Parole chiave: Alcohol Dehydrogenase, Archaeal Protein, Nicotinamide Adenine Dinucleotide, Recombinant Protein, Tryptophan, Amino Acid Substitution, Archaeal Gene, Article, Chemical Structure, Chemistry, Enzyme Active Site, Enzyme Specificity, Enzyme Stability, Enzymology, Genetics, Kinetics, Metabolism, Protein Conformation, Protein Denaturation, Site Directed Mutagenesis, Spectrofluorometry, Sulfolobus Solfataricus, Temperature, X Ray Crystallography, Catalytic Domain, X-Ray, Hydrogen-Ion Concentration, Models, Molecular, Site-Directed, Spectrometry, Fluorescence, Substrate Specificity,
*** IBB - CNR *** Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, 80131, Naples, Italy.
A mutant of the thermostable NAD(+)-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp -> Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD(+) and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.
173 Records (167 escludendo Abstract e Conferenze). Impact factor totale: 638.873 (610.654 escludendo Abstract e Conferenze). Impact factor a 5 anni totale: 628.562 (595.077 escludendo Abstract e Conferenze).