Crystallographic Studies of Retinal Dehydrogenase Type II. Audrey L. Lamb, Xianshu Wang, Joseph L. Napoli and Marica E. Newcomer, Department of Biochemistry, Vanderbilt University, Nashville, TN 37232
Metabolism, differentiation, reproduction, embryogenesis, hematopoesis, bone development, and vision are all dependent on vitamin A (retinol). For all but the visual processes, the biologically active form of the vitamin is retinoic acid. In vivo, retinoic acid is produced from retinol in a two step process: retinol is oxidized to retinal which is in turn oxidized to retinoic acid. The latter step of this process is catalyzed in mouse embryonic eye, mouse embryonic trunk/spinal cord and rat testis by retinal dehydrogenase type II. Xhao and colleagues have suggested that retinal dehydrogenase type II localization and the subsequent production of retinoic acid by the enzyme allows for nested gene activation and pattern formation in the early embryo. Retinal dehydrogenase type II belongs to the superfamily of aldehyde dehydrogenases. This superfamily is subdivided into three classes. Class I is comprised of the tetrameric non-specific cytosolic isozymes (subunit molecular weight 55kD). The isozymes of class II are the same size and quaternary structure as the class I enzymes but are mitochondrial. Class III aldehyde dehydrogenases are smaller, dimeric and primarily tumor-specific, inducible or found in the endoplasmic reticulum. Classes I and II isoenzymes share approximately 70% sequence identity. However, within a class, these enzymes are approximately 95% identical. Retinal dehydrogenase type II is a homotetramer of 55kd monomers which is approximately 70% identical to both the class I or class II enzymes. Crystals of retinal dehydrogenase type II are grown in the presence of sodium acetate and sodium chloride. The protein was very sensitive to oxidation and preventative measures were taken limit oxygen exposure. A single RalDH2 crystal was used to collect a data set to 2.7 Å (92.2% complete, Rmerge=8.8%). The data was processed in the space group P2 with cell dimensions of a=85 , b=150 , c=110 , (=110ø. The volume of the asymmetric unit is 660,000 3. If a tetramer comprises the asymmetric unit, this yields a Vm value of 2.97 Å 3/dalton and a probable solvent content of 59%. Heavy atom derivatives are being scanned.