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J Biol Chem, Vol. 273, Issue 47, 30888-30896, November 20, 1998


Molecular Cloning, Overexpression, and Characterization of Steroid-inducible 3alpha -Hydroxysteroid Dehydrogenase/Carbonyl Reductase from Comamonas testosteroni
A NOVEL MEMBER OF THE SHORT-CHAIN DEHYDROGENASE/REDUCTASE SUPERFAMILY*

Eric Möbus and Edmund MaserDagger

From the Department of Pharmacology and Toxicology, School of Medicine, Philipps University of Marburg, Karl-von-Frisch-Strasse 1, D-35033 Marburg, Germany

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

3alpha -Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha -HSD/CR) from Comamonas testosteroni, a bacterium that is able to grow on steroids as the sole carbon source, catalyzes the oxidoreduction at position 3 of a variety of C19-27 steroids and the carbonyl reduction of a variety of nonsteroidal aldehydes and ketones. The gene of this steroid-inducible 3alpha -HSD/CR was cloned by screening a C. testosteroni gene bank with a homologous DNA probe that was obtained by polymerase chain reaction with two degenerative primers based on the N-terminal sequence of the purified enzyme. The 3alpha -HSD/CR gene is 774 base pairs long, and the deduced amino acid sequence comprises 258 residues with a calculated molecular mass of 26.4 kDa. A homology search revealed that amino acid sequences highly conserved in the short-chain dehydrogenase/reductase (SDR) superfamily are present in 3alpha -HSD/CR. Two consensus sequences of the SDR superfamily were found, an N-terminal Gly-X-X-X-Gly-X-Gly cofactor-binding motif and a Tyr-X-X-X-Lys segment (residues 155-159 in the 3alpha -HSD/CR sequence) essential for catalytic activity of SDR proteins. 3alpha -HSD/CR was overexpressed and purified to homogeneity, and its activity was determined for steroid and nonsteroidal carbonyl substrates. These results suggest that inducible 3alpha -HSD/CR from C. testosteroni is a novel member of the SDR superfamily.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Enzymatic modification of steroids by hydroxysteroid dehydrogenases has an important role in the regulation of steroid-mediated gene transcription since the balance between active and inactive steroids determines the amount of the signal that reaches the receptor in the target tissue. 3alpha -Hydroxysteroid dehydrogenase (3alpha -HSD)1 catalyzes the reversible interconversion of hydroxy and oxo groups at position 3 of the steroid nucleus. In mammalian tissues, 3alpha -HSD works in concert with 5alpha - and 5beta -reductases to generate the 3alpha ,5alpha - and 3alpha ,5beta -tetrahydroxysteroids, respectively, thereby acting as molecular switch in steroid hormone receptor activation (1, 2).

In bacteria, however, the role of steroid dehydrogenases remains obscure, although there is increasing evidence that much of the machinery needed for steroid hormone action found in vertebrates is also present in unicellular organisms. For instance, steroid hormone-binding proteins have been found in yeast (3-5) and procaryotes (6, 7), and mammalian steroid receptors function in a hormone-dependent manner when expressed in Saccharomyces cerevisiae (8-10). Moreover, certain hydroxysteroid dehydrogenases (such as Streptomyces hydrogenans 3alpha ,20beta -HSD and Comamonas testosteroni 3beta ,17beta -HSD) that show similarities to mammalian steroid dehydrogenases involved in steroid activation/inactivation are present in procaryotes (11, 12). It thus appears that the elements for transcriptional activation of genes by steroids were present in organisms that evolved ~2 billion years ago.

On the other hand, certain bacteria are able to grow on steroids as the sole carbon source by the expression of a set of steroid-catabolizing enzymes, including hydroxysteroid dehydrogenases, in the presence of the steroid substrate (13-15). C. testosteroni (formerly Pseudomonas testosteroni (16)) is a Gram-negative bacterium that belongs to the beta  group of the Proteobacteria, corresponding to rRNA superfamily III. Comamonas strains have been isolated from soil, mud, and water, but also from various samples from the clinical environment. These strictly aerobic, nonfermentative, chemoorganotrophic bacteria rarely attack sugars, but grow well on organic acids and amino acids (17). Moreover, C. testosteroni strains are able to use steroids as the sole carbon source and thus may represent an important component in the mineralization of these stable compounds. Interestingly, catabolic genes for steroid degradation are not constitutively expressed, but are induced by their respective steroid substrates (13, 14, 18). Hence, steroids play a particularly important role in certain procaryotes, as they may simultaneously serve both as signal molecules and carbon source.

The same applies for procaryotic hydroxysteroid dehydrogenases, such as 3alpha -HSD from C. testosteroni. On the one hand, these enzymes have their role in procaryotic steroid hormone signal transduction and, on the other hand, participate in steroid degradation.

Procaryotic 3alpha -HSDs have been described in Eubacterium sp. (19) and C. (P.) testosteroni (20, 21). Interestingly, 3alpha -HSD from C. testosteroni is not a constitutive enzyme, but its expression is induced by steroids such as testosterone and progesterone (13, 14, 18, 22). In a previous investigation, steroid-inducible 3alpha -HSD has been purified from C. testosteroni and shown to mediate the oxidoreduction at position 3 of the steroid nucleus of a great variety of C19-27 steroids (14). This reaction is of significance in the initiation of the complete degradation of these relatively inert substrates (18). Surprisingly, this enzyme was also capable of catalyzing the carbonyl reduction of nonsteroidal xenobiotic aldehydes and ketones (14). Based on this pluripotent substrate specificity, the enzyme was named 3alpha -hydroxysteroid dehydrogenase/carbonyl reductase (3alpha -HSD/CR). Further studies revealed that the substrate pluripotency of 3alpha -HSD/CR, as well as its inducibility, not only increases the resistance of C. testosteroni to the steroid antibiotic fusidic acid, but also enhances the metabolic capacity of insecticide degradation in this organism (22).

To further the knowledge about the enzymology of steroid hormone metabolism and steroid hormone-regulated gene expression in bacteria, we have identified and characterized, on the molecular level, 3alpha -HSD from C. testosteroni strain ATCC 11996. In this paper, we present the gene isolation, gene cloning, sequencing, overexpression, purification, and biochemical characterization of this steroid-inducible enzyme. Primary structure analysis revealed that this enzyme is a novel member of the short-chain dehydrogenase/reductase (SDR) superfamily. Important structural features that are found in 3alpha -HSD/CR and that are inferred from three-dimensional structures of other SDR enzymes are discussed. Finally, our studies could serve as a model system for future investigations on the steroid-dependent molecular regulation of hydroxysteroid dehydrogenase expression in procaryotes.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Restriction enzymes, antibiotics (ampicillin, carbenicillin, chloramphenicol, kanamycin, and tetracycline), and isopropyl-1-thio-beta -D-galactopyranoside were purchased from Angewandte Gentechnologie Systeme GmbH (Heidelberg, Germany). Deoxyribonucleotides were from Amersham Pharmacia Biotech, and steroids and metyrapone (2-methyl-1,2-di-(3-pyridyl)-1-propanone) were from Sigma. The insecticide NKI 42255 (2-(1-imidazolyl)-1-(4-methoxyphenyl)-2-methyl-1-propanone) and its reduced alcohol metabolite NKI 42455 (2-(1-imidazolyl)-1-(4-methoxyphenyl)-2-methyl-1-propanol) were kindly provided by I. Bélai (Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary). Metyrapol (2-methyl-1,2-di-(3-pyridyl)-1-propanol) was a kind gift of G. F. Kahl (Department of Toxicology, University of Göttingen, Göttingen, Germany). C. testosteroni strain ATCC 11996 was from the Deutsche Sammlung für Mikroorganismen. All other chemicals were of the highest commercially available quality.

Strains and Growth Conditions-- C. testosteroni strain ATCC 11996 was grown at 30 °C in LB medium under the conditions described previously (20). Escherichia coli strains XL1-Blue MRF', INF alpha F', and BL21(DE3) pLysS were grown in LB medium at 37 °C. When necessary, the following antibiotics were added to the growth medium: ampicillin (50 µg/ml), chloramphenicol (34 µg/ml), kanamycin (50 µg/ml), tetracycline (12.5 µg/ml), and carbenicillin (50 µg/ml).

Construction of PCR Primers and Cloning of a 75-Base Pair PCR Product for 3alpha -HSD/CR-- Based on the N-terminal sequence of 3alpha -HSD (13) and according to C. testosteroni codon usage, two primers were synthesized: forward primer (amino acids 1-10), 5'-ATGGACATCATCGTCAT(C/T)(A/G/C)(G/C)(G/C)GGCGGCGC-3'; and reverse primer (amino acids 19-25), 5'-GGC(G/C)GC(G/C)GC(C/T)TCCAG(G/C)ACCTT-3'. A series of 4 × 10 cycles of PCR using different annealing temperatures was run against a preparation of wild-type C. testosteroni strain ATCC 11996 genomic DNA (denaturation at 96 °C for 30 s; annealing at 65, 60, 55, and 50 °C, respectively, for 10 s; and extension at 72 °C for 30 s). The amplified 75-bp PCR product was subcloned into pCRTM2.1 vector in one-shotTM-competent INV alpha F' cells (Invitrogen) and sequenced. The fragment containing the 5'-sequence of the 3alpha -HSD gene was digoxigenin end-labeled and used as a homologous probe for Southern hybridization and C. testosteroni gene bank screening.

Southern Hybridization-- Total DNA from C. testosteroni (ATCC 11996) was isolated by standard methods (23). For Southern blot analysis, DNA was digested with different restriction enzymes. After agarose gel electrophoresis and capillary transfer onto Hybond-N+ nylon membranes (Amersham Pharmacia Biotech), the DNA was hybridized with the digoxigenin-labeled homologous probe of the 3alpha -HSD/CR gene. For detection of the hybridizing fragments, the standard protocol of Boehringer Mannheim was followed, finally resulting in the identification of a hybridizing 5.2-kilobase EcoRI fragment.

Construction of a C. testosteroni EcoRI Gene Bank-- Total DNA from C. testosteroni (ATCC 11996) was digested with EcoRI restriction enzyme, and the fragments were ligated with the lambda ZAP Express system (Stratagene) according to the manufacturer's instruction. The resulting DNA was packaged using Gigapack Gold III (Stratagene) and amplified in E. coli strain XL1-Blue MRF', finally yielding an EcoRI gene bank of 1.7 × 108 plaque-forming units/ml.

Cloning and Sequencing of the 3alpha -Hydroxysteroid Dehydrogenase Gene-- Screening of the EcoRI bank was carried out as follows. After transferring 12,000 plaques onto Hybond-N+ nylon membranes, plaques containing the 3alpha -HSD/CR gene were detected by the homologous probe described above. Positive clones were converted to pBK-CMV phagemids carrying DNA inserts between the EcoRI sites by helper phage superinfection as described in the lambda ZAP Express system manual. Nucleotide sequences of both strands were determined by the dideoxy chain termination method (24) using Sequenase Version 2.0 and Thermo Sequenase (Amersham Pharmacia Biotech).

Overexpression and Purification of 3alpha -Hydroxysteroid Dehydrogenase/Carbonyl Reductase-- The 3alpha -HSD/CR gene was cloned into the NdeI/BamHI restriction sites of the pET15b vector (Novagen), which additionally codes for an N-terminal His tag sequence with an integrated thrombin cleavage site. The corresponding primers for PCR were constructed as follows. The ATG start codon sequence was modified to yield the forward primer containing a NdeI site: 5'-GAGACAACATATGTCCATCATCGTGATA. The TGA stop codon sequence was modified to yield the reverse primer containing a BamHI site: 5'-GCCGGATCCGAGGTCAGAACTGTGTCGG. A 25-cycle PCR was run against plasmid DNA from pBK-E52 under the following conditions: 95 °C for 45 s, 60 °C for 45 s, and 72 °C for 2 min. The resulting PCR product was digested with NdeI and BamHI restriction enzymes to give a 787-bp fragment, which was inserted into the pET15b vector. The insert was sequenced to confirm the correct in-frame DNA sequence and the absence of any mutations.

Transformation of E. coli BL21(DE3) pLysS cells (Stratagene) was performed by heat shock according to the supplier's instruction. Overnight cultures harboring the recombinant pET15b vector were diluted 1:50 into fresh LB medium containing the appropriate antibiotics. Cells were grown to an absorbance of 0.6 at 600 nm, and then recombinant 3alpha -HSD/CR overexpression was induced by addition of isopropyl-1-thio-beta -D-galactopyranoside to a final concentration of 1 mM. After 5 h of induction, cells were harvested and lysed by freezing and ultrasonication (3 × 10 s), and the debris was removed by spinning at 16,000 × g for 10 min. The histidine-tagged enzyme was purified on a Ni2+-Sepharose column (His-Trap kit, Amersham Pharmacia Biotech) with a Amersham Pharmacia ÄKTA Protein Purifier system. Pure protein was eluted by applying a linear gradient of imidazole (0-0.5 M) in 20 mM phosphate buffer, pH 7.5, and 0.5 M NaCl. Fractions containing purified 3alpha -HSD/CR were pooled and desalted, and the buffer was exchanged with 0.5 M Tris-HCl buffer, pH 7.4, using a Vivaspin concentrator (Vivascience).

Enzyme Assays-- Enzyme assays were carried out as described previously (14).

Protein Determination-- Protein concentration was determined by the method of Bradford (25) with Roti-Quant solution (Roth) using bovine serum albumin as a standard. Protein analysis by SDS-polyacrylamide gel electrophoresis was carried out according to Laemmli (26).

Homology Search, Sequence Alignment, and Phylogenetic Analysis-- The BLAST program (27) was used to screen protein and DNA data bases for proteins that shared sequence similarity. Multiple sequence alignments were made according to the CLUSTAL W method (28), and the similarity relationships were calculated applying bootstrap analysis (29).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cloning and Sequencing of the 3alpha -Hydroxysteroid Dehydrogenase Gene-- The amino acid sequence for the 29 N-terminal residues of 3alpha -HSD/CR was determined previously (13). Based on this sequence and according to C. testosteroni codon usage, primers were designed that, following PCR, resulted in the generation of a 75-bp product containing the 5'-sequence of the 3alpha -HSD gene. The 75-bp PCR product was used as a homologous probe in Southern hybridization and gene bank screening.

A C. testosteroni EcoRI gene bank was constructed based on the hybridization of the labeled PCR probe with a 5.2-kilobase EcoRI fragment in Southern analysis. By screening 12,000 plaques of the EcoRI gene bank, three positive clones were obtained. The results from the sequence analysis of one of these clones is shown in Fig. 1. The 3alpha -HSD/CR gene has 774 base pairs coding for a novel protein of 258 amino acids with a predicted molecular mass of 26.4 kDa. The deduced amino acid sequence (Fig. 1) was in agreement with the available N-terminal amino acid sequence derived from the Edman degradation of the isolated 3alpha -HSD/CR protein (13). A consensus ribosomal binding site (AGGAGA) is located 7 bp upstream of the ATG start codon.


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  		Fig. 1.   Nucleotide sequence and deduced amino acid sequence of the 3alpha -hydroxysteroid dehydrogenase/carbonyl reductase gene from C. testosteroni. Numbers to the left correspond to the nucleotide sequence, and numbers to the right refer to the amino acid sequence. Residues corresponding to the N-terminal amino acid sequence obtained from the isolated protein by Edman degradation (13) are underlined. The ribosomal binding site (AGGAGA), 7 bases upstream of the ATG start codon (-7 to -12), is indicated by boldface underlining. The stop codon is indicated by an asterisk.

Sequence Alignments-- Sequence alignment of 3alpha -HSD/CR with other members of the SDR superfamily showed very little homology and identities (Fig. 2), which is a known phenomenon for these proteins (30, 31). However, some consensus sequences of the SDR enzymes are present in the 3alpha -HSD/CR primary structure. The N-terminal Gly-X-X-X-Gly-X-Gly sequence (Gly-8, Gly-12, and Gly-14; numbering according to the 3alpha -HSD/CR sequence of C. testosteroni) (Fig. 2) is one of the amino acid "fingerprints" of the SDR superfamily that characterizes the dinucleotide-binding motif. In addition, it has been proposed that there are three highly conserved residues in the substrate-binding site of the SDR enzymes: Ser, Tyr, and Lys (the catalytic active "Ser-Tyr-Lys triad") (32). Whereas the conserved Tyr-X-X-X-Lys catalytic motif is present in 3alpha -HSD from C. testosteroni (at amino acids 155-159 in the 3alpha -HSD sequence), Ala is at position 144 instead of Ser as in most other SDRs. In Klebsiella aerogenes ribitol dehydrogenase, which serves as one of the model SDR enzymes, and in dihydropteridine reductase, Val and Ala are at this position, respectively (Fig. 2).


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  		Fig. 2.   Sequence alignment of 3alpha -HSD/CR from C. testosteroni and selected SDR proteins. SDR proteins were searched with the BLAST (27) and FASTA (61) programs and aligned with the CLUSTAL W method (28). Gaps (shown as dashes) were introduced in the sequences where necessary to give better alignment. The residues that are identical to one another in the sequences are boxed. Numbers refer to amino acid residues. Accession numbers are given in parentheses (see below). 3/17beta-HSD, 3beta ,17beta -hydroxysteroid dehydrogenase from C. testosteroni (Protein Identification Resource S62216); 20beta-HSD, 3alpha ,20beta -hydroxysteroid dehydrogenase from S. hydrogenans (Swiss-Prot P19992); 7alpha-HSD, 7alpha -hydroxysteroid dehydrogenase from E. coli (Swiss-Prot P25529); CR, carbonyl reductase from mouse lung (Protein Identification Resource S69141); NodG, NodG protein from Rhizobium meliloti (Swiss-Prot P06234); FixR, FixR protein from B. japonicum (Swiss-Prot P05406); 3alpha-HSD, 3alpha -hydroxysteroid dehydrogenase from C. testosteroni (this study); PDH, 15-hydroxyprostaglandin dehydrogenase from human placenta (Swiss-Prot P15428); RDH, ribitol dehydrogenase from K. aerogenes (Swiss-Prot P00335); 17beta-HSD, 17beta -hydroxysteroid dehydrogenase from human placenta (Protein Data Bank 1BHS); 11beta-HSD, 11beta -hydroxysteroid dehydrogenase from mouse liver (Swiss-Prot P50172); ADH, alcohol dehydrogenase from Drosophila lebanonensis (Swiss-Prot P10807); DR, dihydropteridine reductase from rat liver (Swiss-Prot P11348).

A comparison of the confirmations of five SDR primary structures (bacterial 3alpha ,20beta -HSD (33), human type 1 17beta -HSD (32), rat liver dihydropteridine reductase (34), bacterial 7alpha -HSD (35), and mouse lung carbonyl reductase (36)) for which x-ray coordinates have been reported revealed that although there are only some fully conserved residues common to the five structures, the three-dimensional conformation is highly conserved (37). Some of these residues are also found in the 3alpha -HSD sequence (Gly-8, Gly-12, Gly-14, Val-85, Asn-86, Gly-89, Tyr-155, Ser-158, Lys-159, Ala-170, Pro-185, Gly-186, and Thr-190), and in some cases, the variant residues in 3alpha -HSD/CR are conservative substitutions, i.e. Ser-7 for Thr-7. Two members of the catalytic triad proposed for the mechanism of action of SDR enzymes (Tyr-155 and Lys-159) are included in all five, and Ser-138 (numbering according to 3beta ,17beta -HSD) is present in four of the five structures (replaced by Ala in dihydropteridine reductase and 3alpha -HSD/CR from this study) (Fig. 2).

Phylogenetic Analysis-- Calculations based upon the alignment in Fig. 2 give an evolutionary tree as depicted in Fig. 3. According to the bootstrap analysis, 3alpha -HSD/CR from C. testosteroni is seemingly related to FixR protein from Bradyrhizobium japonicum. Interestingly, this procaryotic nodulation protein serves as signal molecule in the communication between bacteria and plants.


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  		Fig. 3.   Phylogenetic tree of selected SDR proteins. The tree was constructed by the neighbor-joining method using the CLUSTAL W program (28). Bootstrap analysis (29) was performed by generating 1000 reiterations. Branch lengths are arbitrary, and bootstrap values are indicated for each node. DH, dehydrogenase; Red, reductase. For other abbreviations of protein names, see legend to Fig. 2.

Overexpression-- Cloning of the 3alpha -HSD gene from C. testosteroni strain ATCC 11996 into the vector pET15b and subsequent overexpression resulted in a fusion protein with an N-terminal His tag sequence. Recombinant 3alpha -HSD/CR could be purified in one step using metal chelate chromatography, and the purity of the protein was >98% pure as judged by Coomassie Blue staining of an SDS-polyacrylamide gel (Fig. 4). After cleavage of the thrombin site, the molecular mass of the recombinant protein as seen on the SDS gel was identical to that predicted from the genomic sequence. However, in our previous study (14), we proposed a molecular mass of 28 kDa rather than the correct value of 26.4 kDa.


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  		Fig. 4.   Overexpression and purification of 3alpha -hydroxysteroid dehydrogenase/carbonyl reductase from C. testosteroni. The state of purity of overexpressed 3alpha -HSD/CR is shown on a reducing 10% SDS-polyacrylamide gel. First lane, molecular mass markers; second lane, total cell lysate supernatant; third lane, sample load flow-through fraction; fourth lane, column wash; fifth lane, 3alpha -HSD/CR eluted from a nickel-Sepharose column; sixth lane, molecular mass markers. The amount of protein loaded per lane was 20 µg for the total cell lysate supernatants and 10 µg for the purified 3alpha -HSD/CR preparation.

Kinetic Analysis-- The expressed 3alpha -HSD/CR was active when tested with a variety of C19-27 steroid substrates. Enzyme kinetic constants (Km and Vmax) for 3-oxosteroid reduction and 3alpha -hydroxysteroid dehydrogenation were determined with the recombinant protein and compared with the values obtained with native 3alpha -HSD from C. testosteroni in a previous study (14). The recombinant 3alpha -HSD enzyme efficiently catalyzed the 3-oxo reduction and 3alpha -hydroxy dehydrogenation of steroids of the androstane and bile acid series with Km values in the low micromolar range (18-42 µM) under the conditions employed. Moreover, the steroid antibiotic fusidic acid turned out to be a substrate of 3alpha -HSD with a Km value of only 6.1 µM. Interestingly, testosterone and progesterone, which are potent inducers of 3alpha -HSD/CR from C. testosteroni, were not substrates of this enzyme.

On the other hand, the recombinant enzyme catalyzed the carbonyl reduction of nonsteroidal aldehyde and ketone compounds. Although the Km values for the model substrates of the carbonyl-reducing enzymes (metyrapone and p-nitrobenzaldehyde as well as the insecticide NKI 42255) are relatively high (1.8-3.3 µM), their intrinsic clearance values are comparable to those of the steroid substrates. No reverse oxidation of the formed hydroxy derivatives p-nitrobenzalcohol and NKI 42455 could be observed.

Overall, the substrate specificity of the recombinant enzyme is similar to that of native 3alpha -HSD. Moreover, the kinetic constants for the substrates are essentially identical (14).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In this work, we have identified and characterized 3alpha -HSD/CR from C. testosteroni as a new member of the SDR superfamily. The SDR proteins are a growing superfamily of NAD(P)(H)-dependent non-metallo-oxidoreductases that are ~250-350 amino acids in length and that bind NAD(P)(H) with a Rossman fold motif (30, 31). Found in mammals, amphibians, plants, yeast, protozoans, and bacteria, SDRs metabolize a range of substrates including aliphatic aldehydes and ketones, monosaccharides, steroids, prostaglandins, flavonoids, polycyclic aromatic hydrocarbons, and retinoids (30, 31, 38). Several of these SDR substrates are known to serve as important intra- or intercellular signal molecules in pro- and eucaryotes, which is especially true for steroids, retinoids, and flavonoids (38-40). Despite a residue identity level of only 20-30% between different SDR members, the three-dimensional structures so far analyzed (32, 33, 35, 36) reveal a highly similar architecture with a one-domain folding pattern. In addition, sequence alignments, chemical modifications, site-directed mutagenesis, and crystallographic analyses revealed two mainly conserved patterns, an N-terminal Gly-X-X-X-Gly-X-Gly motif related to the binding of the cosubstrate NAD(H) or NADP(H) and a highly conserved Tyr-X-X-X-Lys segment (residues 155-159) assigned to the catalytic center (31, 41, 42). The highly similar overall fold and the conserved Tyr-X-X-X-Lys motif found in almost all SDR structures thus far determined suggest a similar reaction mechanism for the individual proteins (30, 31).

In the reaction mechanism proposed on the basis of these findings, the conserved Tyr residue acts as catalytic base, facilitated by the adjacent protonated Lys residue, which lowers the pKa of the phenolic Tyr OH group, whereas the nicotinamide ring of the cofactor is oriented for pro-S-hydrogen transfer (31, 36, 37, 43). The mutation of the Tyr residue in this motif in SDRs led to inactive enzymes (44-46). However, the exact roles of the active-site residues have not yet clarified, especially concerning the function of the conserved Ser residue, which is located in the vicinity of the conserved Tyr and Lys residues. In most SDR proteins, the Ser hydroxyl group appears to be able to form hydrogen bonds with the reaction product (as in mouse lung carbonyl reductase (36)) and/or with the hydroxyl group of the conserved Tyr residue (47). The role of this Ser residue most likely might derive from a stabilization by hydrogen bonding with the substrate, reaction intermediate, and product. This concept is supported by the work of Oppermann et al. (43), in which replacement of Ser-138 with Thr in 3beta ,17beta -hydroxysteroid dehydrogenase from C. testosteroni yielded an active protein with identical catalytic constants. However, some SDR proteins, like C. testosteroni 3alpha -HSD from this study, rat dihydropteridine reductase, or K. aerogenes ribitol dehydrogenase, contain neither Ser nor Thr, but contain Ala or Val at this position. Obviously, a hydroxyl is important for the reaction to take place in some SDR proteins, whereas in other SDR proteins, including 3alpha -HSD from this study, a modified reaction mechanism is involved in catalysis since Ser is not conserved in the latter subgroup. Therefore, the concept of adding Ser to the previously recognized Tyr and Lys residues to form a catalytic triad in the SDR superfamily is not supported by our findings with 3alpha -HSD from C. testosteroni.

Glycine is the most conserved residue in the SDR superfamily. In the enzymes with known tertiary structure, the unaltered glycine positions largely correspond to bands in the tertiary structure and therefore also support overall conserved conformational properties in the SDR superfamily. In the N-terminal part of 3alpha -HSD/CR, three highly conserved glycine residues (Gly-8, Gly-12, and Gly-14) correspond to the spacing expected for Gly residues at the NAD-binding site of many dehydrogenases. Adjacent portions of most SDRs reveal further conservative replacements and similar secondary structure predictions for alternating beta /alpha -structures in most enzymes (Fig. 2) (30, 31).

Mutations carried out with Thr-13 and Asn-87 of 3beta ,17beta -HSD from C. testosteroni (numbering according to the 3beta ,17beta -HSD sequence) reveal differential effects on the reaction direction in this SDR protein. A Thr right-arrow Ala exchange results in a complete loss of the dehydrogenase reaction, whereas the activity in the reductive direction remains unaffected (43). A Thr right-arrow Ser substitution at this position yields a protein with properties indistinguishable from those of the wild type (43). A similar effect was observed with an Asn-87 right-arrow Ala replacement in 3beta ,17beta -HSD from C. testosteroni, where the dehydrogenase activity was decreased by >80%, again with unchanged reductase properties (43). Interestingly, 3alpha -HSD/CR from C. testosteroni of this study has Ser here (Ser-7 in the 3alpha -HSD/CR sequence) instead of Thr, confirming the importance of the hydroxyl side chain at this position to form hydrogen bonds. On the other hand, the role of Asn-87 in SDR proteins is questionable since wild-type 3alpha -HSD from C. testosteroni has Tyr at this position.

The presence of a charged amino acid in certain positions is important in regulating specificity for NADP(H) or NAD(H) in enzymes that are otherwise very similar in their primary structure. In NAD(P)H-dependent SDR enzymes, basic residues at position 17 (e.g. Lys-17 in mouse lung carbonyl reductase and Lys-44 in mouse type 1 11beta -HSD) confer specificity to NADPH by forming electrostatic interactions with the ribose 2'-phosphate (36). Moreover, an adjacent arginine, such as Arg-39 in mouse lung carbonyl reductase and Arg-66 in type 1 11beta -HSD (48-50), may give an attractive coulombic interaction with the 2'-phosphate of NADP(H), promoting the binding of this molecule (36).

In NAD(H)-dependent enzymes such as 3beta ,17beta -HSD (51), these basic residues (Ser-17 and Ile-39) are not strictly conserved and are preceded at position 38 by an aspartic acid residue. This configuration, Asp and Ile (Asp-32 and Ile-33 in the 3alpha -HSD/CR sequence), is also found in 3alpha -HSD/CR from C. testosteroni (Fig. 2). The Asp-32 carboxylate might result in electrostatic repulsion of the 2'-phosphate moiety, thereby conferring specificity to NAD(H) (52, 53). Chen et al. (52) converted this Asp in Drosophila alcohol dehydrogenase (Asp-37) to Asn and found that the mutated enzyme had a 60-fold increase in affinity for NADP+, presumably because the negative repulsion between the carboxylate of Asp-37 and the 2'-phosphate group of adenosine was removed. This has been confirmed with mouse lung carbonyl reductase, where a Thr right-arrow Asp mutation at this position resulted in increases of >200-fold in the Km values for NADP(H) and decreases of >7-fold in those for NAD(H) (54).

In all members of the SDR superfamily, the N-terminal cofactor-binding motif and the Tyr-X-X-X-Lys motif are well conserved, which is remarkable considering that the bacterial and mammalian proteins diverged at least 2 billion years from a common ancestor. The SDR superfamily may thus represent a case of divergent evolution from a multifunctional ancestor protein. Too little is known about the gene structures of SDRs to assess the mechanism of divergence, but the common constellation of active-site residues and the highly conserved NAD(P)(H)-binding pocket imply gene duplication and subsequent evolution of substrate specificity. On the other hand, it is not surprising that, other than Tyr-155 and Lys-159 (numbering according to 3alpha -HSD/CR from C. testosteroni), there are no conserved residues in the catalytic cleft since each member of the SDR family has selectivity for its individual steroid, prostaglandin, sugar, or alcohol substrate. Residues in the C-terminal end display a large degree of sequence variation among the SDR proteins, with no clear pattern relating to substrate specificity (30). Interestingly, the Tyr-X-X-X-Lys motif can also be present in proteins of the aldo/keto-reductase superfamily (55). For example, this motif is found in rat liver 3alpha -HSD, in which Tyr-205 and Lys-209 are the corresponding residues, although these residues do not have any role in catalysis.

The hydroxysteroid dehydrogenases that belong to the SDR superfamily catalyze the (under certain circumstances reversible) interconversion of potent steroid hormones into their inactive metabolites, thereby regulating the amount of hormone that can bind and activate nuclear steroid receptors. This role has been well documented for type 2 11beta -HSD (EC 1.1.1.146) (56) and type 1 17beta -HSD (EC 1.1.1.62) (32), which regulate the occupancy of mineralocorticoid and estrogen receptors, respectively, and which belong to the SDR protein superfamily. Therefore, like other SDR proteins, 3alpha -HSD from C. testosteroni may also have an important function in the conversion of signaling molecules (steroids) to either the active or inactive state. The fact that testosterone and progesterone do not appear to be substrates of 3alpha -HSD is intriguing since it turned out in previous investigations that both steroids strongly induce the expression of 3alpha -HSD in C. testosteroni. On the other hand, C. testosteroni has been described to use testosterone as the sole carbon source. It is therefore hypothesized that testosterone and progesterone require (a rate-limiting) enzymatic conversion before they can enter the steroid catabolic pathway. This metabolic delay may provide enough time for these steroids to serve as positive regulators in the expression of the involved steroid catabolic enzymes. This idea is supported by the fact that it is difficult to isolate steroid-derived intermediates since they are so rapidly further metabolized and mineralized (57, 58).

On the other hand, it was shown previously that testosterone induction leads to a 5-6-fold elevation of resistance of C. testosteroni against the steroid antibiotic fusidic acid (=ramycin) (59), a secretion product of the fungus Fusidium coccineum. Furthermore, testosterone-induced bacterial cells exhibit a significantly faster uptake and metabolism of NKI 42255 (59), a synthetic compound that is derived from the cytochrome P450 inhibitor metyrapone. In the present study, we have demonstrated that steroid-inducible 3alpha -HSD/CR mediates both the 3alpha -hydroxyl oxidation of fusidic acid and the carbonyl reduction of several nonsteroidal xenobiotics, including the novel heteroaromatic insecticide NKI 42255 (60). We therefore suggest that 3alpha -HSD/CR may act as an initiating enzyme of steroid-inducible metabolic pathways that constitute important defense strategies of certain bacteria against natural and synthetic toxicants.

In conclusion, we have identified and characterized, on the molecular level, 3alpha -HSD from C. testosteroni, revealing this enzyme to be a novel member of the SDR superfamily. The SDR superfamily represents an interesting challenge to understanding structure/function relationships. Although the catalytic mechanism and mode of NAD(P)(H) binding in SDRs are understood, it remains to be elucidated how these proteins discriminate between steroids and other substrates, and how positional selectivity and stereoselectivity between different steroids are achieved. In addition, the architecture of these proteins may prove useful for studying the evolution of substrate specificity and how a common scaffold can catalyze a similar reaction with a broad range of substrates. Further studies of SDR sequences from phylogenetically diverse organisms should help to identify the structural features that determine substrate and cosubstrate specificity and that favor oxidative or reductive direction of enzyme function and to explore the role of multimeric assembly in the action of the SDR enzymes. In view of the central role that these enzymes have in regulating the action of their cognate substrates, such information is important for understanding and treating many steroid hormone-dependent processes in eucaryotes and procaryotes.

    ACKNOWLEDGEMENTS

The excellent technical assistance of Jutta Friebertshäuser and Eva Braun is gratefully acknowledged.

    FOOTNOTES

* This work was supported by European Community Program Biotech 2 under Contract BIO4-97-2123 and by Deutsche Forschungsgemeinschaft Grant SFB 395.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF092031.

Dagger To whom correspondence should be addressed. Tel.: 49-6421-28-5465; Fax: 49-6421-28-5600; E-mail: maser{at}mailer.uni-marburg.de.

The abbreviations used are: HSD, hydroxysteroid dehydrogenase; 3alpha -HSD/CR, 3alpha -hydroxysteroid dehydrogenase/carbonyl reductase; SDR, short-chain dehydrogenase/reductase; PCR, polymerase chain reaction; bp, base pair(s).
    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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