title: Site-directed mutagenesis of putative active-site residues in squalene-hopene cyclase.
abstract: Squalene-hopene cyclase (SHC) catalyzes the complex polycylization of squalene to hopene, similar to the cyclization of oxidosqualene to sterols. Sequence analysis of SHC revealed a highly conserved aspartate-rich motif (DDTA), comparable to the DCTA motif of oxidosqualene cyclases, which is supposed to be part of the active site. In order to determine the importance of the motif in squalene cyclization, the conserved residues Asp376 and Asp377 in the DDTA motif of SHC from Alicyclobacillus acidocaldarius were individually replaced by glutamate, glutamine, glycine, and arginine. With the exception of the [Glu376]SHC mutant, all other substitutions resulted in almost or complete loss of enzyme activity. Compared to that of the wild-type enzyme, the specific activity of the [Glu376]SHC mutant enzyme was reduced to 10%, accompanied by a significant decrease in the apparent Vmax, whereas the apparent K(m) remained unchanged, CD measurements indicated that mutations did not affect the secondary structure. It is proposed that Asp376 and Asp377 are crucial for catalysis and may act as point charges to stabilize intermediate cations. Moreover, for squalene-hopene cyclase, a high content of alpha-helical conformation could be found, providing the first structural information for a triterpene cyclase.
authors.affiliation: Mikrobiologie/Biotechnologie, Universität Tübingen, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Feil
authors.forename: R
authors.initials: R
authors.lastname: Süssmuth
authors.forename: G
authors.initials: G
authors.lastname: Jung
authors.forename: K
authors.initials: K
authors.lastname: Poralla
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D004789:Enzyme Activation; D004795:Enzyme Stability; D019751:Intramolecular Transferases; D007535:Isomerases / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
chemical_list: D007535:Isomerases; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase