Plant polyketide synthases: a chalcone synthase-type enzyme which performs a condensation reaction with methylmalonyl-CoA in the biosynthesis of C-methylated chalcones.

Heterologous screening of a cDNA library from Pinusstrobus seedlings identified clones for two chalcone synthase (CHS) related proteins (PStrCHS1 and PStrCHS2, 87.6% identity). Heterologous expression in Escherichia coli showed that PStrCHS1 performed the typical CHS reaction, that it used starter CoA-esters from the phenylpropanoid pathway, and that it performed three condensation reactions with malonyl-CoA, followed by the ring closure to the chalcone. PstrCHS2 was completely inactive with these starters and also with linear CoA-esters. Activity was detected only with a diketide derivative (N-acetylcysteamine thioester of 3-oxo-5-phenylpent-4-enoic acid) that corresponded to the CHS reaction intermediate postulated after the first condensation reaction. PstrCHS2 performed only one condensation, with 6-styryl-4-hydroxy-2-pyrone derivatives as release products. The enzyme preferred methylmalonyl-CoA against malonyl-CoA, if only methylmalonyl-CoA was available. These properties and a comparison with the CHS from Pinus sylvestris suggested for PstrCHS2 a special function in the biosynthesis of secondary products. In contrast to P. sylvestris, P. strobus contains C-methylated chalcone derivatives, and the methyl group is at the position predicted from a chain extension with methylmalonyl-CoA in the second condensation of the biosynthetic reaction sequence. We propose that PstrCHS2 specifically contributes the condensing reaction with methylmalonyl-CoA to yield a methylated triketide intermediate. We discuss a model that the biosynthesis of C-methylated chalcones represents the simplest example of a modular polyketide synthase.

Urocanic acid analogues and the suppression of the delayed type hypersensitivity response to Herpes simplex virus.

Ultraviolet irradiated urocanic acid (4-imidazoleacrylic acid) containing a mixture of cis- and trans-isomers has been shown previously to induce suppression of the delayed type hypersensitivity (DTH) response to Herpes simplex virus type 1 (HSV-1) in a murine model of infection. The cis-isomer of urocanic acid was prepared and the cis- and trans-isomers of 2-methylurocanic acid. 2-pyrroleacrylic acid, 2-furanacrylic acid, 2-thiopheneacrylic acid, 3-thiopheneacrylic acid as well as dihydrourocanic acid and histamine. Each was applied at concentrations of 1 and 50 micrograms per mouse to the shaved dorsal skin and the mice were infected subcutaneously with HSV 5 h later. After 8-10 days the DTH response to the virus was measured by an ear swelling test. It was found that cis-urocanic acid was effective in suppressing the DTH response at levels of 1 microgram per mouse or less. The cis- and trans-isomers of 2-furanacrylic acid, 2-pyrroleacrylic acid and 2-thiopheneacrylic acid were also effective, with the cis- form generally being more active than trans, and 2-pyrroleacrylic acid being particularly potent. Cis- and trans-3-thiopheneacrylic acid, on the other hand, were only marginally immunosuppressive while neither isomer of 2-methylurocanic acid had any suppressive ability. Dihydrourocanic acid and histamine were also shown to suppress the DTH response. Thus the structural features necessary for urocanic acid and its analogues to act as mediators of UV-induced immunosuppression could be deduced and implications for their mechanism of action discussed.

Quantification of urocanic acid isomers in murine skin during development and after irradiation with UVB light.

Urocanic acid has been postulated as the photoreceptor mediator of immunosuppression induced by ultraviolet-B (UVB) irradiation. We have shown previously that transplanted epidermal cells from neonatal mice, irradiated mice or mice skin painted with cis-urocanic acid suppress the immune responses to herpes simplex virus. Dorsal skin from foetal mice at 3 weeks gestation and from neonatal mice within 1 day of birth were assayed for the presence of cis- and trans-urocanic acid and compared with the amounts in the ears of 2, 4, 6 and 8-week old mice. Foetal mice had a low skin urocanic acid content (11.9 ng/mg wet weight), neonatal mice 227 ng/mg, while the other ages had at least 340 ng/mg. Neonatal mice were found to have 11.4% urocanic acid as the cis-isomer, whereas foetal mice had undetectable amounts and all remaining ages had about 4%. Irradiation of 7-week-old mice with 96 mJ/cm2 UVB light resulted in the presence within the ears of 31.1% urocanic acid as the cis-isomer. This level was maintained for at least 16 h, then declined slowly until, after 7 days, 16.2% was in the cis-form. Nonirradiated ears contained 4.7% cis-isomer. It is known that UVB irradiation of mice suppresses the delayed type hypersensitivity response to HSV. The suppression was found to be dependent on the time interval between irradiation and infection with virus; this had to be longer than 5 h and less than 14 days.(ABSTRACT TRUNCATED AT 250 WORDS)