Functional analyses of Tyr420 and Leu607 of Alicyclobacillus acidocaldarius squalene-hopene cyclase. Neoachillapentaene, a novel triterpene with the 1,5,6-trimethylcyclohexene moiety produced through folding of the constrained boat structure.
The functions of Tyr420 and Leu607 were analyzed by constructing various site-directed mutants. The mutation at position 420 into Ala and Gly gave bicyclic alpha-and gamma-polypodatetraene in significant amounts, but with a trace amount of tricyclic malabaricatriene. The kinetic data for and the product distribution of the Y420F mutant indicate that the major function of Tyr420 is to stabilize the 6/6-fused bicyclic cation. Mutation experiments on Leu607 demonstrate that the appropriate steric bulk size at position 607 is required to strongly bind with the product-like conformation formed during the polycyclization process. Introduction of the bulkiest Trp residue into 420 or 607 led to the production of a novel monocyclic triterpene having the (5R,6R)-1,5,6-trimethylcyclohexene ring, named neoachillapentaene, indicating that the enzymatic cyclization proceeded via a constrained boat structure. Folding of the squalene molecule into a boat conformation by squalene cyclase has not been reported before.
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D014315:Triterpenes / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D014315:Triterpenes; D014443:Tyrosine; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase; D007930:Leucine