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.
نویسندگان
چکیده
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.
منابع مشابه
Structure and function of a squalene cyclase.
The crystal structure of squalene-hopene cyclase from Alicyclobacillus acidocaldarius was determined at 2.9 angstrom resolution. The mechanism and sequence of this cyclase are closely related to those of 2,3-oxidosqualene cyclases that catalyze the cyclization step in cholesterol biosynthesis. The structure reveals a membrane protein with membrane-binding characteristics similar to those of pro...
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Site-directed mutagenesis experiments on all the conserved residues of Phe and Tyr in all the known squalene-hopene cyclases (SHCs) were carried out to identify the active site residues of thermophilic Alicyclobacillus acidocaldarius SHC. The following functions are proposed on the basis of kinetic data and trapping of the prematurely cyclized products: (1) The Y495 residue probably amplifies t...
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ورودعنوان ژورنال:
- Bioscience, biotechnology, and biochemistry
دوره 62 2 شماره
صفحات -
تاریخ انتشار 1998