3I4C

Crystal structure of Sulfolobus Solfataricus ADH(SsADH) double mutant (W95L,N249Y)

Summary for 3I4C

• Entry DOI: 10.2210/pdb3i4c/pdb
• Related: 1JVB 1NTO 1NVG 1R37
• Descriptor: NAD-dependent alcohol dehydrogenase, ZINC ION (3 entities in total)
• Functional Keywords: archaeon, zinc, nad(h)-dependent, mutant, oxidoreductase, metal-binding, methylation, nad
• Biological source: Sulfolobus solfataricus
• Total number of polymer chains: 6
• Total formula weight: 226334.17

Authors

Esposito, L.,Pennacchio, A.,Zagari, A.,Rossi, M.,Raia, C.A. (deposition date: 2009-07-01, release date: 2009-07-21, Last modification date: 2023-11-01)

Primary citation

Pennacchio, A.,Esposito, L.,Zagari, A.,Rossi, M.,Raia, C.A.
Role of Tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase
Extremophiles, 13:751-761, 2009

PubMed Abstract: 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.

PubMed: 19588068
DOI: 10.1007/s00792-009-0256-0

Experimental method

X-RAY DIFFRACTION (2 Å)