proteins STRUCTURE O FUNCTION O BIOINFORMATICS STRUCTURE NOTE Structural and biochemical insights into 7b-hydroxysteroid dehydrogenase stereoselectivity Simone Savino, 1 Erica Elisa Ferrandi, 2 Federico Forneris, 1 Stefano Rovida, 1 Sergio Riva, 2 Daniela Monti, 2 * and Andrea Mattevi 1 * 1 Department of Biology and Biotechnology, University of Pavia, via Ferrata 9, Pavia 27100, Italy 2 Istituto di Chimica del Riconoscimento Molecolare, CNR, via Mario Bianco 9, Milano 20131, Italy ABSTRACT Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo- and regio-selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7b-hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active- site accessibility, the bases of the specificity for NADP 1 , and the general architecture of the steroid binding site. Comparison with 7a-hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C-terminal extension reshapes the substrate site of the b-selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859–865. V C 2016 Wiley Periodicals, Inc. Key words: biocatalysis; short-chain dehydrogenase; steroid; stereoselectivity; NADP. INTRODUCTION Hydroxysteroid dehydrogenases (HSDHs) are NAD(P)H-dependent enzymes belonging to the short- chain dehydrogenase/reductase family. They perform oxi- doreduction of the hydroxyl groups present on the core structure (gonane) of steroid molecules [Fig. 1(A,B)]. 1 The gonane consists of 17 carbon atoms arranged in three cyclohexane and one cyclopentane rings, and HSDHs have been shown to display very high regioselec- tivity for the hydroxyl groups at different positions, for example, at C-3, C-7, and C-12 of bile acids. 2 Moreover, for each one of these positions, HSDHs usually show high stereoselectivity for either the hydroxyl group above (b configuration) or below (a configuration) the plane of the steroid molecule [with reference to standard abso- lute configuration; see Fig. 1(A,B)]. In this framework, 7b-hydroxysteroid dehydrogenase (7b-HSDH; EC 1.1.1.201) catalyzes the NAD(P) 1 -dependent reversible oxidation of ursodeoxycholic acid to 7-keto-lithocholic acid, thus showing an exclusive specificity for the hydroxyl group in b configuration at the C-7 position Additional Supporting Information may be found in the online version of this article. Abbreviations: HSDH, hydroxysteroid dehydrogenase; 7b-HSDH, 7b-hydroxys- teroid dehydrogenase; 7a-HSDH, 7a-hydroxysteroid dehydrogenase. *Correspondence to: Andrea Mattevi; Department of Biology and Biotechnology, University of Pavia, via Ferrata 9, 27100 Pavia, Italy. E-mail: andrea.mattevi@ unipv.it or Daniela Monti; Istituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco 9, 20131 Milano, Italy. E-mail: daniela.monti@icrm.cnr.it Received 26 January 2016; Revised 7 March 2016; Accepted 12 March 2016 Published online 23 March 2016 in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/prot.25036 V V C 2016 WILEY PERIODICALS, INC. PROTEINS 859