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PDBsum entry 3i4c
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Oxidoreductase
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PDB id
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3i4c
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References listed in PDB file
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Key reference
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Title
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Role of tryptophan 95 in substrate specificity and structural stability of sulfolobus solfataricus alcohol dehydrogenase.
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Authors
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A.Pennacchio,
L.Esposito,
A.Zagari,
M.Rossi,
C.A.Raia.
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Ref.
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Extremophiles, 2009,
13,
751-761.
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PubMed id
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Abstract
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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.
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