STRUCTURAL STUDIES OF RAT LIVER 3[[alpha]]-HYDROXYSTEROID/DIHYDRODIOL DEHYDROGENASE. Melanie J. Bennett^*, Brian P. Schlegel^t, Joseph M. Jez^*, Trevor M. Penning⁺, Mitchell Lewis^*, ^*The Johnson Research Foundation, Department of Biochemistry and Biophysics and ⁺Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

Rat liver 3[[alpha]]-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3[[alpha]]-HSD) inactivates circulating steroid hormones and is involved in polycyclic aromatic hydrocarbon (PAM) carcinogenesis. This enzyme is a member of the aldo-keto reductase (AKR) superfamily, and its structure is likely to provide a paradigm for other mammalian HSDs in the family. The previously determined apoenzyme structure¹ showed that the protein folds into an [[alpha]]/[[beta]] barrel. Now, the structure of 3[[alpha]]-HSD complexed with NADPH has been determined at 2.7 Å resolution. This binary complex model reveals the mode of cofactor binding and provides insight into some features of substrate binding and catalysis. The model supports a catalytic mechanism in which Tyr 55 is the general acid, and we present evidence that the structurally distinct short-chain alcohol dehydrogenase (SCAD) family may have convergently evolved a similar catalytic mechanism. We also make predictions about substrate binding to 3[[alpha]]-HSD based on (1) a fortuitous crystal packing contact that may mimic a portion of a bound steroid hormone or PAH trans-dihydrodiol, (2) an active site water molecule that may indicate the position of the carbonyl or hydroxyl oxygen in a substrate, and (3) site-directed mutagenesis data. We are currently working to obtain crystal structures with a variety of bound inhibitors that will allow us to test these predictions and provide direct information about how the enzyme recognizes and reacts with apolar substrates.

1. Hoog, S. S. et al. (1994), Proc. Natl. Acad. Sci., USA 91, 2517-2521.

Supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship, DRG-1298