Squalene synthase: Analysis of the enzyme-catalyzed rearrangement of presqualene diphosphate and analysis of the squalene synthase peptide sequence

The squalene synthase (SQSase)-catalyzed solvolysis of presqualene diphosphate (PSPP) was investigated. In the absence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) SQSase catalyzes the formation of several C30 hydrocarbons and carbinols from PSPP. Dehydrosqualene (29), 10-hydroxybotryococcene (31), and (R)-12-hydroxysqualene (30) were previously identified. Minor products of the reaction are now identified as the C30 analog of artemisia alcohol (33) and the allylic isomer of 12-hydroxysqualene (34). The absolute configurations and the ratio of the minor and major enantiomers of 30 and 31 were determined by comparisons to synthetic standards. The ( R) and (S) enantiomers of 12-hydroxysqualene formed in a 93:7 ratio, and the (S) and (R) enantiomers of 21 formed in a ratio of >93:7. The stereochemical course of the reaction that gave 29 was investigated with (R)- and (S)-[1-2H]-farnesyl diphosphate. This elimination reaction proceeded with the same stereoselectivity as the biosynthesis of (E,Z,E)-phytoene.;The effect of an NADPH analog on the SQSase-catalyzed solvolysis of PSPP was investigated with 1,4,5,6-tetrahydronicotinamide adenine dinucleotide phosphate (NADPH3). The reaction in the presence of NADPH 3 allowed the identification of rillingol (46), which formed from the direct reaction of water with the tertiary cyclopropylcarbinyl cation (22) that is proposed to be the direct precursor to squalene. This provided the first direct evidence for a SQSase-catalyzed cyclopropylcyclopropyl-carbinyl cationic rearrangement.;Isotope effects on the product partitioning of the SQSase-catalyzed solvolysis reaction were investigated with FPP labeled with deuterium at four separate positions. The results from these experiments indicate that the solvolysis products formed from a common intermediate and provide evidence in support of cyclopropylcarbinyl cationic rearrangement mechanism.;Sequence analysis of SQSase, phytoene synthase (PHYSase), dehydrosqualene synthase (DHSQase), farnesyl diphosphate synthase (FPSase), and chrysanthemyl diphosphate synthase (CDSase) was performed. The analysis showed that these enzymes share a common evolutionary history. A secondary structure prediction for SQSase and PHYSase was also performed. The predicted secondary structures of SQSase and PHYSase are similar and are homologous to the x-ray crystal structure of avian FPSase.