Polyhydroxynaphthalene reductase involved in melanin biosynthesis in Magnaporthe grisea

Abstract

During the biosynthesis of fungal melanin, tetrahydroxynaphthalene reductase catalyzes the NADPH-dependent reduction of 1,3,6,S-tetrahydroxynaphthalene (T,HN) into (+)-scytalone and 1,3,8-trihydroxynaphthalene into (-)-vermelone. The enzyme from Magnaporthe grisea, the fungus responsible for rice blast disease, has been purified to homogeneity. It is a tetramer of four identical 30-kDa subunits. A full-length cDNA clone of about 1 kb encoding T4HN reductase has been isolated from a cDNA library constructed in the LZAP I1 vector and characterized. The clone contains a 846-bp open reading frame. Translation of the DNA sequence gave a 282-residue amino acid sequence with a calculated molecular mass of 29.9 kDa. Sequences corresponding to the aminoterminal part and three internal proteolytic peptides were present in the translated sequence. T4HN reductase exhibits characteristics of the short-chain alcohol dehydrogenase family. The reductase shares 56% identity with a putative ketoreductase involved in aflatoxin biosynthesis in Aspergillus parasiticus.Melanin, a high-molecular-mass black pigment, is synthesized by numerous pathogenic fungi [l, 21 such as Verticillium dahliae, Cochliobolus rniyabeanus and Magnaporthe grisea, the agent of rice blast disease. Mutants of those pathogens lacking the capability to synthesize melanin lose their ability to penetrate the host leaf and, by the way, their pathogenicity [3, 41. The melanin biosynthesis is thus a good target for the design of antifungal agents. The biosynthesis of melanin has been elucidated with mutants accumulating shunt products and exhibiting typical phenotypes (albino, orange) [5]. Fungal melanin is derived from a pentaketide intermediate that is cyclized into 1,3,6,8-tetrahydroxynaphthalene (T,HN) (Fig. 1). The last steps consist of a series of reductions and dehydrations, leading to 1,8-dihydroxynaphthalene via (+)scytalone, 2,3,8-trihydroxynaphthalene (T,HN) and (-)vermelone. Polymerization of 1 ,8-dihydroxynaphthalene yields melanin [6]. In contrast with the intermediates, the enzymes carrying out these transformations are much less characterized. Despite the cloning by complementation of a melanin biosynthetic gene of Colletotrickum lagenariuin [7] presumably involved in the pentaketide cyclization step and the recent isolation of a gene cluster involved in melanin biosynthesis in Alternaria alternata [S], neither the sequences nor the corresponding enzymes are available. Recent studies have focused on the dehydration step. The complete purification of scytalone dehydratase from C. miyabeanus [9], as well as preliminary crystallographic studies on the M . grisea enzyme [lo], has been reported. However, no reductase had been described until now, although inhibitors of this enzyme are already known and largely employed as fungicides, such as tricyclazole [ l l , 121. Experiments carried out with crude extracts from M . grisea have shown that a single enzyme performs the two reduction steps [13] and that this reductase is NADPH-dependent [14]. We prese...

Isolation and Characterization of the Versicolorin B Synthase Gene from Aspergillus parasiticus

“…However, whereas the involvement of ver-1 in aflatoxin biosynthesis has been securely established in vivo, the function of its gene product has defied demonstration in vitro (48,56). VidalCros et al (34) observed a 56% protein sequence identity between VER-1 and scytalone reductase, an enzyme that catalyzes an aryl dehydroxylation in the biosynthesis of melanin. Furthermore, a 52% similarity exists between the ver-1 gene product and the presumed ketoreductase from the Streptomyces actIII gene (48).…”

Autophagy vitalizes the pathogenicity of pathogenic fungi

“…[31][32][33] The importance of melanin in infection structures is emphasized by the observation that knockout of genes in the melanin biosynthesis pathway leads to loss of the function of appressorium-mediated penetration and pathogenicity in M. oryzae. 31,34,35 Turgor pressure in the appressoria of melanin-deficient mutants is about 30-70% of that measured in fully melanized appressoria. 10 The force exerted during initial penetration of a single appressorium is substantial.…”

Phylogeny of Bipolaris inferred from nucleotide sequences of Brn1, a reductase gene involved in melanin biosynthesis.

“…This secondary metabolite is theorized to be important to the survival of these fungal groups (Sussman, 1968). Several genes involved in the melanin biosynthetic pathway have been analyzed, especially 1,3,8-trihydroxynaphthalene reductase genes: THR1 in Colletotrichum lagenarium (Perpetua et al, 1996), ThnR in Magnaporthe grisea (Vidal-Cros et al, 1994), Brn1 in Cochliobolus heterostrophus (ϭteleomorph of Bipolaris maydis) (Shimizu et al, 1997). Amino acid sequences of proteins encoded by these genes showed high homology through the entire sequence.…”