Functional analysis of the DXDDTA motif in squalene-hopene cyclase by site-directed mutagenesis experiments: initiation site of the polycyclization reaction and stabilization site of the carbocation intermediate of the initially cyclized A-ring.
In order to clarify the function of the DXDDTA motif in squalene-hopene cyclase and to identify the acidic amino acid residues crucial for the catalysis, site-directed mutagenesis experiments were carried out. The following results were found: (1) residues D374 and D376 work for the initiation of polyolefin cyclization which arises from the proton attack on the terminal double bond; (2) residue D377 stabilizes C-10 carbocation of the initially cyclized A-ring intermediate, leading to subsequent B-ring closure, which was further verified by isolating the partially cyclized monocyclic product; (3) residues D313 and D447 outside the DXDDTA motif were identified as new active sites; (4) the H451 residue is likely to work in the protonated form to enhance the acidity of the carboxyl groups of D374 and/or D376.
Sato
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T
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Japan.
Hoshino
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013696:Temperature
D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase