A structural account of substrate and inhibitor specificity differences between two naphthol reductases

Two short chain dehydrogenase/reductases mediate naphthol reduction reactio ns in fungal melanin biosynthesis. An X-ray structure of 1,3,6,8-tetrahydro xynaphthalene reductase (4HNR) complexed with NADPH and pyroquilon was dete rmined for examining substrate and inhibitor specificities that differ from those of 1,3,8-trihydroxynaphthalene reductase (3HNR). The 1.5 Angstrom re solution structure allows for comparisons with the 1.7 Angstrom resolution structure of 3HNR complexed with the same ligands. The sequences of the two proteins are 46% identical, and they have the same fold. The 30-fold lower affinity of the 4HNR-NADPH complex for pyroquilon (a commercial fungicide that targets 3HNR) in comparison to that of the 3HNR-NADPH complex can be e xplained by unfavorable interactions between the anionic carboxyl group of the C-terminal Ile282 of 4HNR and CH and CH2 groups of the inhibitor that a re countered by favorable inhibitor interactions with 3HNR. 1,3,8-Trihydrox ynaphthalene (3HN) and 1,3,6,8-tetrahydroxynaphthalene (4HN) were modeled o nto the cyclic structure of pyroquilon in the 4-HNR-NADPH-pyroquilon comple x to examine the 300-fold preference of the enzyme for 4HN over 3HN. The mo dels suggest that the C-terminal carboxyl group of Ile282 has a favorable h ydrogen bonding interaction with the C6 hydroxyl group of 4HN and an unfavo rable interaction with the C6 CH group of 3HN. Models of 3HN and 4HN in the 3HNR active site suggest a favorable interaction of the sulfur atom of the C-terminal Met283 with the C6 CH group of 3HN and an unfavorable one with the C6 hydroxyl group of 4HN, accounting for the 4-fold difference in subst rate specificities. Thus, the C-terminal residues of the two naphthol reduc tase are determinants of inhibitor and substrate specificities.