer carbon atom.
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The polyketide origin of DIF-1 was first suggested by its structure, with an aromatic ring and alternating oxy-substitutions (50), and is strongly supported by the inhibition of DIF-1 biosynthesis by cerulenin, a general polyketide synthase inhibitor. The conversion of the proposed polyketide (THPH) into DIF-1 by living cells further supports this view. Recent small scale DNA sequencing projects have identified Dictyostelium genes with good homology to a polyketide condensing enzyme and to a reductase, indicating that Dictyostelium probably does possess polyketide synthases (pksA and pksB),4 although it is not known if these particular ones are involved in DIF-1 biosynthesis.
It is assumed in Fig. 8 that acetate is the starter for the polyketide, which is then extended by the addition of another five acetate groups from malonyl CoA. Two carbonyls would be fully reduced to form the alkanone tail of THPH, and there would be ring closure to form the aromatic ring. An alternative possibility, based on the precedent of aflatoxin biosynthesis, is that the starter is a hexanoyl group that eventually forms the alkanone tail of THPH and is itself made by a specialized fatty acid synthase (51, 52). This starter would then be extended by three more acetate units to form the aromatic ring of THPH.
Both the methylating and chlorinating activities appear in cell lysates at the expected time of development, and their levels are greatly reduced in the HM44 mutant, which makes little DIF-1 (4). This and their substrate specificities leave little doubt that these enzymes are dedicated to converting the polyketide THPH to DIF-1. The chlorinating activity utilizes THPH or monochloro-THPH directly, but the biochemistry of this activity is still ill defined. It appears more complex than other chlorinating enzyme described to date, which are simple soluble enzymes, utilizing H2O2 as oxidant (27, 28, 53, 54) and which do not have the essential soluble and membranous/organellar components of the Dictyostelium activity.
The methyltransferase uses AdoMet as methyl donor at about physiological concentrations (47) and has a substrate specificity converse to that of the chlorinating activity; it does not utilize the polyketide directly but requires it to be chlorinated first. It is also specific for the 4-hydroxyl over the 2,6-hydroxyls of the aromatic ring.
In principle, DIF-1 could be made from THPH either by chlorination followed by methylation or by these reactions in the reverse order. The results show that chlorination must precede methylation in the pathway. Cells will accept THPH or monochloro-THPH for chlorination, but not the methylated version of either compound; the chlorinating activity of cell lysates has precisely the same specificity. Conversely, the methyltransferase will not use THPH but requires it to be chlorinated first.
This work lays the foundation for further elucidating the role of DIF-1 in Dictyostelium development. The most important next step is to identify mutants that are defective in DIF-1 biosynthesis and determine the consequences of this defect for development. The proposed biosynthetic pathway requires a minimum of about nine activities, any of which is a potential mutational target: condensing enzyme, acyl carrier protein, two acyltransferases, ketoreductase, dehydratase, and enoylreductase of the polyketide synthase plus the chlorinating and methyltransferase enzymes. It should now be possible to use the biochemical assays and feeding experiments to identify DIF-1 biosynthetic mutants among existing collections. Alternatively, mutants could be created by reverse genetics if potential biosynthetic genes are identified in the DNA sequence data bases or if one of the biosynthetic enzymes can be purified and cloned. The biochemical assays can also be used to learn more of how DIF-1 production is regulated and which cells make it. Finally, cerulenin provides a new tool for inhibiting DIF-1 biosynthesis.
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ACKNOWLEDGEMENTS |
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I am indebted to D. Hopwood and P. Revill for advice on polyketides.
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FOOTNOTES |
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* This work was supported by the Medical Research Council and the Howard Hughes Medical Institute International Scholars Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to the memory of the late Dr. Mary Berks, good friend and colleague.
To whom correspondence may be addressed. Tel.: 44 01223 402298;
Fax: 44 01223 412142.
1 The abbreviations used are: DIF, differentiation-inducing factor; THPH, (2,4,6-trihydroxyphenyl)-1-hexan-1-one; chloro-THPH, (3-chloro-2,4,6-trihydroxyphenyl)-1-hexan-1-one; dichloro-THPH, (3,5-dichloro-2,4,6-trihydroxyphenyl)-1-hexan-1-one; methoxy-THPH, (2,6-dihydroxy-4-methoxyphenyl)-1-hexan-1-one; AdoMet, S-adenosyl-L-methionine; HPLC, high performance liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid.
2 J. Williams, personal communication.
3 R. R. Kay, unpublished observations.
4 W. F. Loomis, personal communication.
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REFERENCES |
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