1-3-6-8-tetrahydroxynaphthalene and scytolone

Colletotrichum lagenarium is a plant pathogenic fungus, and produces melanin that is an essential factor for appressorial penetration into host tissues. The melanin biosynthesis pathway of C. lagenarium starts with pentaketide synthesis catalyzed by polyketide synthase Pks1p. We previously confirmed that the direct product of Pks1p is 1,3,6,8-tetrahydroxynaphthalene. Thus, melanin biosynthesis in this fungus requires the reduction of 1,3,6,8-tetrahydroxynaphthalene to scytalone. We made a double mutant 9141-144 from the thr1 mutant 9141 that lacks the ability to metabolize 1,3,8-trihydroxynaphthalene. The double mutant 9141-144 could metabolize neither 1,3,6,8-tetrahydroxynaphthalene nor 1,3,8-trihydroxynaphthalene. However melanin production by the double mutant was restored by THR1, indicating that Thr1p can metabolize both compounds in vivo. These results demonstrate that two enzymes, Thr1p and a deduced 1,3,6,8-tetrahydroxynaphthalene-specific reductase, are involved in the first reduction step of the melanin biosynthesis pathway of C. lagenarium.

Topics: Colletotrichum; Fungal Proteins; Gene Expression Regulation, Fungal; Melanins; Mutagenesis; Mutation; Naphthols; Oxidation-Reduction; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors

Mutants of Magnaporthe grisea harboring a defective gene for 1,3, 8-trihydroxynaphthalene reductase retain the capability to produce scytalone, thus suggesting the existence of a second naphthol reductase that can catalyze the reduction of 1,3,6, 8-tetrahydroxynaphthalene to scytalone within the fungal melanin biosynthetic pathway. The second naphthol reductase gene was cloned from M. grisea by identification of cDNA fragments with weak homology to the cDNA of trihydroxynaphthalene reductase. The amino acid sequence for the second naphthol reductase is 46% identical to that of trihydroxynaphthalene reductase. The second naphthol reductase was produced in Esherichia coli and purified to homogeneity. Substrate competition experiments indicate that the second reductase prefers tetrahydroxynaphthalene over trihydroxynaphthalene by a factor of 310; trihydroxynaphthalene reductase prefers trihydroxynaphthalene over tetrahydroxynaphthalene by a factor of 4.2. On the basis of the 1300-fold difference in substrate specificities between the two reductases, the second reductase is designated tetrahydroxynaphthalene reductase. Tetrahydroxynaphthalene reductase has a 200-fold larger K(i) for the fungicide tricyclazole than that of trihydroxynaphthalene reductase, and this accounts for the latter enzyme being the primary physiological target of the fungicide. M. grisea mutants lacking activities for both trihydroxynaphthalene and tetrahydroxynaphthalene reductases do not produce scytalone, indicating that there are no other metabolic routes to scytalone.

Topics: Amino Acid Sequence; Antifungal Agents; Base Sequence; Binding Sites; DNA, Complementary; Escherichia coli; Fungal Proteins; Genotype; Kinetics; Magnaporthe; Melanins; Models, Chemical; Molecular Sequence Data; Naphthols; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Plasmids; Protein Binding; Sequence Homology, Amino Acid; Substrate Specificity; Thermodynamics; Thiazoles; Transformation, Genetic