Retinoic acid biosynthesis catalyzed by retinal dehydrogenases relies on a rate-limiting conformational transition associated with substrate recognition.
ABSTRACT: Retinoic acid (RA), a metabolite of vitamin A, exerts pleiotropic effects throughout life in vertebrate organisms. Thus, RA action must be tightly regulated through the coordinated action of biosynthetic and degrading enzymes. The last step of retinoic acid biosynthesis is irreversibly catalyzed by the NAD-dependent retinal dehydrogenases (RALDH), which are members of the aldehyde dehydrogenase (ALDH) superfamily. Low intracellular retinal concentrations imply efficient substrate molecular recognition to ensure high affinity and specificity of RALDHs for retinal. This study addresses the molecular basis of retinal recognition in human ALDH1A1 (or RALDH1) and rat ALDH1A2 (or RALDH2), through the comparison of the catalytic behavior of retinal analogs and use of the fluorescence properties of retinol. We show that, in contrast to long chain unsaturated substrates, the rate-limiting step of retinal oxidation by RALDHs is associated with acylation. Use of the fluorescence resonance energy transfer upon retinol interaction with RALDHs provides evidence that retinal recognition occurs in two steps: binding into the substrate access channel, and a slower structural reorganization with a rate constant of the same magnitude as the kcat for retinal oxidation: 0.18 vs. 0.07 and 0.25 vs. 0.1 s(-1) for ALDH1A1 and ALDH1A2, respectively. This suggests that the conformational transition of the RALDH-retinal complex significantly contributes to the rate-limiting step that controls the kinetics of retinal oxidation, as a prerequisite for the formation of a catalytically competent Michaelis complex. This conclusion is consistent with the general notion that structural flexibility within the active site of ALDH enzymes has been shown to be an integral component of catalysis.
Project description:A novel bacterial aldehyde dehydrogenase (ALDH) that converts retinal to retinoic acid was first identified in Bacillus cereus The amino acid sequence of ALDH from B. cereus (BcALDH) was more closely related to mammalian ALDHs than to bacterial ALDHs. This enzyme converted not only small aldehydes to carboxylic acids but also the large aldehyde all-trans-retinal to all-trans-retinoic acid with NAD(P)(+) We newly found that BcALDH and human ALDH (ALDH1A1) could reduce all-trans-retinal to all-trans-retinol with NADPH. The catalytic residues in BcALDH were Glu266 and Cys300, and the cofactor-binding residues were Glu194 and Glu457. The E266A and C300A variants showed no oxidation activity. The E194S and E457V variants showed 15- and 7.5-fold higher catalytic efficiency (kcat/Km) for the reduction of all-trans-retinal than the wild-type enzyme, respectively. The wild-type, E194S variant, and E457V variant enzymes with NAD(+) converted 400 μM all-trans-retinal to 210 μM all-trans-retinoic acid at the same amount for 240 min, while with NADPH, they converted 400 μM all-trans-retinal to 20, 90, and 40 μM all-trans-retinol, respectively. These results indicate that BcALDH and its variants are efficient biocatalysts not only in the conversion of retinal to retinoic acid but also in its conversion to retinol with a cofactor switch and that retinol production can be increased by the variant enzymes. Therefore, BcALDH is a novel bacterial enzyme for the alternative production of retinoic acid and retinol.Although mammalian ALDHs have catalyzed the conversion of retinal to retinoic acid with NAD(P)(+) as a cofactor, a bacterial ALDH involved in the conversion is first characterized. The biotransformation of all-trans-retinal to all-trans-retinoic acid by BcALDH and human ALDH was altered to the biotransformation to all-trans-retinol by a cofactor switch using NADPH. Moreover, the production of all-trans-retinal to all-trans-retinol was changed by mutations at positions 194 and 457 in BcALDH. The alternative biotransformation of retinoids was first performed in the present study. These results will contribute to the biotechnological production of retinoids, including retinoic acid and retinol.
Project description:Retinoic acid (RA), the active metabolite of vitamin A, is required for spermatogenesis and many other biological processes. RA formation requires irreversible oxidation of retinal to RA by aldehyde dehydrogenase enzymes of the 1A family (ALDH1A). While ALDH1A1, ALDH1A2, and ALDH1A3 all form RA, the expression pattern and relative contribution of these enzymes to RA formation in the testis is unknown. In this study, novel methods to measure ALDH1A protein levels and intrinsic RA formation were used to accurately predict RA formation velocities in individual human testis samples and an association between RA formation and intratesticular RA concentrations was observed. The distinct localization of ALDH1A in the testis suggests a specific role for each enzyme in controlling RA formation. ALDH1A1 was found in Sertoli cells, while only ALDH1A2 was found in spermatogonia, spermatids, and spermatocytes. In the absence of cellular retinol binding protein (CRBP)1, ALDH1A1 was predicted to be the main contributor to intratesticular RA formation, but when CRBP1 was present, ALDH1A2 was predicted to be equally important in RA formation as ALDH1A1. This study provides a comprehensive novel methodology to evaluate RA homeostasis in human tissues and provides insight to how the individual ALDH1A enzymes mediate RA concentrations in specific cell types.
Project description:All-trans retinoic acid (atRA), which is mainly generated endogenously via two steps of oxidation from vitamin A (retinol), plays an indispensible role in the development of the kidney and many other organs. Enzymes that catalyze the oxidation of retinol to generate atRA, including aldehyde dehydrogenase 1 family (ALDH1)A1, ALDH1A2 and ALDH1A3, exhibit complex expression patterns at different stages of renal development. However, molecular triggers that control these differential expression levels are poorly understood. In this study, we provide in vitro evidence to demonstrate that Wilms' tumor 1 (WT1) negatively regulates the expression of the atRA synthetic enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, in the 293 cell line, leading to significant blockage of atRA production. Furthermore, we demonstrate that the suppression of ALDH1A1 by WT1 can be markedly attenuated by histone deacetylase inhibitors (HDACis). Taken together, we provide evidence to indicate that WT1 and HDACs are strong regulators of endogenous retinoic acid synthetic enzymes in 293 cells, indicating that they may be involved in the regulation of atRA synthesis.
Project description:In vivo pathways of natural retinoid metabolism and elimination have not been well characterized in primary myeloid cells, even though retinoids and retinoid receptors have been strongly implicated in regulating myeloid maturation. With the use of a upstream activation sequence-GFP reporter transgene and retrovirally expressed Gal4-retinoic acid receptor ? in primary mouse bone marrow cells, we identified 2 distinct enzymatic pathways used by mouse myeloid cells ex vivo to synthesize retinoic acid receptor ? ligands from free vitamin A metabolites (retinyl acetate, retinol, and retinal). Bulk Kit(+) bone marrow progenitor cells use diethylaminobenzaldehyde-sensitive enzymes, whereas bone marrow-derived macrophages use diethylaminobenzaldehyde-insensitive enzymes to synthesize natural retinoic acid receptor ?-activating retinoids (all-trans retinoic acid). Bone marrow-derived macrophages do not express the diethylaminobenzaldehyde-sensitive enzymes Aldh1a1, Aldh1a2, or Aldh1a3 but instead, express Aldh3b1, which we found is capable of diethylaminobenzaldehyde-insensitive synthesis of all trans-retinoic acid. However, under steady-state and stimulated conditions in vivo, diverse bone marrow cells and peritoneal macrophages showed no evidence of intracellular retinoic acid receptor ?-activating retinoids, despite expression of these enzymes and a vitamin A-sufficient diet, suggesting that the enzymatic conversion of retinal is not the rate-limiting step in the synthesis of intracellular retinoic acid receptor ?-activating retinoids in myeloid bone marrow cells and that retinoic acid receptor ? remains in an unliganded configuration during adult hematopoiesis.
Project description:All-trans-retinoic acid (atRA) stimulates neurogenesis, dendritic growth of hippocampal neurons, and higher cognitive functions, such as spatial learning and memory formation. Although astrocyte-derived atRA has been considered a key factor in neurogenesis, little direct evidence identifies hippocampus cell types and the enzymes that biosynthesize atRA. Here we show that primary rat astrocytes, but not neurons, biosynthesize atRA using multiple retinol dehydrogenases (Rdh) of the short chain dehydrogenase/reductase gene family and retinaldehyde dehydrogenases (Raldh). Astrocytes secrete atRA into their medium; neurons sequester atRA. The first step, conversion of retinol into retinal, is rate-limiting. Neurons and astrocytes both synthesize retinyl esters and reduce retinal into retinol. siRNA knockdown indicates that Rdh10, Rdh2 (mRdh1), and Raldh1, -2, and -3 contribute to atRA production. Knockdown of the Rdh Dhrs9 increased atRA synthesis ?40% by increasing Raldh1 expression. Immunocytochemistry revealed cytosolic and nuclear expression of Raldh1 and cytosol and perinuclear expression of Raldh2. atRA autoregulated its concentrations by inducing retinyl ester synthesis via lecithin:retinol acyltransferase and stimulating its catabolism via inducing Cyp26B1. These data show that adult hippocampus astrocytes rely on multiple Rdh and Raldh to provide a paracrine source of atRA to neurons, and atRA regulates its own biosynthesis in astrocytes by directing flux of retinol. Observation of cross-talk between Dhrs9 and Raldh1 provides a novel mechanism of regulating atRA biosynthesis.
Project description:Retinoids, which include vitamin A (retinol) and metabolites such as retinoic acid, can inhibit tumor growth and reverse carcinogenesis in animal models of prostate cancer. We analyzed retinoid signaling and metabolism in the TRAMP (transgenic adenocarcinoma mouse prostate) model. We detected increased retinol and retinyl esters in prostates pooled from 24 to 36 week TRAMP transgenic positive mice compared to nontransgenic littermates by HPLC. We used quantitative RT-PCR to measure transcripts for genes involved in retinoid signaling and metabolism, including ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, LRAT, and RARbeta(2), in prostate tissue from TRAMP positive (+) and age-matched littermate control mice ranging from 18 to 36 weeks. Transcript levels of ALDH1A1, a putative stem cell marker, were decreased in ventral and lateral lobes of prostates from TRAMP mice compared to age-matched, nontransgenic mice. ALDH1A2 (RALDH2) mRNA levels in dorsal and anterior lobes of TRAMP+ mice were lower than in age-matched (24 week) nontransgenic mice. We detected lower RARbeta(2) mRNA levels in dorsal prostate lobes of 36 week TRAMP mice relative to nontransgenic mice. We detected high levels of ALDH1A2 protein in the cytoplasm and nucleus in nontransgenic murine prostate paraffin sections, and lower ALDH1A2 protein levels in all prostate lobes of TRAMP mice compared to nontransgenic mice by immunohistochemistry. We also detected much lower cytoplasmic ALDH1A2 protein levels in all human prostate cancer paraffin sections stained (19 total) relative to normal human prostate tissue on the same sections. Our data indicate that this reduction in ALDH1A2 protein is an early event in human prostate cancer.
Project description:In mammals, all-trans retinoic acid (ATRA) is instrumental to spermatogenesis. It is synthesized by two retinaldehyde dehydrogenases (RALDH) present notably in germ cells (GC). In order to determine the contributions of the GC-derived source of ATRA, we have generated mice lacking all RALDH activities in GC. We show that the GC-derived source of ATRA does not perform any specific role despite contributing to two-third of the total amount of ATRA present in the testis. Overall design: Mice carrying loxP-flanked alleles (L2) of Aldh1a1(tm1Nbg), Aldh1a2 and Aldh1a3(tm1Pcn) were crossed with mice bearing the Tg(Stra8-cre)1Reb transgene. Testis were sampled for analysis at the age of 9 weeks.
Project description:There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been considered a specific inhibitor for ALDH1A1 isoform. In this study, we explore the effects of human ALDH isoenzymes, ALDH1A2 and ALDH2, on drug resistance and proliferation, and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay, Western blot, RT-PCR, and Aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors, DEAB and disulfiram, we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore, our TLDA results revealed that ALDH1, ALDH7, ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition, other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells.
Project description:Locally applied retinol is metabolized to retinoic acid in mouse epidermis in vivo. To characterize the oxidation system we investigated the ability of soluble extracts of hairless-mouse epidermis to convert retinol and retinal into retinoic acid. The extracts oxidized retinol to retinoic acid in two steps catalysed by two NAD+-dependent enzymes that were resolved on h.p.l.c. The first enzyme catalyses the reversible oxidation of retinol to retinal and is an alcohol dehydrogenase isoenzyme. The second enzyme oxidizes retinal to retinoic acid. Retinol oxidation by epidermal extracts was inhibited by the alcohol dehydrogenase inhibitor 4-methylpyrazole and by the polyene citral. The toxicity and relatively low potency at inhibiting the epidermal alcohol dehydrogenase isoenzyme curtailed the use of 4-methylpyrazole in vivo. However, citral significantly inhibited retinoic acid formation from retinol in the epidermis in vivo. The ability to inhibit the oxidation of retinol to retinoic acid in mouse epidermis provides a potential method to resolve the roles of retinol and retinoic acid in epithelial function.
Project description:The intracellular concentration of retinoic acid is determined by two sequential oxidation reactions that convert retinol to retinoic acid. We recently demonstrated that retinoic acid synthesis is significantly impaired in glomeruli of HIV-1 transgenic mice (Tg26), a murine model of HIV-associated nephropathy. This impaired retinoic acid synthesis correlates with reduced renal expression of retinol dehydrogenase 9, which catalyzes the rate-limiting step of retinoic acid synthesis by converting retinol to retinal. Because retinoic acid has renal protective effects and can induce podocyte differentiation, we hypothesized that restoration of retinoic acid synthesis could slow the progression of renal disease. Herein, we demonstrate that overexpression of retinol dehydrogenase 9 in cultured podocytes induces the expression of podocyte differentiation markers. Furthermore, we confirm that podocyte-specific overexpression of retinol dehydrogenase 9 in mice with established kidney disease due to either HIV-associated nephropathy or adriamycin-induced nephropathy decreases proteinuria, attenuates kidney injury, and restores podocyte differentiation markers. Our data suggest that restoration of retinoic acid synthesis could be a new approach to treat kidney disease.