ENZYMATIC CHARACTERISTICS OF RETINAL DEHYDROGENASE TYPE-I EXPRESSED IN ESCHERICHIA-COLI

We expressed RalDH(I) in Escherichia coli and have shown that it funct ions in vitro with the complex CRBP-retinal (cellular retinol-binding protein) as substrate, either generated in situ from the complex CRBP- retinol and microsomal retinol dehydrogenases or provided directly as CRBP-retinal. Recombinant RalDH(I) had kinetic constants with CRBP-ret inal of: Hill coefficient 1.8; K-0.5, 0.8 mu M; and V-m 1.5 nmol/min/m g of protein at 25 degrees C. Apo-CRBP inhibited the reaction with CRB P-retinal with an IC50 of 1.4 mu M. Citral inhibited RalDH(I) with an IC50 of similar to 1 mu M compared to an IC50 of similar to 12 mu M fo r RalDH(II), but did not serve as substrate for RalDH(I). RalDH(I) did not catalyze efficiently the dehydrogenation of acetaldehyde, but sho wed higher V-max/K-m values for hexanal, octanal, decanal and benzalde hyde than for either propanal or retinal. These data extend the charac terization of RalDH(I), show that apo-CRBP competes with holo-CRBP as substrate for RalDH(I), and expand insight into the pathways of retino ic acid biogenesis from the most abundant substrates in vivo, retinoid -liganded CRBP. (C) 1997 Elsevier Science B.V.