Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

Abstract

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.

Figure 1. HPLC chromatogram showing LRAT activity

An esterification assay for microsomal LRAT was performed with 2 μM all-trans-retinol (A) or 2 μM holoCRBPI (B) as a substrate and 6.2 μg of microsomes containing LRAT. AU, arbitrary units.

Figure 2. HPLC chromatograms showing MDR and SDR activities with all-trans-retinol and holoCRBPI

Activities were measured with all-trans-retinol, in the free form (A) and bound as holoCRBPI (B–F), using different enzymes: (A) and (B), ADH4; (C), AKR1B10; (D), RDH11; (E), RoDH-4; and (F), RDH5. Different isomer peaks could be identified: 1, 13-cis-retinaldehyde; 2, 9-cis-retinaldehyde; 3, -all-trans-retinaldehyde; 4, 13-cis-retinol; 5, 9-cis-retinol; 6, -all-trans-retinol. AU, arbitrary units.

Figure 3. Retinoid metabolism in primary cultures of (A) human ASMC and (B) HEK-293 cells stably transfected with RoDH-4

Scheme 1. Cellular retinoid metabolism

Levels of CRBPI, LRAT and oxidoreductases influence the retinoid flow either towards the storage pathway or towards retinoic acid synthesis. REH, retinyl ester hydrolase; ALDH, aldehyde dehydrogenase; CYP, cytochrome P450.