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-4H-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 race-mizes 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 pKa of 2.5. At pH 7.0 and 25 degrees C, the kcat for 3HNR catalyzed
DDBO oxidation is 14 s-1 and the K(m) is 5 microM; the kcat for SD catalyzed
DDBO dehydration is 400 s-1 and the K(m) is 15 microM. 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.