Active Site Directed Mutagenesis of 3/17-Hydroxysteroid Dehydrogenase Establishes Differential Effects on Short-Chain Dehydrogenase/Reductase Reactions ² Udo C. T. Oppermann, ‡ Charlotta Filling, ‡ Kurt D. Berndt, ‡ Bengt Persson, ‡ Jordi Benach, § Rudolf Ladenstein, § and Hans Jo ¨rnvall* ,‡ Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden, and Center of Structural Biochemistry, Karolinska Institutet, NOVUM, S-141 57 Huddinge, Sweden ReceiVed July 22, 1996; ReVised Manuscript ReceiVed October 28, 1996 X ABSTRACT: Mutagenetic replacements of conserved residues within the active site of the short-chain dehydrogenase/reductase (SDR) superfamily were studied using prokaryotic 3/17-hydroxysteroid dehydrogenase (3/17-HSD) from Comamonas testosteroni as a model system. The results provide novel data to establish Ser138 as a member of a catalytically important “triad” of residues also involving Tyr151 and Lys155. A Ser f Ala exchange at position 138 results in an almost complete (>99.9%) loss of enzymatic activity, which is not observed with a Ser f Thr replacement. This indicates that an essential factor for catalysis is the ability of side chain 138 to form hydrogen bond interactions. Mutations in the NAD(H) binding region, in strands A, D, and adjacent turns, reveal two additional residues, Thr12 and Asn87, which are important for correct binding of the coenzyme and with a differential effect on the reactions catalyzed. Thus, mutation of Thr12 to Ala results in a complete loss of the 3-dehydrogenase activity, whereas the 3-oxoreductase activity remains unchanged. On the other hand, a T12S substitution yields a protein with unaltered catalytic constants for both reactions, revealing that a specific hydrogen bond is critical for the dehydrogenase activity. Our interpretation of the available crystal structure of 3R/20-HSD from Streptomyces hydrogenans suggests a hydrogen bond in that enzyme between the Thr12 side chain and the backbone NH of Asn87 rather than the coenzyme, indicating that this hydrogen bond to the D strand might determine a crucial difference between the reductive and the oxidative reaction types. Similarly, mutation of Asn87 to Ala results in an 80% reduction of k cat /K m in the dehydrogenase direction but also unchanged 3-oxoreductase properties. It appears that the binding of NAD + to the protein is influenced by local structural changes involving strand D and turn A to RB. Short-chain dehydrogenases/reductases (SDRs) constitute a protein family with highly diverse functions in pro- and eukaryotes [for recent reviews, cf. Krozowski (1994) and Jo ¨rnvall et al. (1995)]. All known three-dimensional struc- tures reveal a highly similar one-domain R/ pattern. Well- conserved primary structure elements are restricted to certain segments in the sequence, indicating a possibly common fold, active site, reaction mechanism, and coenzyme and substrate binding regions (Persson et al., 1991). Essential parts of the coenzyme binding site, as established by X-ray crystal- lography (Ghosh et al., 1991, 1994, 1995), and comparisons (Persson et al., 1991; Jo ¨rnvall et al., 1995), are located in the N-terminal part and consist of a “Rossmann fold” structure (Rossmann et al., 1974) with a conserved but variable (Jo ¨rnvall et al., 1984) Gly-X-X-X-Gly-X-Gly pattern (residues 13-19 in the 3/17-HSD sequence). Compari- sons, chemical modifications, and site-directed mutagenesis of different SDR enzymes have revealed a conserved Tyr- X-X-X-Lys segment (residues 151-155 in the 3/17-HSD sequence) essential for catalytic activity of SDR proteins (Jo ¨rnvall et al., 1981; Krook et al., 1990, 1992; Persson et al., 1991; Obeid & White, 1992; Chen et al., 1993; Ensor & Tai, 1994). However, recent investigations (Ghosh et al., 1994; Jo ¨rnvall et al., 1995) also highlight several other amino acid residues as important in substrate binding and catalytic conversion. Structure alignments of short-chain dehydro- genases/reductases, based on the available data of hydroxy- steroid and prostaglandin dehydrogenases and other SDR enzymes (Ghosh et al., 1994, 1995; Krook et al., 1993, Varughese et al., 1992; Tanaka et al., 1996), demonstrate that these residues are close to the catalytic center but also propose a more complicated architecture of the active site. In order to establish the roles for these residues, which are strictly conserved in most SDR structures thus far known (Ghosh et al., 1994; Jo ¨rnvall et al., 1995), we performed mutagenetic replacement at these positions, i.e. replacement of Thr12, Ser16, Asn87, and Ser138, in the prokaryotic 3/ 17-hydroxysteroid dehydrogenase (3/17-HSD), which is a SDR protein (Yin et al., 1991) derived from the Gram- negative bacterium Comamonas testosteroni ATCC 11996. ² This study was supported by grants from the Swedish Medical Research Council (Projects 13X-3532 and 13X-11210). U.C.T.O. was supported by research scholarships from the DFG (Deutsche Fors- chungsgemeinschaft) and STINT (Stiftelsen fo ¨ r internationalisering av ho ¨gre utbildning och forskning). * Corresponding author: Hans Jo ¨rnvall, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stock- holm, Sweden. Fax: +46-8-33 74 62. ‡ Department of Medical Biochemistry and Biophysics. § Center of Structural Biochemistry. X Abstract published in AdVance ACS Abstracts, December 15, 1996. 1 C. testosteroni is used instead of Pseudomonas testosteroni throughout the text (Marcus & Talalay, 1956; Tamaoka et al., 1987). 34 Biochemistry 1997, 36, 34-40 S0006-2960(96)01803-X CCC: $14.00 © 1997 American Chemical Society