Estudi estructural i cinètic de l'especificitat de coenzim d'una alcohol deshidrogenasa d'amfibi dependent de NADP(H)

Consultable des del TDXTítol obtingut de la portada digitalitzadaEls teixits gàstrics de l'amfibi Rana perezi expressen una alcohol deshidrogenasa (ADH8) dependent de NADP(H), una especificitat de coenzim única dintre de les ADHs de vertebrat. Aquest enzim és actiu amb etanol i retinoides. Han estat resoltes i refinades les estructures tridimensionals de l'ADH8 i del complex binari ADH8-NADP+ a 2,2 i 1,8 Å, respectivament. ADH8 presenta una identitat de seqüència amb ADHs dependents de NAD(H) de vertebrat inferior al 60%. La seva especificitat de coenzim i de substrat suggereixen un paper d'aquest enzim en la reducció d'aldehids més que en l'oxidació d'alcohols. El gran volum del seti d'unió al substrat pot explicar les altes eficiències catalítiques d'ADH8 amb retinoides. La preferència per el NADP(H) sembla ser deguda a la presència en ADH8 de la triada Gly223-Thr224-His225, i al manteniment dels residus conservats Lys228 i Leu200, que defineixen un seti d'unió per el grup fosfat terminal del cofactor. El NADP(H) s'uneix a l'ADH8 amb una conformació extesa. Aquest coenzim es pot superposar amb el NAD(H) unit en complexos d'ADHs dependents de NAD(H). Les desviacions conformacionals més grans corresponen al grup adenina-ribosa de la molècula. No s'observaren diferències adicionals en el seti d'unió al dinucleòtid, fet que explicaria que el NAD(H) pugui també ser utilitzat com a cofactor per ADH8. En el complex crystal·logràfic ADH8-NADP+, el seti d'unió del grup fosfat extra del coenzim està format per Gly223-Thr224-His225, residus específics de l'ADH8, i per les posicions conservades Leu200 i Lys228. Amb l'objectiu d'investigar els mínims determinants estructurals responsables de l'especificitat de coenzim, diferents mutants d'ADH8 en les posicions 223-225 foren dissenyats i cinèticament caracteritzats. El mutant G223D, tot i presentar una càrrega negativa en el seti d'unió al fosfat, encara preferia el NADP(H) com a coenzim, de la mateixa manera que els mutants T224I i H225N. Les eficiències catalítiques amb NADP(H) disminuiren dramàticament en els dobles mutants, G223D/T224I i T224I/H225N, i en el triple mutant G223D/T224I/H225N (kcat/KmNADPH = 760 mM-1min-1), respecte l'enzim silvestre (kcat/KmNADPH = 133330 mM-1min-1). Aquest resultat fou associat amb una més baixa afinitat per el NADP+ i amb un canvi en l'etapa limitant de la reacció. Contràriament, en el triple mutant, l'eficiència catalítica amb NAD(H) incrementà, assolint valors (kcat/KmNADH = 155000 mM-1min-1) similars als de l'enzim silvestre amb NADP(H). La reversió completa de l'especificitat de coenzim de l'ADH8 doncs, fou aconseguida amb únicament la substitució de tres residus consecutius en el seti d'unió del fosfat extra, un resultat sense precedents dintre de la família ADH.Gastric tissues from amphibian Rana perezi express an alcohol dehydrogenase (ADH8) with a distinct specificity for NADP(H) and active with ethanol and retinoids.The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP+ have been determined and refined to resolutions of 2.2 and 1.8 Å, respectively. ADH8 exhibits less than 60% sequence identity with vertebrate NAD(H)-dependent ADHs. Its coenzyme and substrate specificity support a role in aldehyde reduction rather than in alcohol oxidation. The large volume of the substrate-binding pocket can explain the high catalytic efficiency of ADH8 with retinoids. Preference for NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225, and the recruitment of conserved Lys228 and Leu200, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds ADH8 in an extended conformation and can be superimposed with NAD(H) in NAD(H)-dependent ADH complexes, the largest conformational deviations being for the adenine-ribose moiety of molecule. No additional reshaping of the dinucleotide binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. In the crystallographic ADH8-NADP+ complex, a binding pocket for the extra phosphate group of coenzyme is formed by ADH8-specific residues Gly223-Thr224-His225, and the highly conserved Leu200 and Lys228. In order to investigate the minimal structural determinants for coenzyme specificity, several ADH8 mutants involving residues 223 to 225 were engineered and kinetically characterized. The G223D mutant, having a negative charge in the phosphate-binding site, still preferred NADP(H) over NAD(H), as did the T224I and H225N mutants. Catalytic efficiency with NADP(H) dropped dramatically in the double mutants, G223D/T224I and T224I/H225N, and in the triple mutant, G223D/T224I/H225N (kcat/KmNADPH = 760 mM-1min-1), as compared to the wild-type enzyme (kcat/KmNADPH = 133330 mM-1min-1). This was associated with a lower affinity for NADP+ and a change in the rate-limiting step. Conversely, in the triple mutant, catalytic efficiency with NAD(H) increased, reaching values (kcat/KmNADH = 155000 mM-1min-1) similar to those of the wild-type enzyme with NADP(H). The complete reversal of ADH8 coenzyme specificity was therefore attained by the substitution of only three consecutive residues in the phosphate-binding site, an unprecedented achievement within the ADH family

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

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 Å 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%

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level

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

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 Å 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%