Fungal metabolic model for tyrosinemia type 3: molecular characterization of a gene encoding a 4-hydroxy-phenyl pyruvate dioxygenase from Aspergillus nidulans

Abstract

Mutations in the human HPD gene (encoding 4-hydroxyphenylpyruvic acid dioxygenase) cause hereditary tyrosinemia type 3 (HT3). We deleted the Aspergillus nidulans homologue (hpdA). We showed that the mutant strain is not able to grow in the presence of phenylalanine and that it accumulates increased concentrations of tyrosine and 4-hydroxyphenylpyruvic acid, mimicking the human HT3 phenotype.

FIG. 1.

Growth phenotypes of the wild-type and ΔhpdA mutant (HPPD15 and HPPD21) strains. Strains GR5 (wild type), HPPD15, and HPPD21 were grown for 72 h at 37°C in YUU, (YAG supplemented with 1.2 g/liter each of uracil and uridine), MM, MC plus lactose (L), MC plus lactose plus phenylalanine (L+P), or MC plus phenylalanine (P). All media except YUU were supplemented with pyridoxin.

FIG. 2.

HPLC determination of the Tyr and 4-HPP intracellular and supernatant concentrations. (A) HPLC chromatograms, monitored at 274 nm, of culture supernatants from wild-type and ΔhpdA strains submitted to solid-phase extraction onto Octadecyl (C₁₈) SPE columns. Arrows 1 and 2 represent Tyr and 4-HPP, respectively. The separation was achieved by applying the following gradient with 0.02 M acetic acid (solvent A) and different amounts of HPLC-grade methanol (solvent B) as follows: 3% to 35% solvent B from 0 to 5 min, 35% solvent B from 5 to 12 min, 35% solvent B to 3% solvent B from 12 to 13 min, and 3% solvent B from 13 to 15 min. Standard solutions were also submitted to solid-phase extraction onto Octadecyl (C₁₈) SPE columns (Mallinckrodt Baker) and injected into the C₈ column. The intracellular and supernatant concentrations of Tyr (B) and 4-HPP (C) were measured by HPLC determination (A). The concentration of Tyr and 4-hydroxyphenylpyruvate was determined by comparing areas of standards and samples. mAU, milliabsorbance unit.

FIG. 3.

Growth phenotypes of the wild-type strain. Strain GR5 (wild-type strain) was grown for 48 h at 37°C in MC plus lactose with increasing concentrations of either 4-hydroxyphenylpyruvate or Tyr. The control was grown only in MC plus lactose. All the media were supplemented with pyridoxin.