Melanin biosynthesis: a study of polyphenol deoxygenation

Abstract

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.

The Second Naphthol Reductase of Fungal Melanin Biosynthesis inMagnaporthe grisea

“…Scytalone was purified from cultures of Rsy Ϫ mutants of M. grisea (25). The enzymatic synthesis of vermelone from scytalone was accomplished essentially as described (26). UV-visible spectrophotometric analyses were performed on HP 8452A or HP 8453 diode array spectrophotometers (Hewlett Packard).…”

“…7.9 Ϯ 0.7 ϫ 10 Viviani et al (26,27) and Vidal-Cros et al (28) report cloning and characterization of a "tetrahydroxynaphthalene reductase" that varies from the 3HNR we have studied by three amino acids that lie outside the active site. We believe this difference owes to the two M. grisea strains used as the enzyme sources.…”

2,3-Dihydro-2,5-dihydroxy-4H-benzopyran-4-one: A Nonphysiological Substrate for Fungal Melanin Biosynthetic Enzymes

“…Trihydroxynaphthalene, an in vivo substrate of 3HNR and in vivo product of SD, is unstable in aerated solutions where it oxidizes on a time scale of minutes. Catalysis by 3HNR has been studied using the natural substrates tetrahydroxynaphthalene and scytalone under anaerobic conditions (8). If one measures initial velocities over time ranges of seconds rather than minutes, it is not necessary to exclude oxygen from either dehydrations or oxidations of scytalone because the oxidation of products does not contribute 1 significantly to absorbance changes.…”

Polyhydroxynaphthalene reductase involved in melanin biosynthesis in Magnaporthe grisea

“…2 1 5 GGC CCC T?\3 GGT CCT CAG TCA GCC TCT CTT GAG GGC AAG GTT GCC 259 dehydrogenase [42] which transfers the pro-S 4-H of NADPH. 3a,20P-Hydroxysteroid dehydrogenase transfers the p r o 3 4-H of NADH [43] which has been correlated with the syn conformation adopted by the nicotinamide ring according to the X-ray structure.…”