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Endocrinology Vol. 147, No. 4 1589-1590
Copyright © 2006 by The Endocrine Society

Rat 3{alpha}-Hydroxysteroid Dehydrogenase: To Oxidize or Reduce, that Is the Question

Michael E. Baker

Department of Medicine University of California, San Diego La Jolla, California 92093-0693

Address all correspondence and requests for reprints to: Michael E. Baker, Department of Medicine, 0693, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0693. E-mail: mbaker{at}ucsd.edu.

There is a creative tension between basic and clinical research. Basic research seeks answers to fundamental questions about mechanisms of action, whereas clinical science seeks methods for treating diseases. In the best cases, there is an overlap between these two approaches. A good example comes from research on the regulation of steroid concentrations by enzymes, which are partners with steroid receptors in the physiological response to steroids.

An important insight into this regulatory role for enzymes came from several laboratories studying 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), which acts as a dehydrogenase to catalyze the inactivation of cortisol (1, 2, 3). Thus, in vivo, 11ß-HSD2 functions as a gatekeeper for access of cortisol to the glucocorticoid and mineralocorticoid receptors. In addition to uncovering an enzyme-based mechanism for regulating steroid hormone action, this discovery has important clinical applications for diagnosis of people with genetic disposition for hypertension (3, 4) and elucidating unexpected hormone-like actions of dietary compounds (5).

Research on 11ß-HSD stimulated studies on other enzymes that metabolize steroids. As a result, more than 10 17ß-HSDs have been isolated (6, 7). Like 11ß-HSD2, in vivo, 17ß-HSD2 is primarily a dehydrogenase, catalyzing the conversion of the alcohol at C17 on estradiol and testosterone (T) to a ketone. In contrast, 17ß-HSD1 and 17ß-HSD3 are primarily reductases, catalyzing the conversion of the C17 ketone to an alcohol (8, 9).

11ß-HSDs and most 17ß-HSDs belong to the short-chain dehydrogenase/reductase (SDR) family (10), a large and diverse group of enzymes. However, 17ß-HSD5 belongs to the aldo-keto reductase (AKR) family, another large enzyme family (11) (http://www.med.upenn.edu/akr), which includes other enzymes that regulate steroid hormone access to receptors. One of these AKRs is 3{alpha}-HSD, which reduces 5{alpha}-dihydrotestosterone (DHT) to 3{alpha}-androstanediol (Adiol) while concurrently oxidizing reduced nicotinamide adenine dinucleotide (NAD) phosphate (NADPH) to NAD phosphate (NADP+). In prostate, 3{alpha}-HSD regulates DHT levels. In this issue, Papari-Zareei et al. (12) show that mutation of arginine-276 to glutamic acid changes the preference of rat 3{alpha}-HSD from reduction of DHT to dehydrogenation of Adiol, which would have profound effects on cellular DHT levels. Their work is another example of a mechanistic discovery with important clinical applications, which in this case concerns genetic disposition for androgen-dependent diseases such as prostate cancer.

Papari-Zareei et al. (12) focused on altering the interaction of NADP(H) with rat 3{alpha}-HSD as a way of changing its preference for dehydrogenation of Adiol to DHT in cells. For the most part, cosubstrates have been neglected in studies on metabolism of steroids by SDRs and AKRs. Instead, the focus has been on understanding the interaction between steroid and enzyme with the goal of developing steroid analogs that can regulate steroid hormone levels while ignoring the cosubstrate, which is essential for SDRs and AKRs. Papari-Zareei et al. (12) give cosubstrates well-deserved recognition for their importance in catalysis of steroids by dehydrogenases.

In this respect, Papari-Zareei et al. (12) build on an increasing awareness that the high ratios in cells of NADPH/NADP+ and NAD+/NADH regulate the preference for reduction or oxidation of steroids, respectively, by dehydrogenases. In fact, regeneration of NADPH by enzymes such as hexose-6-phosphate dehydrogenase is crucial for maintaining a ratio of NADPH/NADP+ in cells that will promote reduction of cortisone to cortisol by 11ß-HSD1 (13, 14, 15, 16).

To change the preference of 3{alpha}-HSD to dehydrogenation of Adiol by altering the interaction with NADP(H), several mutants of Arg-276 were constructed. Arg-276 was chosen for mutagenesis because the three-dimensional (3D) structure of rat 3{alpha}-HSD cocrystallized with T and NADP+ shows that Arg-276 has a stabilizing interaction with the 2'-phosphate on NADP(H) (17) (Fig. 1AGo). Papari-Zareei et al. (12) mutated Arg-276 to methionine (R276M), glutamic acid (R276E), and glycine (R276G), each of which has different interactions with oxygens on the 2'-phosphate on NADP+ (Fig. 1BGo). Met-276 fits nicely with NADP+; Gly-276 lacks a side chain that can stabilize the 2'-phosphate, whereasGlu-276 produces a coulombic interaction that repels NADP+ and destabilizes its binding and also stabilizes binding of NAD+ (Fig. 2Go). This change in binding of NADPH and NAD+ by the R276E mutant is what would be expected from the 3D structure, because replacement of the 2'-phosphate with a hydroxyl group eliminates the destabilizing coulombic interaction with the side chain on Glu-276 and adds a stabilizing interaction between Glu-276 and the ribose 2'-hydroxyl group.


Figure 1
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  		FIG. 1. 3D model of rat 3{alpha}-hydroxysteroid dehydrogenase with DHT and NADP+. A, Wild-type rat 3{alpha}-HSD showing the interaction of the C3-alcohol on DHT with Tyr-55 and His-117 and 2'-phosphate of NADP+ with Arg-276; B, overlap of mutant rat 3{alpha}-HSDs R276M, R276E, and R276G with wild-type rat 3{alpha}-HSD. Side chains on methionine and glutamic acid are spatially close to the 2'-phosphate on NADP+. Insight II was used to extract the PDB structure 1AFS of rat 3{alpha}-HSD with T and NADP+ and to replace T with DHT. The Biopolymer software was used to replace Arg-276 with methionine, glutamic acid, and glycine.

 

Figure 2
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  		FIG. 2. 3D model of R276E mutant 3{alpha}-HSD with DHT and NAD+. Glu-276 has stabilizing polar interaction with the ribose 2'-hydroxyl. Insight II was used to convert NADP+ to NAD+.

 
This change has an important physiological outcome because NAD+ is plentiful in cells (15, 16). Thus, R276E mutant 3{alpha}-HSD now functions to catalyze the formation of DHT from Adiol (13). This would be expected to have significant effects on androgen homeostasis in prostate and other tissues. Moreover, a similar mutation in other AKRs, such as 17ß-HSD5, could yield an NAD+-dependent dehydrogenase that would change in vivo metabolism of estrogens, androgens, and progestins (18), with profound clinical consequences.


   Acknowledgments
 
I thank C. Chandsawangbhuwana for help in preparing Figs. 1Go and 2Go.


   Footnotes
 
The author has no conflicts of interest to report.

Abbreviations: Adiol, 3{alpha}-Androstanediol; AKR, aldo-keto reductase; 3D, three-dimensional; DHT, 5{alpha}-dihydrotestosterone; 11ß-HSD2, 11ß-hydroxysteroid dehydrogenase type 2; NAD, nicotinamide adenine dinucleotide; NADP, NAD phosphate; NADPH, reduced NADP; SDR, short-chain dehydrogenase/reductase; T, testosterone.

Received December 29, 2005.

Accepted for publication January 5, 2006.


   References
Top
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