Crystallization and preliminary X-ray analysis of NADP(H)-dependent alcohol dehydrogenases fromSaccharomyces cerevisiaeandRana 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-speci®city 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 A Ê resolution and belonged to the trigonal space group P3 1 21 (or to its enantiomorph P3 2 21), with unit-cell parameters a = b = 102.2, c = 149.7 A Ê , = 120 . These crystals were produced by the hangingdrop 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 A Ê , respectively, using synchrotron radiation. These crystals were isomorphous, space group C2, with approximate unitcell parameters a = 122, b = 79, c = 91 A Ê , = 113 and contain one dimer per asymmetric unit, with a volume solvent content of about 50%.

Complete Reversal of Coenzyme Specificity by Concerted Mutation of Three Consecutive Residues in Alcohol Dehydrogenase

“…All reactions were performed in a DNA thermal cycler (MJ Research) with High Fidelity DNA polymerase (Roche Diagnostics) under the following conditions: hot start at 95°C for 1 min, annealing at 55°C for 1 min, extension at 68°C for 12 min (11 cycles to step 2), and a final extension step. All PCR products were purified and cloned into pGEX 4T-2 as for wild-type cDNA (14).…”

“…Protein Expression and Purification-All mutants were expressed using the conditions previously described for the wild-type enzyme (14). Batch-wise purification of wild-type and T224I enzymes followed identical procedures (14).…”

“…Batch-wise purification of wild-type and T224I enzymes followed identical procedures (14). For G223D, H225N, G223D/T224I, T224I/H225N, and G223D/T224I/H225N mutants, the only difference was the matrix used in the last affinity chromatography step (Cibacron Blue 3-GA; Sigma).…”

Apo and Holo Structures of an NADP(H)-dependent Cinnamyl Alcohol Dehydrogenase from Saccharomyces cerevisiae

“…ScAdh6p was purified by the sequential use of DEAE-Sepharose, Red-Sepharose and, finally, by gel-filtration HPLC to eliminate the NADP þ used to elute the enzyme from the affinity column. 10 Samples of ScAdh6p were used, as reported previously, 33 in crystallization screenings with the hanging-drop, vapour-diffusion method, producing trigonal crystals ( Table 1). The best-diffracting crystals were obtained in about two days at room temperature with 1 ml of a 10 mg/ml protein solution and 1 ml of the Table 1).…”

Merging protein, gene and genomic data: the evolution of the MDR-ADH family

“…Although initially ascribed to class IV on the basis of its substrate specificity and gastric localization, as mammalian class IV forms, the three-dimensional structure (Rosell et al, 2003b;Valencia et al, 2003), in vitro behaviour -more as a retinal reductase than alcohol dehydrogenase -and its unique preference among vertebrate ADHs for NADP þ rather than NAD þ , all suggested a separate class, named class VIII. It has been proposed that the triad Gly223-Thr224-His225, together with Leu200 and Lys228, may account for the cofactor preference, also verified by sitedirected mutagenesis at the triad segment (Rosell et al, 2003a).…”