|
(Last
modification: 20. Feb. 2009: Figures, details in text)
MPBD (4-Methyl-5-pentylbenzene-1,3-diol)
biosynthesis in Dictyostelium
discoideum
In the life cycle of the amoeba Dictyostelium, starvation triggers a complex
differentiation process, in which up to 100.000 cells aggregate to form a
multicellular mass; this can migrate towards light and heat.
Differentiation-inducing factors (DIFs) play important roles in this process.
The structures of many molecules were elucidated (Morris
et al., 1987;
Morris et al., 1988; Kay et al., 1988;
Traynor and Kay, 1991;
Kay et al., 1993;
Takaya
et al., 2000; Maeda et al., 2003;
Arai et al., 2005;
Serafimidis and Kay, 2005;
Saito et al., 2006). The structure
of DIF-1 and other differentiation signals possibly synthesized via PKS
reactions are shown in Fig. 1.
Fig. 1.
Morphogenetic factors in Dictyostelium discoideum with a suspected role
of a PKS in the biosynthesis
For DIF-1 it was soon proposed that the key biosynthetic reaction should be carried out by a
polyketide synthase (PKS) (Kay, 1998),
by an enzyme that functionally looked just like a chalcone synthase (CHS), but with
hexanoyl-CoA instead of 4-coumaroyl-CoA as substrate (this is on another page; you really should look at
that as well: Austin et al., 2006,
more...). The dictyopyrones contain a pyrone ring
system, similar to that observed in other natural products or in
byproducts of type III PKS in vitro. It therefore seemed possible that a PKS reaction was
also here a key element in their biosynthesis, but this is still not clear. With MPBD
(4-methyl-5-pentylbenzene-1,3-diol) it seemed
possible from the hydroxylation pattern of the aromatic ring system that an STS-type
reaction was involved, but the presence of the methyl group at the aromatic ring argued
that something additional should be involved. This type III PKS and a model for
the biosynthesis will be discussed here:
This page deals with
the contribution by a Dictyostelium discoideum type III PKS that in
vivo probably carries out a rather unusual reaction: two condensation
reactions with a precursor substrate containing already a ß-keto group, and then
a stilbene synthase (STS) type ring-folding.
Key
publication:
Ghosh et al., 2008
These authors also started out with a bioinformatics
approach based on the availability of the
D. discoideum genome project (Eichinger
et al., 2005). They identified 45 polyketide synthases of the multidomain
type with >2000 amino acids, and they called them DiPKS1 to DiPKS45. These
included of course the previously (Austin et al., 2006,
more...) identified
Steely1 (=
DiPKS1) and Steely2 (= DiPKS37). I am sure this duplicate
nomenclature will lead to some
confusion in the future, and therefore in this page I will use them together. As
reported before, these were the only ones containing a type III PKS domain, and I will not repeat the basic setup of the genes for the two
polyproteins.
The authors then proceeded with functional investigations, after
expressing the type III PKS domains in E. coli. With DiPKS37 (Steely2)
the results confirmed the previous work (Austin et al., 2006): it carried out a
CHS-Type reaction with hexanoyl-CoA. The really interesting new results were
obtained with DiPKS1 (= Steely1). In contrast to the previous work, the
experiments by Ghosh et al. (2008) found not only pyrone products from two
condensations (triketide pyrones), but
Steely1 (=
DiPKS1) synthesized a resorcinol type product resulting from three condensations
and a stilbene synthase (STS)-type ring-folding; see Fig. 2. It is
interesting to note that the product from hexanoyl-CoA is olivetol: this is also
the in vitro product of a PKS activity in crude extracts of hemp (Cannabis
sativa). However, that is not the activity postulated in the biosynthesis of tetrahydrocannabinol:
the product of that enzyme should be the carboxylated form, olivetolic acid:
more...
Fig. 2.
Ghosh et al. (2008):
Biosynthesis of morphogenetic
factors in Dictyostelium discoideum:
the new result is that DiPKS1 (Steely1) can carry out three condensation
reactions, followed by a stilbene synthase (STS) type ring-folding. The acyl
phloroglucinol is the precursor of the differentiation factor DIF-1:
more...
Definitions:
DiPKS37 = Steely2 (biosynthesis
of DIF-1): three condensations, CHS-type ring-folding;
DiPKS1 = Steely1 synthesizes two products in vitro:
5-pentylbenzene-1,3-diol (three condensations, STS-type ring-folding), and a
triketide pyrone (two condensations).
Experiments with other substrates
showed that resorcinolic products were obtained not only with hexanoyl-CoA, but
also with octanoyl-CoA and decanoyl-CoA; longer chain substrates led to pyrones
only. Interesting were also the results with a construct that in addition to the
type III PKS domain also contained the preceding ACP domain, because now the
products were almost exclusively of the resorcinol type. This clearly suggested
that the structure of the protein as a whole is an important factor in
determining the product specificity.
This capacity to synthesize resorcinols was quite intriguing
because it suggested ideas for the biosynthesis of MPBD
(4-Methyl-5-Pentylbenzene-1,3-Diol) which does contain a resorcinol ring-system
(Fig. 1). There are several more interesting findings and discussions
in that publication, but here I will focus only on the model proposed for the
biosynthesis of MPBD, with special interest of course in the mechanism proposed
for the introduction of the methyl group at the aromatic ring-system. Fig. 3
is a summary of the model, and it also compares the reaction of DiPKS1 (Steely1) with that of
DiPKS37 (Steely2) in the biosynthesis of DIF-1.
Some other interesting points for these unusual PKS were
discussed before, and it might be worthwhile to have another look:
more...
Fig. 3.
Ghosh et al. (2008): Models for the
biosynthesis of morphogenetic factors in Dictyostelium discoideum.
DiPKS1 (Steely1): It is proposed that the multidomain PKS part carries
out two rounds of complete extensions involving all reactions leading to a
hexanoyl-moiety. This is followed by a third condensation without the reducing
steps, but with a methylation at the ß-keto stage;
this domain is absent in DiPKS37. That intermediate is then handed over to the
type III PKS domain, which performs two additional
condensations, followed by an STS-type ring-folding to MPBD
(4-methyl-5-pentylbenzene-1,3-diol).
DiPKS37 (Steely2): This is the same as previously proposed by Austin et
al. (2006). In this case, the hexanoyl-moiety is directly transferred to the
type III PKS domain, which then performs three
condensation reactions followed by a CHS-type ring-folding. Further reactions (halogenase,
O-methylation) lead to DIF-1.
Abbreviations: KS, ß-ketoacyl synthase; AT, acyltransferase; DH, dehydratase;
MT, methyltransferase; ER, enoyl reductase; KR, ketoreductase; ACP, acyl
carrier protein; type III PKS = CHS-related protein.
Return
to page top
Direct links to
Return
to page top
.
References
-
Austin, M. B., Saito, T., Bowman, M. E., Haydock,
S., Kato, A., Moore, B. S., Kay, R. R., Noel, J. P., 2006. Biosynthesis of
Dictyostelium discoideum differentiation-inducing factor by a hybrid
type I fatty acid-type III polyketide synthase. Nature Chemical Biology 2,
494-502.
Differentiation-inducing factors (DIFs) are well known to
modulate formation of distinct communal cell types from identical
Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure.
We report complimentary in vivo and in vitro experiments
identifying one of two approximately 3,000-residue D. discoideum
proteins, termed 'steely', as responsible for biosynthesis of the DIF
acylphloroglucinol scaffold. Steely proteins possess six catalytic domains
homologous to metazoan type I fatty acid synthases (FASs) but feature an
iterative type III polyketide synthase (PKS) in place of the expected FAS
C-terminal thioesterase used to off load fatty acid products. This new
domain arrangement likely facilitates covalent transfer of steely
N-terminal acyl products directly to the C-terminal type III PKS active
sites, which catalyze both iterative polyketide extension and cyclization.
The crystal structure of a steely1 C-terminal domain confirms conservation
of the homodimeric type III PKS fold. These findings suggest new
bioengineering strategies for expanding the scope of fatty acid and
polyketide biosynthesis.
Return
More...
-
Ghosh, R., Chhabra, A., Phatale, P. A., Samrat, S.
K., Sharma, J., Gosain, A., Mohanty, D., Saran, S., Gokhale, R. S., 2008.
Dissecting the functional role of polyketide synthases in Dictyostelium
discoideum: biosynthesis of the differentiation regulating factor
4-methyl-5-pentylbenzene-1,3-diol.
Journal of Biological Chemistry 283, 11348-11354.
Dictyostelium discoideum exhibits the largest repository of polyketide
synthase (PKS) proteins of all known genomes. However, the functional
relevance of these proteins in the biology of this organism remains largely
obscure. On the basis of computational, biochemical, and gene expression
studies, we propose that the multifunctional Dictyostelium PKS (DiPKS)
protein DiPKS1 could be involved in the biosynthesis of the differentiation
regulating factor 4-methyl-5-pentylbenzene-1,3-diol (MPBD). Our cell-free
reconstitution studies of a novel acyl carrier protein Type III PKS
didomain from DiPKS1 revealed a crucial role of protein-protein
interactions in determining the final biosynthetic product. Whereas the
Type III PKS domain by itself primarily produces acyl pyrones, the presence
of the interacting acyl carrier protein domain modulates the catalytic
activity to produce the alkyl resorcinol scaffold of MPBD. Furthermore, we
have characterized an O-methyltransferase (OMT12) from Dictyostelium
with the capability to modify this resorcinol ring to synthesize a variant
of MPBD. We propose that such a modification in vivo could in fact
provide subtle variations in biological function and specificity. In
addition, we have performed systematic computational analysis of 45
multidomain PKSs, which revealed several unique features in DiPKS proteins.
Our studies provide a new perspective in understanding mechanisms by which
metabolic diversity could be generated by combining existing functional
scaffolds.
Return to text
-
Eichinger, L., Pachebat, J. A., Glockner, G.,
Rajandream, M. A., Sucgang, R., Berriman, M., Song, J., Olsen, R.,
Szafranski, K., Xu, Q., Tunggal, B., Kummerfeld, S., Madera, M., Konfortov,
B. A., Rivero, F., Bankier, A. T., Lehmann, R., Hamlin, N., Davies, R.,
Gaudet, P., Fey, P., Pilcher, K., Chen, G., Saunders, D., Sodergren, E.,
Davis, P., Kerhornou, A., Nie, X., Hall, N., Anjard, C., Hemphill, L.,
Bason, N., Farbrother, P., Desany, B., Just, E., Morio, T., Rost, R.,
Churcher, C., Cooper, J., Haydock, S., van, D. N., Cronin, A., Goodhead,
I., Muzny, D., Mourier, T., Pain, A., Lu, M., Harper, D., Lindsay, R.,
Hauser, H., James, K., Quiles, M., Madan, B. M., Saito, T., Buchrieser, C.,
Wardroper, A., Felder, M., Thangavelu, M., Johnson, D., Knights, A.,
Loulseged, H., Mungall, K., Oliver, K., Price, C., Quail, M. A.,
Urushihara, H., Hernandez, J., Rabbinowitsch, E., Steffen, D., Sanders, M.,
Ma, J., Kohara, Y., Sharp, S., Simmonds, M., Spiegler, S., Tivey, A.,
Sugano, S., White, B., Walker, D., Woodward, J., Winckler, T., Tanaka, Y.,
Shaulsky, G., Schleicher, M., Weinstock, G., Rosenthal, A., Cox, E. C.,
Chisholm, R. L., Gibbs, R., Loomis, W. F., Platzer, M., Kay, R. R.,
Williams, J., Dear, P. H., Noegel, A. A., Barrell, B., Kuspa, A., 2005. The
genome of the social amoeba Dictyostelium discoideum. Nature 435,
43-57.
Return
-
Arai, A., Goto, Y., Hasegawa, A., Hosaka, K.,
Kikuchi, H., Oshima, Y., Tanaka, S., Kubohara, Y., 2005. Dictyopyrones,
novel alpha-pyronoids isolated from Dictyostelium spp., promote stalk cell
differentiation in Dictyostelium discoideum. Differentiation 73,
377-384.
Return
-
Kay, R. R., 1998. The biosynthesis of
differentiation-inducing factor, a chlorinated signal molecule regulating
Dictyostelium development. Journal of Biological Chemistry 273,
2669-2675.
Return
-
Kay, R. R., Berks, M., Traynor, D., Taylor, G. W.,
Masento, M. S., Morris, H. R., 1988. Signals controlling cell
differentiation and pattern formation in Dictyostelium. Dev. Genet. 9,
579-587.
Return
-
Kay, R. R., Large, S., Traynor, D., Nayler, O., 1993.
A localized differentiation-inducing-factor sink in the front of the
Dictyostelium slug. Proceedings of the National Academy of Sciences of
the United States of America 90, 487-491.
Return
-
Maeda, Y., Kikuchi, H., Sasaki, K., Amagai, A.,
Sekiya, J., Takaya, Y., Oshima, Y., 2003. Multiple activities of a novel
substance, dictyopyrone C isolated from Dictyostelium discoideum, in
cellular growth and differentiation. Protoplasma 221, 185-192.
Return
-
Morris, H. R., Taylor, G. W., Masento, M. S.,
Jermyn, K. A., Kay, R. R., 1987. Chemical structure of the morphogen
differentiation inducing factor from Dictyostelium discoideum.
Nature 328, 811-814.
Return
-
Morris, H. R., Masento, M. S., Taylor, G. W.,
Jermyn, K. A., Kay, R. R., 1988. Structure elucidation of two
differentiation inducing factors (DIF-2 and DIF-3) from the cellular slime
mould Dictyostelium discoideum. Biochemical Journal 249, 903-906.
Return to text
-
Saito, T., Taylor, G. W., Yang, J. C., Neuhaus, D.,
Stetsenko, D., Kato, A., Kay, R. R., 2006. Identification of new
differentiation inducing factors from Dictyostelium discoideum.
Biochimica et Biophysica Acta 1760, 754-761.
Return to text
-
Serafimidis, I., Kay, R. R., 2005. New
prestalk and prespore inducing signals in Dictyostelium. Developmental
Biology 282, 432-441.
Return to text
-
Takaya, Y., Kikuchi, H., Terui, Y., Komiya, J.,
Furukawa, K. I., Seya, K., Motomura, S., Ito, A., Oshima, Y., 2000. Novel
acyl alpha-pyronoids, dictyopyrone A, B, and C, from Dictyostelium cellular
slime molds. Journal of Organic Chemistry 65, 985-989.
Return
-
Traynor, D., Kay, R. R., 1991. The DIF-1
signaling system in Dictyostelium. Metabolism of the signal. Journal
of Biological Chemistry 266, 5291-5297.
Return
Return
to page top
. |
