Document Detail


Role of tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase.
MedLine Citation:
PMID:  19588068     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Angela Pennacchio; Luciana Esposito; Adriana Zagari; Mosè Rossi; Carlo A Raia
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2009-07-09
Journal Detail:
Title:  Extremophiles : life under extreme conditions     Volume:  13     ISSN:  1433-4909     ISO Abbreviation:  Extremophiles     Publication Date:  2009 Sep 
Date Detail:
Created Date:  2009-09-03     Completed Date:  2009-11-16     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9706854     Medline TA:  Extremophiles     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  751-61     Citation Subset:  IM; S    
Affiliation:
Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, 80131, Naples, Italy.
Data Bank Information
Bank Name/Acc. No.:
PDB/3I4C
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MeSH Terms
Descriptor/Qualifier:
Alcohol Dehydrogenase / chemistry*,  genetics,  metabolism*
Amino Acid Substitution
Archaeal Proteins / chemistry*,  genetics,  metabolism*
Catalytic Domain
Crystallography, X-Ray
Enzyme Stability
Genes, Archaeal
Hydrogen-Ion Concentration
Kinetics
Models, Molecular
Mutagenesis, Site-Directed
NAD / metabolism
Protein Conformation
Protein Denaturation
Recombinant Proteins / chemistry,  genetics,  metabolism
Spectrometry, Fluorescence
Substrate Specificity
Sulfolobus solfataricus / enzymology*,  genetics
Temperature
Tryptophan / chemistry
Chemical
Reg. No./Substance:
0/Archaeal Proteins; 0/Recombinant Proteins; 53-84-9/NAD; 73-22-3/Tryptophan; EC 1.1.1.1/Alcohol Dehydrogenase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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