Identification of amino acids related to catalytic function of Sulfolobus solfataricus P1 carboxylesterase by site-directed mutagenesis and molecular modeling

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Title
Identification of amino acids related to catalytic function of Sulfolobus solfataricus P1 carboxylesterase by site-directed mutagenesis and molecular modeling
Author(s)
Y H Choi; Y N Lee; Young-Jun ParkSung Jin Yoon; H B Lee
Bibliographic Citation
BMB Reports, vol. 49, no. 6, pp. 349-354
Publication Year
2016
Abstract
The archaeon Sulfolobus solfataricus P1 carboxylesterase is a thermostable enzyme with a molecular mass of 33.5 kDa belonging to the mammalian hormone-sensitive lipase (HSL) family. In our previous study, we purified the enzyme and suggested the expected amino acids related to its catalysis by chemical modification and a sequence homology search. For further validating these amino acids in this study, we modified them using site-directed mutagenesis and examined the activity of the mutant enzymes using spectrophotometric analysis and then estimated by homology modeling and fluorescence analysis. As a result, it was identified that Ser151, Asp244, and His274 consist of a catalytic triad, and Gly80, Gly81, and Ala152 compose an oxyanion hole of the enzyme. In addition, it was also determined that the cysteine residues are located near the active site or at the positions inducing any conformational changes of the enzyme by their replacement with serine residues.
Keyword
CarboxylesteraseMolecular modelingSite-directed mutagenesisSulfolobus solfataricus P1Thermostability
ISSN
1225-8687
Publisher
Korea Soc-Assoc-Inst
DOI
http://dx.doi.org/10.5483/BMBRep.2016.49.6.077
Type
Article
Appears in Collections:
Division of Research on National Challenges > Environmental diseases research center > 1. Journal Articles
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