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Last modification: 24. November 2009)

 

Proposal: Biosynthesis of Bis-5-Alkylresorcinols

(Key reference: Miyanaga and Horinouchi (2009)

 

   These are interesting compounds; they contain two resorcinol ring systems (Fig. 1). They have not been described very often, but a few reports showed their presence in various plants (Lytollis et al., 1995; Deng et al., 1999; Suzuki et al., 1999; Chaturvedula et al., 2002).

   The biosynthesis could be quite intriguing. One could think that there should be two STS-type PKS reactions: the first with a long-chain fatty acid CoA-ester, and the second with the product (a long-chain alkylresorcinol), after oxidation of the terminal methyl group to the carboxylate and ligation with CoASH (Fig. 1). Although other possibilities are not excluded, this seems to be most likely pathway since long-chain alkylresorcinols are well-known and wide-spread, and it seems reasonabl to think that they might be precursors (see the excellent review by Kozubek and Tyman, 1999). To the best of my knowledge, the enzyme activity has not been demonstrated with extracts from plants containing such compounds.

 

Fig. 1.
Model for the biosynthesis of a bis-5-alkylresorcinol. 

 

    How would one go about testing that?
    There several examples for type III PKS using long-chain fatty acid CoA-ester as substrates to synthesize alkylresorcinols, e.g. the alkylresorcinol synthase ArsB from the bacterium Azotobacter vinelandii (more...), the alkylresorcinol synthase (ARS) in the biosynthesis of sorgoleone in the plant Sorghum bicolor (more...), the fungal enzyme from Neurospora crassa which is discussed either as 2'-oxoalkylresorcylic acid synthase (ORAS) or  alkylresorcinol synthase (ARS) (more...), and finally an enzyme from the plant Physcomitrella patens (more...). However, protein candidates for the production of bis-5-alkylresorcinols have not been described, and the reaction has not even been demonstrated in vitro. In addition, there were no long-chain substrates containing already a resorcinol ring system for the second reaction, and that is obviously  the part of the greatest interest.

    The authors therefore had to make two compromises. The first was to use type III enzymes already known to synthesize alkylresorcinols: they chose the ArsB from Azotobacter vinelandii, the ORAS from Neurospora crassa, and a third protein, an unpublished enzyme from rice (Oryza sativa)  that is cited as alkylresorcylic acid synthase (ARAS2). The reactions of ArsB and ORAS are summarized in Fig. 2. 

 

Fig. 2.

Simplified scheme of the main reactions carried out by ArsB, the alkylresorcinol synthase from Azotobacter vinelandii (more...), and ORAS, the 2'-oxoalkylresorcylic acid synthase from Neurospora crassa (more...). Note that ArsB synthesizes alkylresorcinols, while the products of ORAS are resorcylic acids (from three or four condensations) which are non-enzymatically converted into the resorcinols. The colours mark the condensation reactions.

 

   The second compromise was with the substrate. The authors synthesized an artificial compound that would be suitable to mimic a possible substrate: a dicarboxylic acid activated at both carboxyl groups by coupling with N-acetylcysteamine (NAC) (Fig. 3), a CoA-replacement used in many previous in vitro experiments. Note that this substrate is not the one predicted to be likely in vivo (see Fig. 2), but it could be useful to investigate the potential of type III PKS for such reactions. 

    The principal results are summarized in Fig. 3. The most clear-cut data were obtained with ArsB and the substrate hexandecanedioyl-diNAC, i.e. with 16 carbons, essentially the starting backbone predicted for the synthesis of the bis-5-alkylresorcinol in Hakea trifurcata (Fig.1 ). The major product was indeed the bis-5-alkylresorcinol, and the time course showed that the tetraketide resorcinol (one STS-type reaction) was the first product which was then converted into the bis-5-alkylresorcinol in a second reaction. The results with other NAC dithioesters (internal alkyl chain length 10, 12, or  16 carbons) were similar, but a shorter internal chain length only led to the tetraketide resorcinol. The results with ORAS and ARAS2 were more complex. They could synthesize the bis-5-alkylresorcinols, but also a fairly high percentage of other products (see Fig. 3: pyrones/resorcinols from a sequential reaction with two and three malonyl-CoA, and a mixed product from a sequential reaction with four and three malonyl-CoAs). As ORAS and ARAS2 are resorcylic acid synthases, it is a bit unexpected that no resorcylic acids were detected at all as products, but the authors argued that this might be a consequence of low efficiency in their formation and fast non-enzymatic decarboxylation.

   What do we learn from these experiments? Well, they show that STS-type enzymes with a preference for long-chain substrates can also accept long-chain substrates that do contain already a resorcinol ring-system. As noted above, it seems hardly likely that a dicarboxylic acid activated with two CoASH (corresponding to the di-NAC compounds used here) are the substrates in vivo, but the data do suggest type III PKS as good candidates for the biosynthesis. It will be interesting to see whether both reactions are carried out by one enzyme (as it was the case in the experiments reported here), or whether there are two different, but related proteins specialized for either the first or the second STS-type reaction.

 

Fig. 3.
NAC-dithioester as substrate, and main reactions with ArsB, ORAS, and ARAS2.

The coloured dots mark the carbons introduced by the condensation reactions with malonyl-CoA.

 

 

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References

 

  • Miyanaga, A., Horinouchi, S., 2009. Enzymatic synthesis of bis-5-alkylresorcinols by resorcinol-producing type III polyketide synthases. J. Antibiot. (Tokyo) 62, 371-376.
    No enzyme systems responsible for the biosynthesis of structurally and biosynthetically intriguing bis-5-alkylresorcinols produced by plants have been identified. Herein, we show that bacterial, fungal and plant alkylresorcinol-producing type III polyketide synthases (PKSs), such as ArsB in the Gram-negative bacterium Azotobacter vinelandii, ORAS in the fungus Neurospora crassa and ARAS2 in the rice plant Oryza sativa, can synthesize bis-5-alkylresorcinol from alkanedioic acid N-acetylcysteamine dithioester as a starter substrate and from malonyl-CoA as an extender substrate by two-step conversion. Plants presumably use a type III PKS for the biosynthesis of bis-5-alkylresorcinols.
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  • Kozubek, A., Tyman, J. H. P., 1999. Resorcinolic lipids, the natural non-isoprenoid phenolic amphiphiles and their biological activity. Chemical Reviews 99, 1-25.
    No Abstract.
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  • Lytollis, W., Scannell, R. T., An, H., Murty, V. S., Reddy, K. S., Barr, J. R., Hecht, S. M., 1995. 5-Alkylresorcinols from Hakea trifurcata, that cleave DNA. Journal of the American Chemical Society 117, 12683-12690.
    No Abstract.
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  • Deng, J. Z., Starck, S. R., Hecht, S. M., 1999. Bis-5-alkylresorcinols from Panopsis rubescens that inhibit DNA polymerase ß. Journal of Natural Products 62, 477-480.
    Bioassay-guided fractionation of Panopsis rubescens, using an assay to detect DNA polymerase ß inhibition, led to the isolation of two new bis-5-alkylresorcinols (1 and 2), in addition to one known bis-5-alkylresorcinol (3). The structures of 1-3 were established as 1,3-dihydroxy-5-[14'-(3,5- dihydroxyphenyl)-cis-4'-tetradecenyl]benzene (1), 1,3-dihydroxy-5-[14'- (3,5-dihydroxyphenyl)-cis-7'-tetradecenyl]benzene (2), and 1,3-dihydroxy- 5-[14'-(3,5-dihydroxyphenyl)tetradecenyl]benzene (3), respectively, by spectroscopic and chemical analyses. Compounds 1-3 exhibited potent inhibition of calf thymus DNA polymerase ß, with IC50 values of 7.5, 6.5, and 5.8 µM, respectively.
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  • Suzuki, Y., Esumi, Y., Yamaguchi, I., 1999. Structures of 5-alkylresorcinol-related analogues in rye. Phytochemistry 52, 281-289.
    A series of 5-(2'-oxo)alkylresorcinols, 5-(2'- and 4'- hydroxy)alkylresorcinols, and 5,5'-(alkadiyl)diresorcinols were isolated from rye, each as a mixture of homologues. The identification of these alkylresorcinol-related analogues was achieved by analyses of the 1H-NMR and MS spectra. From the identity of the analogues found, a possible biosynthetic pathway to the 2'-oxoalkylresorcinols is proposed, based on a correlation of the carbon-chain lengths between the alkylresorcinols and 2'- oxoalkylresorcinols.
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  • Chaturvedula, V. S. P., Schilling, J. K., Miller, J. S., Andriantsiferana, R., Rasamison, V. E., Kingston, D. G. I., 2002. New cytotoxic bis 5-alkylresorcinol derivatives from the leaves of Oncostemon bojerianum from the Madagascar rainforest. Journal of Natural Products 65, 1627-1632.
    Bioassay-directed fractionation of a CH2Cl2-MeOH extract of the leaves of Oncostemon bojerianum resulted in the isolation of eight new 5-alkylresorcinols, named oncostemonols A-H (1-8), and two known derivatives, (8'Z)-1,3-dihydroxy-5-[16'-(3' ',5' '-dihydroxyphenyl)-8'-hexadecen-1'-yl]benzene (9) and (8'Z)-1,3-dihydroxy-5-[14'-(3' ',5' '-dihydroxyphenyl)-8'-tetradecen-1'-yl]benzene (10). The structures of the new compounds 1-8 were elucidated on the basis of extensive 1D and 2D NMR spectroscopic interpretation and chemical derivatization. All the compounds exhibited cytotoxic activity against the A2780 ovarian cancer cell line.
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