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Lecithin retinol acyltransferase (LRAT) plays a major role in the vertebrate visual cycle. Indeed, it is responsible for the esterification of all-trans retinol into all-trans retinyl esters, which can then be stored in microsomes or further metabolized to produce the chromophore of rhodopsin. In the present study, a detailed characterization of the enzymatic properties of truncated LRAT (tLRAT) has been achieved using in vitro assay conditions. A much larger tLRAT activity has been obtained compared to previous reports and to an enzyme with a similar activity. In addition, tLRAT is able to hydrolyze phospholipids bearing different chain lengths with a preference for micellar aggregated substrates. It therefore presents an interfacial activation property, which is typical of classical phospholipases. Furthermore, given that stability is a very important quality of an enzyme, the influence of different parameters on the activity and stability of tLRAT has thus been studied in detail. For example, storage buffer has a strong effect on tLRAT activity and high enzyme stability has been observed at room temperature. The thermostability of tLRAT has also been investigated using circular dichroism and infrared spectroscopy. A decrease in the activity of tLRAT was observed beyond 70°C, accompanied by a modification of its secondary structure, i.e. a decrease of its α-helical content and the appearance of unordered structures and aggregated β-sheets. Nevertheless, residual activity could still be observed after heating tLRAT up to 100 °C. The results of this study highly improved our understanding of this enzyme.

The aldo-keto reductase (AKR) human type 3 3alpha-hydroxysteroid dehydrogenase (h3alpha-HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3alpha-HSD3-NADP(H) at 1.9 A resolution. During the refinement process, we found acetate and citrate molecules deeply engulfed in the steroid-binding cavity. Superimposition of this structure with the h3alpha-HSD3-NADP(H)-testosterone/acetate ternary complex structure reveals that one of the mobile loops forming the binding cavity operates a slight contraction movement against the citrate molecule while the side chains of many residues undergo numerous conformational changes, probably to create an optimal binding site for the citrate. These structural changes, which altogether cause a reduction of the substrate-binding cavity volume (from 776 A(3) in the presence of testosterone/acetate to 704 A(3) in the acetate/citrate complex), are reminiscent of the "induced-fit" mechanism previously proposed for the aldose reductase, another member of the AKR superfamily. We also found that the replacement of residues Arg(301) and Arg(304), localized near the steroid-binding cavity, significantly affects the 3alpha-HSD activity of this enzyme toward 5alpha-DHT and completely abolishes its 17beta-HSD activity on 4-dione. All these results have thus been used to reevaluate the binding mode of this enzyme for androgens.

The 5beta-reductases (AKR1D1-3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 5beta-reduction of the C4-C5 double bond found into Delta4-3-ketosteroids, including steroid hormones and bile acids. Multiple-sequence alignments and mutagenic studies have already identified one of the residues presumably located at their active site, Glu 120, as the major molecular determinant for the unique activity displayed by 5beta-reductases. To define the exact role played by this glutamate in the catalytic activity of these enzymes, biochemical and structural studies on human 5beta-reductase (h5beta-red) have been undertaken. The crystal structure of h5beta-red in a ternary complex with NADP (+) and 5beta-dihydroprogesterone (5beta-DHP), the product of the 5beta-reduction of progesterone (Prog), revealed that Glu 120 does not interact directly with the other catalytic residues, as previously hypothesized, thus suggesting that this residue is not directly involved in catalysis but could instead be important for the proper positioning of the steroid substrate in the catalytic site. On the basis of our structural results, we thus propose a realistic scheme for the catalytic mechanism of the C4-C5 double bond reduction. We also propose that bile acid precursors such as 7alpha-hydroxy-4-cholesten-3-one and 7alpha,12alpha-dihydroxy-4-cholesten-3-one, when bound to the active site of h5beta-red, can establish supplementary contacts with Tyr 26 and Tyr 132, two residues delineating the steroid-binding cavity. These additional contacts very likely account for the higher activity of h5beta-red toward the bile acid intermediates versus steroid hormones. Finally, in light of the structural data now available, we attempt to interpret the likely consequences of mutations already identified in the gene encoding the h5beta-red enzyme which lead to a reduction of its enzymatic activity and which can progress to severe liver function failure.

The mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-HSD) is the unique known member of the aldo-keto reductase (AKR) superfamily able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta4) into epi-testosterone (epi-T), the 17alpha-epimer of testosterone. Structural and mutagenic studies had already identified one of the residues delineating the steroid-binding cavity, A24, as the major molecular determinant for the stereospecificity of m17alpha-HSD. We report here a ternary complex crystal structure (m17alpha-HSD:NADP(+):epi-T) determined at 1.85 A resolution that confirms this and reveals a unique steroid-binding mode for an AKR enzyme. Indeed, in addition to the interactions found in all other AKRs (van der Waals contacts stabilizing the core of the steroid and the hydrogen bonds established at the catalytic site by the Y55 and H117 residues with the oxygen atom of the ketone group to be reduced), m17alpha-HSD establishes with the other extremity of the steroid nucleus an additional interaction involving K31. By combining direct mutagenesis and kinetic studies, we found that the elimination of this hydrogen bond did not affect the affinity of the enzyme for its steroid substrate but led to a slight but significant increase of its catalytic efficiency (k(cat)/K(m)), suggesting a role for K31 in the release of the steroidal product at the end of the reaction. This previously unobserved steroid-binding mode for an AKR is similar to that adopted by other steroid-binding proteins, the hydroxysteroid dehydrogenases of the short-chain dehydrogenases/reductases (SDR) family and the steroid hormone nuclear receptors. Mutagenesis and structural studies made on the human type 3 3alpha-HSD, a closely related enzyme that shares 73% amino acids identity with the m17alpha-HSD, also revealed that the residue at position 24 of these two enzymes directly affects the binding and/or the release of NADPH, in addition to its role in their 17alpha/17beta stereospecificity.

Androgens exert their effects by binding to the highly specific androgen receptor (AR). In addition to natural potent androgens, AR binds a variety of synthetic agonist or antagonist molecules with different affinities. To identify molecular determinants responsible for this selectivity, we have determined the crystal structure of the human androgen receptor ligand-binding domain (hARLBD) in complex with two natural androgens, testosterone (Testo) and dihydrotestosterone (DHT), and with an androgenic steroid used in sport doping, tetrahydrogestrinone (THG), at 1.64, 1.90, and 1.75 A resolution, respectively. Comparison of these structures first highlights the flexibility of several residues buried in the ligand-binding pocket that can accommodate a variety of ligand structures. As expected, the ligand structure itself (dimension, presence, and position of unsaturated bonds that influence the geometry of the steroidal nucleus or the electronic properties of the neighboring atoms, etc.) determines the number of interactions it can make with the hARLBD. Indeed, THG--which possesses the highest affinity--establishes more van der Waals contacts with the receptor than the other steroids, whereas the geometry of the atoms forming electrostatic interactions at both extremities of the steroid nucleus seems mainly responsible for the higher affinity measured experimentally for DHT over Testo. Moreover, estimation of the ligand-receptor interaction energy through modeling confirms that even minor modifications in ligand structure have a great impact on the strength of these interactions. Our crystallographic data combined with those obtained by modeling will be helpful in the design of novel molecules with stronger affinity for the AR.

RPE65 is the major component of the retinal pigment epithelium (RPE) microsomal membrane, and it plays a critical role in the binding of retinoids involved in the visual cycle. To understand how RPE65 binds to membranes, we have expressed and purified soluble fragments of human RPE65 fused to glutathione S-transferase (GST). The interaction between two fragments of RPE65 (F1 and F2 which include residues 1-125 and 126-250, respectively) and lipid monolayers has been studied by surface pressure, ellipsometry, and surface rheology measurements. Surface pressure and ellipsometry clearly showed a rapid adsorption of F2 to lipid monolayers whereas the kinetics of binding of F1 was much slower. Furthermore, the data suggest that the F2 fragment inserts into the lipid monolayer. Surface rheology showed a clear increase in monolayer rigidity only in the presence of F2, thereby demonstrating high intermolecular interactions of this fragment. This observation is further supported by the GST pull-down assays which demonstrated that F2 cosediments with full-length RPE65, suggesting that RPE65 has the propensity to form clusters or oligomers. The structure homology modeling of RPE65 based on a related family member, apocarotene 15',15'-oxygenase, further suggests that a hydrophobic patch located in the F2 region might be responsible for membrane binding. The present work shows that F2 interacts much stronger with lipid monolayers than does F1, which suggests that the region of RPE65 located between residues 126-250 should be very important for its membrane binding. Moreover, given that these fragments are not acylated, these data also suggest that an effective binding of RPE65 to membranes can be achieved without palmitoylation. Furthermore, GST pull-down assays also indicated that F2 interacts with 11-cis-retinol dehydrogenase, which supports previous data suggesting that it could act as a partner of RPE65.

The recently discovered CLECSF6 protein displays the features of a receptor involved in the down-modulation of leukocyte activation. Although CLECSF6 has been the focus of the interest of many researchers lately, a Western blot characterization of the protein is still lacking. This highly reduces our ability to gain full knowledge of the biological relevance of this protein in cell responses. We produced two rabbit polyclonal antisera that detected a glycosylated protein at approximately 35kDa in neutrophils. Four different CLECSF6 mRNA species have been discovered to date. When deglycosylated, the protein displayed the molecular weight expected for the longest CLECSF6 form. Neutrophil membrane fractions were strongly enriched in the protein. We showed a down-modulation of the expression of this protein in neutrophils treated with granulocyte-macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF-alpha), lipopolysaccharide (LPS), and interleukin (IL)-4. This work supports the hypothesis that CLECSF6 is involved in the control of inflammation in neutrophils.

Human 20alpha-hydroxysteroid dehydrogenase (h20alpha-HSD; AKR1C1) catalyzes the transformation of progesterone (Prog) into 20alpha-hydroxy-progesterone (20alpha-OHProg). Although h20alpha-HSD shares 98% sequence identity with human type 3 3alpha-HSD (h3alpha-HSD3, AKR1C2), these two enzymes differ greatly in their activities. In order to explain these differences, we have solved the crystal structure of h20alpha-HSD in a ternary complex with NADP(+) and 20alpha-OHProg at 1.59A resolution. The steroid is stabilized by numerous hydrophobic interactions and a hydrogen bond between its O20 and the N(epsilon ) atom of His222. This new interaction prevents the formation of a hydrogen bond with the cofactor, as seen in h3alpha-HSD3 ternary complexes. By combining structural, direct mutagenesis and kinetic studies, we found that the H(222)I substitution decreases the K(m) value for the cofactor 95-fold. With these results, we hypothesize that the rotation of the lateral chain of His222 could be a mediating step between the transformation of Prog and the release of the cofactor. Moreover, crystal structure analysis and direct mutagenesis experiments lead us to identify a new residue involved in the binding of Prog. Indeed, the R(304)L substitution leads to a 65-fold decrease in the K(m) value for Prog reduction. We thus propose that Prog is maintained in a new steroid-binding site composed mainly of residues found in the carboxy-terminal region of the protein.

The recent cloning of several human and monkey UDP-glucuronosyltransferase (UGT) 2B proteins has allowed the characterization of these steroid metabolic enzymes. However, relatively little is known about the structure-function relationship, and the potential post-translational modifications of these proteins. The mammalian UGT2B proteins contain at least one consensus asparagine-linked glycosylation site NX(S/T). Endoglycosidase H digestion of the human and monkey UGT2B proteins demonstrates that only UGT2B7, UGT2B15, UGT2B17, and UGT2B20 are glycosylated. Although UGT2B15 and UGT2B20 contain three and four potential glycosylation sites, respectively, site-directed mutagenesis revealed that both proteins are glycosylated at the same first site. In both proteins, abolishing glycosylation decreased glucuronidation activity; however, the K(m) values and the substrate specificities were not affected. Despite the similarities between UGT2B15 and UGT2B20, UGT2B20 is largely more labile than UGT2B15. Treating HK293 cells stably expressing UGT2B20 with cycloheximide for 2 h decreased the enzyme activity by more than 50%, whereas the activity of UGT2B15 remained unchanged after 24 h. The UGT2B20 protein is unique in having an isoleucine at position 96 instead of an arginine as found in all the other UGT2B enzymes. Changing the isoleucine in UGT2B20 to an arginine stabilized enzyme activity, while the reciprocal mutation in UGT2B15 R96I produced a more labile enzyme. Secondary structure predictions of UGT2B proteins revealed a putative alpha-helix in this region in all the human and monkey proteins. This alpha-helix is shortest in UGT2B20; however, the helix is lengthened in UGT2B20 I96R. Thus, it is apparent that the length of the putative alpha-helix between residues 84 and 100 is a determining factor in the stability of UGT2B enzyme activity. This study reveals the extent and importance of protein glycosylation on UGT2B enzyme activity and that the effect of residue 96 on UGT2B enzyme stability is correlated to the length of a putative alpha-helix.

Background The steroid hormone 17β-estradiol is important in the genesis and development of human breast cancer. Its intracellular concentration is regulated by 17β-hydroxysteroid dehydrogenase, which catalyzes the reversible reduction of estrone to 17β-estradiol. This enzyme is thus an important target for inhibitor design. The precise localization and orientation of the substrate and cofactor in the active site is of paramount importance for the design of such inhibitors, and for an understanding of the catalytic mechanism. Results The structure of recombinant human 17β-hydroxysteroid dehydrogenase of type 1 (17β-HSD1) in complex with estradiol at room temperature has been determined at 1.7 a resolution, and a ternary 17βHSD1–estradiol–NADP + complex at −150°C has been solved and refined at 2.20 a resolution. The structures show that estradiol interacts with the enzyme through three hydrogen bonds (involving side chains of Ser142, Tyr155 and His221), and hydrophobic interactions between the core of the steroid and nine other residues. The NADP + molecule binds in an extended conformation, with the nicotinamide ring close to the estradiol molecule. Conclusions From the structure of the complex of the enzyme with the substrate and cofactor of the oxidation reaction, the orientation of the substrates for the reduction reaction can be deduced with confidence. A triangular hydrogen-bond network between Tyr155, Ser142 and O17 from estradiol probably facilitates the deprotonation of the reactive tyrosine, while the conserved Lys159 appears not to be directly involved in catalysis. Both the steroid-binding site and the NADPH-binding site can be proposed as targets for the design of inhibitors.

In our efforts to develop compounds with therapeutic potential as antiandrogens, we synthesized a series of 5alpha-androstane-3alpha,17beta-diol derivatives with a fixed side-chain length of 3-methylenes at C-16alpha, but bearing a diversity of functional groups at the end. Among these, the chloride induced the best antiproliferative activity on androgen-sensitive Shionogi cells. Substituting the OH at C-3 by a methoxy group showed the importance of the OH. Moreover, its transformation into a ketone increased the androgen receptor (AR) binding but decreased the antiproliferative activity and induced a proliferative effect on Shionogi cells. These results confirm the importance of keeping a 5alpha-androstane-3alpha,17beta-diol nucleus instead of a dihydrotestosterone nucleus. Variable side-chain lengths of 2-, 3-, 4-, and 6-methylenes at C-16alpha were investigated and the optimal length was found to be 3-methylenes. Although exhibiting a weak AR binding affinity, 16alpha-(3'-chloropropyl)-5alpha-androstane-3alpha,17beta-diol (15) provided an antiproliferative activity on Shionogi cells similar to that of pure non-steroidal antiandrogen hydroxy-flutamide (77% and 67%, respectively, at 0.1 microM). The new steroidal compound, 15, thus constitutes a good starting point for development of future antiandrogens with a therapeutic potential against prostate cancer.

Very recently, the mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, has been characterized and identified as the unique enzyme able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta4) into epitestosterone (epi-T), the 17alpha-epimer of testosterone. Indeed, the other AKR enzymes that significantly reduce keto groups situated at position C17 of the steroid nucleus, the human type 3 3alpha-HSD (h3alpha-HSD3), the human and mouse type 5 17beta-HSD, and the rabbit 20alpha-HSD, produce only 17beta-hydroxy derivatives, although they possess more than 70% amino acid identity with m17alpha-HSD. Structural comparisons of these highly homologous enzymes thus offer an excellent opportunity of identifying the molecular determinants responsible for their 17alpha/17beta-stereospecificity. Here, we report the crystal structure of the m17alpha-HSD enzyme in its apo-form (1.9 A resolution) as well as those of two different forms of this enzyme in binary complex with NADP(H) (2.9 A and 1.35 A resolution). Interestingly, one of these binary complex structures could represent a conformational intermediate between the apoenzyme and the active binary complex. These structures provide a complete picture of the NADP(H)-enzyme interactions involving the flexible loop B, which can adopt two different conformations upon cofactor binding. Structural comparison with binary complexes of other AKR1C enzymes has also revealed particularities of the interaction between m17alpha-HSD and NADP(H), which explain why it has been possible to crystallize this enzyme in its apo form. Close inspection of the m17alpha-HSD steroid-binding cavity formed upon cofactor binding leads us to hypothesize that the residue at position 24 is of paramount importance for the stereospecificity of the reduction reaction. Mutagenic studies have showed that the m17alpha-HSD(A24Y) mutant exhibited a completely reversed stereospecificity, producing testosterone only from Delta4, whereas the h3alpha-HSD3(Y24A) mutant acquires the capacity to metabolize Delta4 into epi-T.

Background Epi-testosterone (epiT) is the 17α-epimer of testosterone. It has been found at similar level as testosterone in human biological fluids. This steroid has thus been used as a natural internal standard for assessing testosterone abuse in sports. EpiT has been also shown to accumulate in mammary cyst fluid and in human prostate. It was found to possess antiandrogenic activity as well as neuroprotective effects. So far, the exact pathway leading to the formation of epiT has not been elucidated. Results In this report, we describe the isolation and characterization of the enzyme 17α-hydroxysteroid dehydrogenase. The name is given according to its most potent activity. Using cells stably expressing the enzyme, we show that 17α-HSD catalyzes efficienty the transformation of 4-androstenedione (4-dione), dehydroepiandrosterone (DHEA), 5α-androstane-3,17-dione (5α-dione) and androsterone (ADT) into their corresponding 17α-hydroxy-steroids : epiT, 5-androstene-3β,17α-diol (epi5diol), 5α-androstane-17α-ol-3-one (epiDHT) and 5α-androstane-3α,17α-diol (epi3α-diol), respectively. Similar to other members of the aldo-keto reductase family that possess the ability to reduce the keto-group into hydroxyl-group at different position on the steroid nucleus, 17α-HSD could also catalyze the transformation of DHT, 5α-dione, and 5α-pregnane-3,20-dione (DHP) into 3α-diol, ADT and 5α-pregnane-3α-ol-20-one (allopregnanolone) through its less potent 3α-HSD activity. We also have over-expressed the 17α-HSD in Escherichia coli and have purified it by affinity chromatography. The purified enzyme exhibits the same catalytic properties that have been observed with cultured HEK-293 stably transfected cells. Using quantitative Realtime-PCR to study tissue distribution of this enzyme in the mouse, we observed that it is expressed at very high levels in the kidney. Conclusion The present study permits to clarify the biosynthesis pathway of epiT. It also offers the opportunity to study gene regulation and function of this enzyme. Further study in human will allow a better comprehension about the use of epiT in drug abuse testing; it will also help to clarify the importance of its accumulation in breast cyst fluid and prostate, as well as its potential role as natural antiandrogen.

The human ELAC2 gene was the first candidate prostate cancer susceptibility gene identified by linkage analysis and positional cloning. DNA sequence indicates a protein of 826 amino acids encoded by 24 exons. In the present study, we characterized the coding sequence of chimpanzee and gorilla ELAC2 orthologs by direct sequencing of genomic fragments, and of cynomolgus monkey and rat orthologs by screening cDNA libraries. The orthologs characterized in the chimpanzee, gorilla and cynomolgus monkey also encode proteins of 826 amino acids, sharing 98.9%, 98.5% and 93.7% sequence identity with the human protein. Our analyses of the mouse ELAC2 gene identified two alternative mRNA transcripts. One is translated into a protein of 824 a.a. (mouse ELAC2), whereas the other one encodes a protein of 831 amino acids (mouse ELAC2A) resulting from an alternatively spliced form of 25 exons. The rat ELAC2 gene ortholog also expressed two similar alternatively spliced transcripts. These two forms are ubiquitously expressed in mouse and rat tissues. The highest levels of expression of the ELAC2 form are observed in the testis while the lowest levels are seen in the prostate and in the muscle. However, it is of interest to note that the relative abundance of the rat and mouse ELAC2 transcripts, measured by real-time quantitative PCR, is higher than the respective ELAC2A forms in all surveyed tissues except for the prostate and the muscle. The ELAC2A transcript levels are 4.1 to 5.0-fold higher than the ELAC2 levels in the prostate of rat and mouse, respectively. A fine analysis of the conserved domains on the primary structure of ELAC2 orthologs revealed the presence of a putative beta-CASP domain shared by the PSO2 (SNM1) DNA interstrand cross-link repair proteins, and the 73-kDa subunit of mRNA 3' end cleavage and polyadenylation specificity factor (CPSF73) as well as Artemis proteins, thus suggesting a potential interaction of ELAC2 gene product with nucleic acids and more specifically with RNA targets. Taken together, these data offer useful tools to further study the regulation and cellular function of ELAC2 gene in experimental models and provide further insight concerning conserved amino acid motifs that could have biological significance.

Single crystals of human placental 17β-hydroxysteroid dehydrogenase, an enzyme that plays an important role in the interconversion of estrogens, were obtained as the NADP + complex. These are the first crystals suitable for complete X-ray structure analysis ever reported for a steroid-converting enzyme from a human source. The crystals were grown by vapor diffusion at pH 7·5 with polyethyleneglycol (4000) as the precipitating agent. They have a monoclinic space group C 2 and unit cell parameters are a = 123·03 A, b = 45·03 A, c = 61·29 A, and β = 99·1°. A complete set of diffraction data to 2·9 A has been collected on native crystals.

The first crystallographic structure of human type 3 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD3, AKR1C2), an enzyme playing a critical role in steroid hormone metabolism, has been determined in complex with testosterone and NADP at 1.25-A resolution. The enzyme's 17beta-HSD activity was studied in comparison with its 3alpha-HSD activity. The enzyme catalyzes the inactivation of dihydrotestosterone into 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) as well as the transformation of androstenedione into testosterone. Using our homogeneous and highly active enzyme preparation, we have obtained 150-fold higher 3alpha-HSD specificity as compared with the former reports in the literature. Although the rat and the human 3alpha-HSDs share 81% sequence homology, our structure reveals significantly different geometries of the active sites. Substitution of the Ser(222) by a histidine in the human enzyme may compel the steroid to adopt a different binding to that previously described for the rat (Bennett, M. J., Albert, R. H., Jez, J. M., Ma, H., Penning, T. M., and Lewis, M. (1997) Structure 5, 799-T812). Furthermore, we showed that the affinity for the cofactor is higher in the human 3alpha-HSD3 than the rat enzyme due to the presence of additional hydrogen bonds on the adenine moiety and that the cofactor is present under its reduced form in the active site in our preparation.

Progesterone plays an essential role in the maintenance of the pregnancy of most mammals. 20alpha-Hydroxysteroid dehydrogenase (20alpha-HSD) catalyses the inactivation of progesterone into its inactive form, 20alpha-hydroxyprogesterone, and could thus be involved in progesterone withdrawal and in the control of gestation. In this report, the purification and crystallization of recombinant human and rabbit 20alpha-HSDs (h20alpha-HSD and rb20alpha-HSD) are described, two highly homologous enzymes possessing, in addition to their common 20alpha-HSD activity, different activities and substrate specificities. Complete diffraction data sets have been collected for crystals of rb20alpha-HSD in complex with NADP(H) and with either dihydrotestosterone (1.8 A), progesterone (1.7 A) or 4-androstenedione (1.8 A). All these crystals belong to the monoclinic space group P2(1). A partial data set has also been collected for a crystal of h20alpha-HSD (P2(1)2(1)2(1)) in complex with NADP(H) and progesterone.

In androgen-sensitive target tissues, 3 alpha-hydroxysteroid dehydrogenase regulates the androgen receptor (AR) activity by catalyzing the inactivation of 5 alpha-dihydrotestosterone (the most natural potent androgen) to 5 alpha-androstane-3 alpha,17 beta-diol. In this report, the crystallization of a human prostatic type 3 3 alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily, is described. Two different crystal forms of the complex between the human type 3 3 alpha-HSD, NADP(+) and testosterone have been obtained using PEG as precipitant. Crystal form I, which diffracts to 1.6 A, belongs to the monoclinic space group P2(1), with unit-cell parameters a = 55.07, b = 87.15, c = 76.88 A, beta = 107.37 degrees and two subunits in the asymmetric unit. A complete data set has been collected at 1.8 A. Crystal form II, which diffracts to 2.6 A, belongs to the rhombohedral space group R32, with unit-cell parameters a = b = 143.59, c = 205.86 A, alpha = beta = 90, gamma = 120 degrees and two subunits in the asymmetric unit.

Type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD1), a member of the short chain dehydrogenase reductase (SDR) family, is responsible for the synthesis of 17beta-estradiol, the biologically active estrogen involved in the genesis and development of human breast cancers. Here, we report the crystal structures of the H221L 17beta-HSD1 mutant complexed to NADP+ and estradiol and the H221L mutant/NAD+ and a H221Q mutant/estradiol complexes. These structures provide a complete picture of the NADP+-enzyme interactions involving the flexible 191-199 loop (well ordered in the H221L mutant) and suggest that the hydrophobic residues Phe192-Met193 could facilitate hydride transfer. 17beta-HSD1 appears to be unique among the members of the SDR protein family in that one of the two basic residues involved in the charge compensation of the 2'-phosphate does not belong to the Rossmann-fold motif. The remarkable stabilization of the NADP+ 2'-phosphate by the enzyme also clearly establishes its preference for this cofactor relative to NAD+. Analysis of the catalytic properties of, and estradiol binding to, the two mutants suggests that the His221-steroid O3 hydrogen bond plays an important role in substrate specificity.

Estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) plays a pivotal role in the synthesis of estrogens. We overproduced human placental estrogenic 17 beta-HSD using a baculovirus expression system for the study of the enzyme mechanism. A cDNA encoding the entire open reading frame of human 17 beta-HSD was inserted into the genome of Autographa californica nuclear polyhedrosis virus and expressed in Spodoptera frugiperda (Sf9) insect cells. Metabolic labeling and Western blot analysis using polyclonal antibodies raised against native human 17 beta-HSD indicated that a molecule with an apparent mass of 35 kDa was maximally expressed 60 h after infection. At that time interval, intracellular 17 beta-HSD activity reached 0.26 U/mg of protein in crude homogenate, about 70 times the level measured in human placenta. Purification of recombinant 17 beta-HSD was achieved by a single affinity fast liquid protein chromatography step yielding 24 mg of purified 17 beta-HSD protein per liter of suspension culture, with a specific activity of about 8 mumol/min/mg of protein for conversion of estradiol into estrone, at pH 9.2. In addition, the recombinant protein purified from infected Sf9 cells was assembled as a dimer with molecular mass and specific activity identical to those of the enzyme purified directly from placenta. The present data show that the baculovirus expression system can provide active 17 beta-HSD that is functionally identical to its natural counter-part and easy to purify in quantities suitable for its physico-chemical studies.