Overexpression of squalene-hopene cyclase by the pET vector in Escherichia coli and first identification of tryptophan and aspartic acid residues inside the QW motif as active sites.
An overexpression system for squalene-hopene cyclase (SHC) was constructed by using the pET3a vector, which is responsible for high expression with help from the strong T7 promoter when incorporated into E. coli BL21(DE3). Site-directed mutagenesis experiments prove that two amino acid residues of tryptophan and aspartic acid inside the QW-motif 5 resided as active sites.
Sato
T
T
Graduate School of Science and Technology, Faculty of Agriculture, Niigata University, Japan.
Kanai
Y
Y
Hoshino
T
T
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D001406:Bacillaceae / Q000201:enzymology / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D019751:Intramolecular Transferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013185:Squalene / Q000032:analysis; D014170:Transformation, Genetic; D014315:Triterpenes / Q000032:analysis; D014364:Tryptophan / Q000737:chemistry*
D004269:DNA, Bacterial; D014315:Triterpenes; C028765:diploptene; D001224:Aspartic Acid; D007544:Isopropyl Thiogalactoside; D013185:Squalene; D014364:Tryptophan; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase