Role of tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase.
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 A 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.
Pennacchio
Angela
A
Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, 80131, Naples, Italy.
Esposito
Luciana
L
Zagari
Adriana
A
Rossi
Mosè
M
Raia
Carlo A
CA
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D019766:Genes, Archaeal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D009243:NAD; D014364:Tryptophan; D000426:Alcohol Dehydrogenase