Catalytic function of the residues of phenylalanine and tyrosine conserved in squalene-hopene cyclases.
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 the D376 acidity, which is assumed to work as a proton donor for initiating the polycyclization cascade, but its role is moderate. (2) Y609 possibly assists the function of F365, which has previously been assigned to exclusively stabilize the C-8 carbocation intermediate through cation-pi interaction. The Y609A mutant produced a partially cyclized bicyclic triterpene. (3) Y612 works to stabilize both the C10 and C8 carbocations, this being verified by the finding that mono- and bicyclic products were formed with the Y612A mutant. (4) F129 was first identified to play a crucial role in catalysis. (5) The three residues, Y372, Y474 and Y540, are responsible for reinforcing the protein structure against thermal denaturation, Y474 being located inside QW motif 3.
Sato
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Department of Applied Biological Chemistry, Faculty of Agriculture, and Graduate School of Science and Technology, Niigata University, Ikarashi, Japan.
Hoshino
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D016937:Gram-Positive Endospore-Forming Rods / Q000201:enzymology* / Q000235:genetics; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D010649:Phenylalanine; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase