Primary structure of rat liver D-.beta.-hydroxybutyrate dehydrogenase from cDNA and protein analyses: a short-chain alcohol dehydrogenase

We report a mouse cDNA that encodes a 317-amino acid short-chain dehydrogenase which recognizes as substrates 9-cis-retinol, 11-cis-retinol, 5␣-androstan-3␣,17␤-diol, and 5␣-androstan-3␣-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 3␣-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 5␣-androstan-3␣,17␤-diol. These data also illustrate the multifunctional nature of short-chain dehydrogenases and provide a potential mechanism for androgen-retinoid interactions.The retinol (vitamin A) metabolite all-trans-retinoic acid modulates the transcription of multiple genes in diverse cells during embryogenesis and post-natally by activating three retinoic acid receptors, RAR␣, 1 RAR␤, and RAR␥ (1-3). An isomer of all-trans-retinoic acid, 9-cis-retinoic acid also binds with these three receptors with K d values in vitro similar to those of all-trans-retinoic acid. 9-cis-Retinoic acid, but not all-transretinoic acid, activates three other members of the steroid hormone/thyroid hormone/vitamin D/retinoid receptor superfamily, the RXRs ␣, ␤, and ␥ (4 -6). RARs and RXRs function as heterodimers. RXRs also serve as partners for other members of the superfamily, such as thyroid hormone, vitamin D, and peroxisome proliferator-activated receptors, and can modulate gene expression as homodimers (2, 3). This multiplicity of receptors, receptor partners, and ligands suggests a mechanism for the pleiotropic effects of retinoids, but much depends on the loci of all-trans-retinoic acid and 9-cis-retinoic acid biosynthesis. Even though 9-cis-retinoic acid occurs in vivo, and has been identified as an endogenous ligand of RXR, its concentrations are lower than all-trans-retinoic acid, and it has been found only in a few tissues to date, compared with the ubiquitous distribution of all-trans-retinoic acid (7,8). Rapid conversion in vivo into the receptor inactive isomer 9,13-di-cis-RA most likely limits 9-cis-retinoic acid concentrations and effects (9, 10).All-trans-retinoic acid undergoes isomerization in cultured cells...

Section: Cis-retinol/androgen Short-chain Dehydrogenasementioning

confidence: 99%

“…The rat liver D-␤-hydroxybutyrate dehydrogenase (35), the RoDH isozymes, and CRAD represent SDRs with six cysteine residues, whereas bovine 11-cis-RoDH has seven (28,29).…”

Section: Cis-retinol/androgen Short-chain Dehydrogenasementioning

confidence: 99%

cDNA Cloning and Characterization of a cis-Retinol/3α-Hydroxysterol Short-chain Dehydrogenase

Chai

Zhai

Napoli

1997

“…Mitochondrial R-␤-hydroxybutyrate dehydrogenase (BDH) 2 has been extensively studied (1)(2)(3)(4)(5) and constitutes a paradigm of lipid regulation of enzymatic function; however, a crystallographic structural characterization has not been achieved to date. The enzyme belongs to the short chain dehydrogenase/reductase superfamily (6 -8), an evolutionarily conserved family of oxidoreductases found in all forms of life.…”

mentioning

confidence: 99%

Characterization of Human DHRS6, an Orphan Short Chain Dehydrogenase/Reductase Enzyme

Guo

Lukacik

Papagrigoriou

et al. 2006

Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD ؉ -dependent conversion of (R)-hydroxybutyrate to acetoacetate with K m values of about 10 mM, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 Å in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg 144 , Arg 188 , and Arg 205 ) was found to bind a sulfate molecule at the active site. Docking analysis of R-␤-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.Hepatic ketone body formation and utilization of these compounds by peripheral tissues with high energy demands is essential in humans and other mammals for survival during times of starvation and extended fasting. The compounds categorized as ketone bodies comprise acetoacetate, R-␤-hydroxybutyrate, and acetone, the last being a nonmetabolizable decarboxylation product of acetoacetate. The liver produces and excretes ketone bodies during times when the amount of acetyl-CoA exceeds the oxidative capacity of hepatic mitochondria. Ketone body formation is thus necessary to maintain ␤-oxidation by supplying free CoA. This metabolic situation occurs when high amounts of fatty acids derived from peripheral tissues during starvation or fasting are oxidized by ␤-oxidation pathways. In exacerbated disease states, such as ketoacidotic coma, extremely high amounts of fatty acids are oxidized as a consequence of insulin resistance in diabetes mellitus. In this situation, a potentially life-threatening metabolic acidosis occurs through the high amounts of protons provided by acetoacetate and R-␤-hydroxybutyrate, exceeding the serum bicarbonate buffer capacity. Serum levels of 5-10 mM ketone bodies are reached under these circumstances as compared with levels of 1 mM in normal states. The liver synthesizes acetoacetate through the enzymes thiolase, hydroxymethylglutaryl-CoA synthase, and hydroxymethylglutaryl-CoA lyase from three molecules of acetyl-CoA, thus producing 1 mol of acetoacetate, 1 mol of acetyl-CoA, and 2 mol of free CoA. Acetoacetate is further reduced to R-␤-hydroxybutyrate through mito...

“…These include the residues typical of an shortchain dehydrogenase/reductase: the G(X) 3 GXG cofactor binding site (Gly 36 ), N-terminal to the active site; the sequence LXNNAG (Leu 109 ); and the active site Y(X) 3 K (Tyr 176 ), C-terminal to the cofactor binding site. One of the two substitutions, D107W, represents a common substitution in short-chain dehydrogenase/reductase; the second, A191R, also occurs in the short-chain dehydrogenase/reductases 11␤-hydroxysteroid and D-␤-hydroxybutyrate dehydrogenases of rat (23,24). RoDH(II) differs from RoDH(I) in 57 amino acid residues; most are nonconservative substitutions.…”

Section: Resultsmentioning

confidence: 99%

Cloning of a cDNA for a Second Retinol Dehydrogenase Type II

Chai¹,

Zhai²,

Popescu³

et al. 1995

A retinol dehydrogenase, RoDH(1), which recognizes holo-cellular retinol-binding protein (CRBP) as substrate, has been cloned, expressed, and identified as a short-chain dehydrogenase/reductase (Chai, X., Boerman, M. H. E. M., Zhai, Y., and Napoli, J. L. (1995) J. Biol. Chem. 270, 3900-3904). This work reports the cloning and expression of a cDNA encoding a RoDH isozyme, RoDH(II). The predicted amino acid sequence verifies RoDH(II) as a short-chain dehydrogenase/reductase, 82% identical with RoDH(I). RoDH(II) recognized the physiological form of retinol as substrate, CRBP, with a Km of 2 mM. Similar to microsomal RoDH and RoDH(I), RoDH(II) had higher activity with NADP rather than NAD, was stimulated by ethanol and phosphatidyl choline, was not inhibited by the medium-chain alcohol dehydrogenase inhibitor 4-methylpyrazole, but was inhibited by phenylarsine oxide and the short-chain dehydrogenase/reductase inhibitor carbenoxolone. Northern blot analysis detected RoDH(I) and RoDH(II) mRNA only in rat liver, but RNase protection assays revealed RoDH(I) and RoHD(II) mRNA in kidney, lung, testis, and brain. These data indicate that short-chain dehydrogenases/reductase isozymes expressed tissue-distinctively catalyze the first step of retinoic acid biogenesis from the physiologically most abundant substrate, CRBP.