Crystal structure of pentaerythritol tetranitrate reductase: "Flipped" binding geometries for steroid substrates in different redox states of the enzyme

Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explo sive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including ste roids; some steroids act as substrates whilst others are inhibitors. Unders tanding the basis of reactivity with cyclic enones requires structural info rmation for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase a t 1.5 Angstrom resolution establishes a close structural similarity to the beta/alpha -barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-di one and prednisone (both substrates) the steroids are stacked over the si-f ace of the flavin in an orientation different from that reported for old ye llow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-andros tadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinami de coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indica tes la addition at the steroid C2 atom. These studies rule out lateral moti on of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme. (C) 2001 Academic Press.