title: Identification of amino acids related to catalytic function of Sulfolobus solfataricus P1 carboxylesterase by site-directed mutagenesis and molecular modeling.
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. [BMB Reports 2016; 49(6): 349-354].
authors.affiliation: Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 24341, Korea.
authors.forename: Yun-Ho
authors.initials: YH
authors.lastname: Choi
authors.affiliation: Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 24341, Korea.
authors.forename: Ye-Na
authors.initials: YN
authors.lastname: Lee
authors.affiliation: Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea.
authors.forename: Young-Jun
authors.initials: YJ
authors.lastname: Park
authors.affiliation: Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea.
authors.forename: Sung-Jin
authors.initials: SJ
authors.lastname: Yoon
authors.affiliation: Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 24341, Korea.
authors.forename: Hee-Bong
authors.initials: HB
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D055162:Biocatalysis; D043182:Carboxylesterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D005453:Fluorescence; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D048229:Sulfolobus solfataricus / Q000201:enzymology*
chemical_list: D000596:Amino Acids; D050505:Mutant Proteins; D043182:Carboxylesterase; D003545:Cysteine