BACKGROUND: Trihydroxynaphthalene reductase catalyzes two intermediate steps in the fungal melanin biosynthetic pathway. The enzyme, a typical short-chain dehydrogenase, is the biochemical target of three commercial fungicides. The fungicides bind preferentially to the NADPH form of the enzyme. RESULTS: Three X-ray structures of the Magnaporthe grisea enzyme complexed with NADPH and two commercial and one experimental fungicide were determined at 1.7 A (pyroquilon), 2.0 A (2,3-dihydro-4-nitro-1H-inden-1-one, 1), and 2.1 A (phthalide) resolutions. The chemically distinct inhibitors occupy similar space within the enzyme's active site. The three inhibitors share hydrogen bonds with the side chain hydroxyls of Ser-164 and Tyr-178 via a carbonyl oxygen (pyroquilon and 1) or via a carbonyl oxygen and a ring oxygen (phthalide). Active site residues occupy similar positions among the three structures. A buried water molecule that is hydrogen bonded to the NZ nitrogen of Lys-182 in each of the three structures likely serves to stabilize the cationic form of the residue for participation in catalysis. CONCLUSIONS: The pro S hydrogen of NADPH (which is transferred as a hydride to the enzyme's naphthol substrates) is directed toward the carbonyl carbon of the inhibitors that mimic an intermediate along the reaction coordinate. Modeling tetrahydroxynaphthalene and trihydroxynaphthalene in the active site shows steric and electrostatic repulsion between the extra hydroxyl oxygen of the former substrate and the sulfur atom of Met-283 (the C-terminal residue), which accounts, in part, for the 4-fold greater substrate specificity for trihydroxynaphthalene over tetrahydroxynaphthalene.
BACKGROUND:Trihydroxynaphthalene reductase catalyzes two intermediate steps in the fungal melanin biosynthetic pathway. The enzyme, a typical short-chain dehydrogenase, is the biochemical target of three commercial fungicides. The fungicides bind preferentially to the NADPH form of the enzyme. RESULTS: Three X-ray structures of the Magnaporthe grisea enzyme complexed with NADPH and two commercial and one experimental fungicide were determined at 1.7 A (pyroquilon), 2.0 A (2,3-dihydro-4-nitro-1H-inden-1-one, 1), and 2.1 A (phthalide) resolutions. The chemically distinct inhibitors occupy similar space within the enzyme's active site. The three inhibitors share hydrogen bonds with the side chain hydroxyls of Ser-164 and Tyr-178 via a carbonyl oxygen (pyroquilon and 1) or via a carbonyl oxygen and a ring oxygen (phthalide). Active site residues occupy similar positions among the three structures. A buried water molecule that is hydrogen bonded to the NZ nitrogen of Lys-182 in each of the three structures likely serves to stabilize the cationic form of the residue for participation in catalysis. CONCLUSIONS: The pro S hydrogen of NADPH (which is transferred as a hydride to the enzyme's naphthol substrates) is directed toward the carbonyl carbon of the inhibitors that mimic an intermediate along the reaction coordinate. Modeling tetrahydroxynaphthalene and trihydroxynaphthalene in the active site shows steric and electrostatic repulsion between the extra hydroxyl oxygen of the former substrate and the sulfur atom of Met-283 (the C-terminal residue), which accounts, in part, for the 4-fold greater substrate specificity for trihydroxynaphthalene over tetrahydroxynaphthalene.