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...

Using both primary- and tertiary-structure comparisons, we have established new structural similarities shared by reductases, epimerases and dehydrogenases not previously known to be related. Despite the low sequence identity (down to 10%), short consensus segments are identified. We show that the sequence, the active site and the supersecondary structure are well conserved in these proteins. New homologues (the protochlorophyllide reductases) are detected, and we define a new superfamily composed of single-domain dinucleotide-binding enzymes. Rules for the cofactor-binding specificity are deduced from our sequence alignment. The involvement of some amino acids in catalysis is discussed. Comparison with two-domain dehydrogenases allows us to distinguish two general mechanisms of divergent evolution.

The 1,3,6,8-tetrahydroxynaphthaIene (T, H N) reductase of Verticillium dahliae has been studied in a cell-free system. The use of specifically labelled 4(R)and [4(S)-?H] NADPH in the reduction of T4HN to scytalone reveals that the label is specifically transferred in the case of [4(S)-2H] NADPH whereas no deuterium transfer occurs with the 4R-isomer. This establishes that the T4HN reductase of V. dahliae is a NADPH-dependent dehydrogenase and that it belongs to class B.The deoxygenation of phenols is an important step in the biosynthesis of polyketide derived products. During the biosynthesis of melanin, a dark pigment produced by ascomycetes Pyricularia oryzae or Verticillium dahliue, two successive deoxygenations occur, leading from 1,3,6,8-tetrahydroxynaphthalene (T4HN) f to 1,8-dihydroxynaphthalene (DHN),* the last identified precursor of melanin (Scheme I). Biosynthetic studies with mutants have established that the deoxygenation is achieved in two steps: a reduction stage in which T4HN is reduced to scytalone, followed by a dehydration stage leading to TjHN (the same process leads from T,HN to DHN uia vermelone). To date, little is known concerning the reduction step: the enzymes have not been purified or characterized but Wheeler,' working on crude cell-free extracts from V. dahliae, mentioned that NADPH was necessary. Since diphenols can exist as a mixture of phenolic and ketonic forms: two mechanisms could be considered for the reduction: (i) a hydride transfer on the ketonic forms, catalysed by a NADP+dependent dehydrogenase; or (ii) a reduction catalysed by a T4HN scytalone Ho vermelone melanin DHN Scheme 1. Biosynthesis of melanin.

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