Crystallization and preliminary X-ray analysis of NADP(H)-dependent alcohol dehydrogenases from Saccharomyces cerevisiae and Rana perezi

Abstract

Different crystal forms diffracting to high resolution have been obtained for two NADP(H)-dependent alcohol dehydrogenases, members of the medium-chain dehydrogenase/reductase superfamily: ScADHVI from Saccharomyces cerevisiae and ADH8 from Rana perezi. ScADHVI is a broad-specificity enzyme, with a sequence identity lower than 25% with respect to all other ADHs of known structure. The best crystals of ScADHVI diffracted beyond 2.8 resolution and belonged to the trigonal space group P3121 (or to its enantiomorph P3221), with unit-cell parameters a = b = 102.2, c = 149.7 Å, γ = 120°. These crystals were produced by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. Packing considerations together with the self-rotation function and the native Patterson map seem to indicate the presence of only one subunit per asymmetric unit, with a volume solvent content of about 80%. ADH8 from R. perezi is the only NADP(H)-dependent ADH from vertebrates characterized to date. Crystals of ADH8 obtained both in the absence and in the presence of NADP+ using polyethylene glycol and lithium sulfate as precipitants diffracted to 2.2 and 1.8 Å, respectively, using synchrotron radiation. These crystals were isomorphous, space group C2, with approximate unit-cell parameters a = 122, b = 79, c = 91 Å, β = 113° and contain one dimer per asymmetric unit, with a volume solvent content of about 50%.