Biochemical and molecular studies on phenylalanine ammonia lyase in the phytopathogenic fungus Ustilago maydis.

摘要

Phenylalanine ammonia-lyase (PAL) is the entrypoint enzyme into phenylpropanoid metabolism in plants. Little is known about PAL in fungi. In order to explore the role PAL plays in the growth and survival of fungi, the structure and regulation of fungal PAL were investigated in the phytopathogen Ustilago maydis, the causal agent of corn smut.;PAL, purified from liquid-cultured cells of U. maydis, was estimated as 320 kDa and its subunit molecular mass was 80 kDa. The enzyme was most active at pH 8.8-9.2 and 30;A PAL clone was isolated from a U. maydis genomic library and 3047 bp of its nucleotide sequence was determined. It contained 495 bp of ;In U. maydis, PAL is constitutively produced at a low level but its regulation can be influenced by aromatic amino acids. L-tryptophan apparently induced the lyase enzyme. The inducibilty of PAL by L-tryptophan was also demonstrated in six other U. maydis strains and three Ustilago species tested. The enzyme is most readily induced during the early stationary phase of growth and the induced activity remains relatively constant during stationary stage. PAL induction was repressed by glucose but not by its reaction product t-cinnamic acid. Induction did not require de novo protein synthesis, suggesting that some form of post-translational protein modification or a metabolic effect may be the basis of the induction of Ustilago PAL by L-tryptophan. PAL was detected only in cell extracts and not in the growth medium.;A putative biosynthetic pathway of Ustilago melanin was deduced from the examination of the metabolic fate of L-phenylalanine. Overall, the pattern of regulation of PAL induction in U. maydis was very different from patterns known for plants and other fungi. These results, together with evidence for genetic divergence, are consistent with a unique role for PAL in U. maydis. It remains to be determined whether this role is essential for survival and pathogenicity of this plant pathogen. (Abstract shortened by UMI.)