2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one:: A nonphysiological substrate for fungal melanin biosynthetic enzymes

We have synthesized an alternate substrate for trihydroxynaphthalene reductase (3HNR) and scytalone dehydratase (SD), two enzymes in the fungal melanin biosynthetic pathway. The oxidation of 2,3-dihydro-2,5-dihydroxy-4R-benzopyran-4-one (DDBO) to 4,5-dihydroxy-2H-benzopyran-2-one (DBO) with concomitant reduction of NADP(+) is catalyzed by 3HNR. DDBO is dehydrated by SD to 5-hydroxy-4H-1-benzopyran-4-one (HBO). These reactions can be monitored using continuous spectrophotometric assays. DDBO racemizes rapidly, so chiral synthesis to mimic the natural substrate is not required. DDBO, DBO, and HBO are stable in aerated aqueous solution, in contrast to the rapidly autooxidizing trihydroxynaphthalene, a physiological substrate for 3HNR and product of SD. Unlike the natural substrates, DDBO, DBO, and HBO do not change protonation state between pH's 4 and 9. Oxidation of DDBO is effectively irreversible at pH 7, as DBO deprotonates with a pK(a) of 2.5. At pH 7.0 and 25 degrees C, the k(cat) for 3HNR catalyzed DDBO oxidation is 14 s(-1) and the K-m is 5 mu M; the k(cat) for SD catalyzed DDBO dehydration is 400 s(-1) and the K-m is 15 mu M. Based on these kinetic constants, DDBO is a better substrate than the natural substrate scytalone for both 3HNR and SD at neutral pH. An explanation for the preference of DDBO over scytalone in the oxidation and dehydration reactions is offered. (C) 1998 Academic Press.