Publications

• cDNA cloning and characterization of a cis-retinol/3alpha-hydroxysterol short-chain dehydrogenase.

  Chai X., Zhai Y., Napoli J.L.

  We report a mouse cDNA that encodes a 317-amino acid short-chain dehydrogenase which recognizes as substrates 9-cis-retinol, 11-cis-retinol, 5alpha-androstan-3alpha,17beta-diol, and 5alpha-androstan-3alpha-ol-17-one. This cis-retinol/androgen dehydrogenase (CRAD) shares closest amino acid similari ... >> More

  We report a mouse cDNA that encodes a 317-amino acid short-chain dehydrogenase which recognizes as substrates 9-cis-retinol, 11-cis-retinol, 5alpha-androstan-3alpha,17beta-diol, and 5alpha-androstan-3alpha-ol-17-one. This cis-retinol/androgen dehydrogenase (CRAD) shares closest amino acid similarity with mouse retinol dehydrogenase isozymes types 1 and 2 (86 and 91%, respectively). Recombinant CRAD uses NAD+ as its preferred cofactor and exhibits cooperative kinetics for cis-retinoids, but Michaelis-Menten kinetics for 3alpha-hydroxysterols. Unlike recombinant retinol dehydrogenase isozymes, recombinant CRAD was inhibited by 4-methylpyrazole, was not stimulated by ethanol, and did not require phosphatidylcholine for optimal activity. CRAD mRNA was expressed intensely in kidney and liver, in contrast to retinol dehydrogenase isozymes, which show strong mRNA expression only in liver. CRAD mRNA expression was widespread (relative abundance): kidney (100) > liver (92) > small intestine (9) = heart (9) > retinal pigment epithelium and sclera (4.5) > brain (2) > retina and vitreous (1.6) > spleen (0.7) > testis (0.6) > lung (0.4). CRAD may catalyze the first step in an enzymatic pathway from 9-cis-retinol to generate the retinoid X receptor ligand 9-cis-retinoic acid and/or may regenerate dihydrotestosterone from its catabolite 5alpha-androstan-3alpha,17beta-diol. These data also illustrate the multifunctional nature of short-chain dehydrogenases and provide a potential mechanism for androgen-retinoid interactions. << Less

  J. Biol. Chem. 272:33125-33131(1997) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Characterization of a novel type of human microsomal 3alpha-hydroxysteroid dehydrogenase. Unique tissue distribution and catalytic properties.

  Chetyrkin S.V., Belyaeva O.V., Gough W.H., Kedishvili N.Y.

  We report characterization of a novel member of the short chain dehydrogenase/reductase superfamily. The 1513-base pair cDNA encodes a 319-amino acid protein. The corresponding gene spans over 26 kilobase pairs on chromosome 2 and contains five exons. The recombinant protein produced using the bac ... >> More

  We report characterization of a novel member of the short chain dehydrogenase/reductase superfamily. The 1513-base pair cDNA encodes a 319-amino acid protein. The corresponding gene spans over 26 kilobase pairs on chromosome 2 and contains five exons. The recombinant protein produced using the baculovirus system is localized in the microsomal fraction of Sf9 cells and is an integral membrane protein with cytosolic orientation of its catalytic domain. The enzyme exhibits an oxidoreductase activity toward hydroxysteroids with NAD(+) and NADH as the preferred cofactors. The enzyme is most efficient as a 3alpha-hydroxysteroid dehydrogenase, converting 3alpha-tetrahydroprogesterone (allopregnanolone) to dihydroprogesterone and 3alpha-androstanediol to dihydrotestosterone with similar catalytic efficiency (V(max) values of 13-14 nmol/min/mg microsomal protein and K(m) values of 5-7 microm). Despite approximately 44-47% sequence identity with retinol/3alpha-hydroxysterol dehydrogenases, the enzyme is not active toward retinols. The corresponding message is abundant in human trachea and is present at lower levels in the spinal cord, bone marrow, brain, heart, colon, testis, placenta, lung, and lymph node. Thus, the new short chain dehydrogenase represents a novel type of microsomal NAD(+)-dependent 3alpha-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. << Less

  J. Biol. Chem. 276:22278-22286(2001) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Further characterization of human microsomal 3alpha-hydroxysteroid dehydrogenase.

  Chetyrkin S.V., Hu J., Gough W.H., Dumaual N., Kedishvili N.Y.

  This manuscript reports further characterization of the recently discovered human short-chain alcohol dehydrogenase, proposed to oxidize 3alpha-androstanediol to dihydrotestosterone in testis and prostate (M. G. Biswas and D. W. Russell, 1997, J. Biol. Chem. 272, 15959-15966). Enzyme expressed usi ... >> More

  This manuscript reports further characterization of the recently discovered human short-chain alcohol dehydrogenase, proposed to oxidize 3alpha-androstanediol to dihydrotestosterone in testis and prostate (M. G. Biswas and D. W. Russell, 1997, J. Biol. Chem. 272, 15959-15966). Enzyme expressed using the Baculovirus System localized in the microsomal fraction and catalyzed oxidation and reduction of the functional groups on steroids at carbons 3 and 17. Autoradiography assays revealed that the enzyme was most efficient as a 3alpha-hydroxysteroid oxidoreductase. High affinity of the enzyme for NADH (Km of 0.18 microM), lack of stereospecificity in the reductive direction, and poor efficiency for 3beta-versus 3alpha-hydroxyl oxidation could account for the observed transient accumulation of 3beta-stereoisomers in the oxidative reaction. Consistent with the 65% sequence identity with RoDH dehydrogenases, the enzyme oxidized all-trans-retinol with the Km value of 3.2 microM and Vmax value of 1.2 nmol/min per milligram microsomes. 13-cis-Retinol and all-trans-retinol bound to the cellular retinol-binding protein were not substrates. Neurosteroid allopregnanolone was a better substrate than all-trans-retinol with the Km and Vmax values of 0.24 microM and 14.7 nmol/min per milligram microsomes. Northern blot analysis revealed that the corresponding mRNA was present in adult human brain (caudate nucleus, amygdala, hippocampus, substantia nigra, thalamus) and spinal cord in addition to other tissues. The message was also detected in fetal lung, liver, and brain. Antibodies against the enzyme recognized a protein of approximately 35 kDa in the particulate fraction of human tissues. This study presents new information about enzymatic properties, substrate specificity, and tissue distribution of this enzyme, and provides a better insight into its possible physiological function(s). << Less

  Arch. Biochem. Biophys. 386:1-10(2001) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis.

  Yang J.K., Park M.S., Waldo G.S., Suh S.W.

  One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form. We have applied the directed evolution technique and prepared soluble mutants of the Mycobacterium tuberculosis Rv2002 gene product, the wild type of which had been expressed as ... >> More

  One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form. We have applied the directed evolution technique and prepared soluble mutants of the Mycobacterium tuberculosis Rv2002 gene product, the wild type of which had been expressed as inclusion bodies in Escherichia coli. A triple mutant I6TV47MT69K (Rv2002-M3) was chosen for structural and functional characterizations. Enzymatic assays indicate that the Rv2002-M3 protein has a high catalytic activity as a NADH-dependent 3alpha, 20beta-hydroxysteroid dehydrogenase. We have determined the crystal structures of a binary complex with NAD(+) and a ternary complex with androsterone and NADH. The structure reveals that Asp-38 determines the cofactor specificity. The catalytic site includes the triad Ser-140Tyr-153Lys-157. Additionally, it has an unusual feature, Glu-142. Enzymatic assays of the E142A mutant of Rv2002-M3 indicate that Glu-142 reverses the effect of Lys-157 in influencing the pKa of Tyr-153. This study suggests that the Rv2002 gene product is a unique member of the SDR family and is likely to be involved in steroid metabolism in M. tuberculosis. Our work demonstrates the power of the directed evolution technique as a general way of overcoming the difficulties in overexpressing the target proteins in soluble form. << Less

  Proc. Natl. Acad. Sci. U.S.A. 100:455-460(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Human 3alpha-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones.

  Penning T.M., Burczynski M.E., Jez J.M., Hung C.F., Lin H.K., Ma H., Moore M., Palackal N., Ratnam K.

  The kinetic parameters, steroid substrate specificity and identities of reaction products were determined for four homogeneous recombinant human 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) isoforms of the aldo-keto reductase (AKR) superfamily. The enzymes correspond to type 1 3alpha-HSD (AKR1 ... >> More

  The kinetic parameters, steroid substrate specificity and identities of reaction products were determined for four homogeneous recombinant human 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) isoforms of the aldo-keto reductase (AKR) superfamily. The enzymes correspond to type 1 3alpha-HSD (AKR1C4), type 2 3alpha(17beta)-HSD (AKR1C3), type 3 3alpha-HSD (AKR1C2) and 20alpha(3alpha)-HSD (AKR1C1), and share at least 84% amino acid sequence identity. All enzymes acted as NAD(P)(H)-dependent 3-, 17- and 20-ketosteroid reductases and as 3alpha-, 17beta- and 20alpha-hydroxysteroid oxidases. The functional plasticity of these isoforms highlights their ability to modulate the levels of active androgens, oestrogens and progestins. Salient features were that AKR1C4 was the most catalytically efficient, with k(cat)/K(m) values for substrates that exceeded those obtained with other isoforms by 10-30-fold. In the reduction direction, all isoforms inactivated 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one; 5alpha-DHT) to yield 5alpha-androstane-3alpha,17beta-diol (3alpha-androstanediol). However, only AKR1C3 reduced Delta(4)-androstene-3,17-dione to produce significant amounts of testosterone. All isoforms reduced oestrone to 17beta-oestradiol, and progesterone to 20alpha-hydroxy-pregn-4-ene-3,20-dione (20alpha-hydroxyprogesterone). In the oxidation direction, only AKR1C2 converted 3alpha-androstanediol to the active hormone 5alpha-DHT. AKR1C3 and AKR1C4 oxidized testosterone to Delta(4)-androstene-3,17-dione. All isoforms oxidized 17beta-oestradiol to oestrone, and 20alpha-hydroxyprogesterone to progesterone. Discrete tissue distribution of these AKR1C enzymes was observed using isoform-specific reverse transcriptase-PCR. AKR1C4 was virtually liver-specific and its high k(cat)/K(m) allows this enzyme to form 5alpha/5beta-tetrahydrosteroids robustly. AKR1C3 was most prominent in the prostate and mammary glands. The ability of AKR1C3 to interconvert testosterone with Delta(4)-androstene-3,17-dione, but to inactivate 5alpha-DHT, is consistent with this enzyme eliminating active androgens from the prostate. In the mammary gland, AKR1C3 will convert Delta(4)-androstene-3,17-dione to testosterone (a substrate aromatizable to 17beta-oestradiol), oestrone to 17beta-oestradiol, and progesterone to 20alpha-hydroxyprogesterone, and this concerted reductive activity may yield a pro-oesterogenic state. AKR1C3 is also the dominant form in the uterus and is responsible for the synthesis of 3alpha-androstanediol which has been implicated as a parturition hormone. The major isoforms in the brain, capable of synthesizing anxiolytic steroids, are AKR1C1 and AKR1C2. These studies are in stark contrast with those in rat where only a single AKR with positional- and stereo-specificity for 3alpha-hydroxysteroids exists. << Less

  Biochem. J. 351:67-77(2000) [PubMed] [EuropePMC]

  This publication is cited by 15 other entries.

• Mouse 17alpha-hydroxysteroid dehydrogenase (AKR1C21) binds steroids differently from other aldo-keto reductases: identification and characterization of amino acid residues critical for substrate binding.

  Faucher F., Cantin L., Pereira de Jesus-Tran K., Lemieux M., Luu-The V., Labrie F., Breton R.

  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 testoste ... >> More

  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. << Less

  J. Mol. Biol. 369:525-540(2007) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Studies on a Tyr residue critical for the binding of coenzyme and substrate in mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21): structure of the Y224D mutant enzyme.

  Dhagat U., Endo S., Mamiya H., Hara A., El-Kabbani O.

  Mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) is the only aldo-keto reductase that catalyzes the stereospecific reduction of 3- and 17-ketosteroids to the corresponding 3(17)alpha-hydroxysteroids. The Y224D mutation of AKR1C21 reduced the K(m) value for NADP(H) by up to 80-fold and compl ... >> More

  Mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) is the only aldo-keto reductase that catalyzes the stereospecific reduction of 3- and 17-ketosteroids to the corresponding 3(17)alpha-hydroxysteroids. The Y224D mutation of AKR1C21 reduced the K(m) value for NADP(H) by up to 80-fold and completely reversed the 17alpha stereospecificity of the enzyme. The crystal structure of the Y224D mutant at 2.3 A resolution revealed that the mutation resulted in a change in the conformation of the flexible loop B, including the V-shaped groove, which is a unique feature of the active-site architecture of wild-type AKR1C21 and is formed by the side chains of Tyr224 and Trp227. Furthermore, mutations (Y224F and Q222N) of residues involved in forming the safety belt for binding of the coenzyme showed similar alterations in kinetic constants for 3alpha-hydroxy/3-ketosteroids and 17-hydroxy/ketosteroids compared with the wild type. << Less

  Acta Crystallogr. D 66:198-204(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of two isoforms of mouse 3(17)alpha-hydroxysteroid dehydrogenases of the aldo-keto reductase family.

  Ishikura S., Usami N., Nakajima S., Shiraishi H., El-Kabbani O., Hara A.

  Mouse kidney contains two 3(17)alpha-hydroxysteroid dehydrogenases (HSDs) that show essentially the same properties except for their isoelectric points. However, the structural differences and physiological roles of the two enzymes remain unknown. In this study, we have isolated cDNAs for the two ... >> More

  Mouse kidney contains two 3(17)alpha-hydroxysteroid dehydrogenases (HSDs) that show essentially the same properties except for their isoelectric points. However, the structural differences and physiological roles of the two enzymes remain unknown. In this study, we have isolated cDNAs for the two 3(17)alpha-HSDs from a total RNA sample of mouse kidney by reverse transcription-PCR. The identity of the cDNAs was confirmed by characterization of the recombinant enzymes that showed the same molecular weights, pI values, pH optima, substrate specificity and inhibitor sensitivity as those of the enzymes from mouse kidney. We also found that the recombinant enzymes reduce precursors of neuroactive progesterone derivatives, 5alpha-dihydrotestoserone, deoxycorticosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate and estrone at low Km values of 0.3-2 microM. The two enzymes belonged to the aldo-keto reductase (AKR) family, and their 323-amino acid sequences differed only by five amino acids. The sequences of the two isoforms are identical to those of proteins that are predicted to be encoded in a gene for AKR1C21 in the database of the mouse genome. However, the mRNAs for the two isoforms were expressed in mouse kidney and other tissues, in which their expression levels were different. The results indicate an important role of 3(17)alpha-HSD in controlling the concentrations of various steroid hormones in the mouse tissues, and suggest the existence of two genes for the two isoforms of the enzyme. << Less

  Biol. Pharm. Bull. 27:1939-1945(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.