Recombinant Squalene Synthase. A Mechanism for the Rearrangement of Presqualene Diphosphate to Squalene

Squalene synthase (SQase) catalyzes the condensation of two molecules of farnesyl diphosphate (<b>FPP</b>) to form presqualene diphosphate (<b>PSPP</b>) and the subsequent rearrangement and NADPH-dependent reduction of <b>PSPP</b> to squalene (<b>SQ</b>). These reactions are the first committed steps in cholesterol biosynthesis. When recombinant SQase was incubated with <b>FPP</b> in the presence of dihydroNADPH (NADPH<sub>3</sub>, an unreactive analogue lacking the 5,6-double bond in the nicotinamide ring), three products were formed:  dehydrosqualene (<b>DSQ</b>), a C<sub>30</sub> analogue of phytoene; 10(<i>S</i>)-hydroxysqualene <b>(HSQ</b>), a hydroxy analogue of squalene; and rillingol (<b>ROH</b>), a cyclopropylcarbinyl alcohol formed by addition of water to the tertiary cyclopropylcarbinyl cation previously proposed as an intermediate in the rearrangement of <b>PSPP</b> to <b>SQ</b> (Poulter, C. D. <i>Acc. Chem. Res</i>. <b>1990</b>, <i>23</i>, 70−77). The structure and absolute stereochemistry of the tertiary cyclopropylcarbinyl alcohol were established by synthesis using two independent routes. Isolation of <b>ROH</b> from the enzyme-catalyzed reaction provides strong evidence for a cyclopropylcarbinyl−cyclopropylcarbinyl rearrangement in the biosynthesis of squalene. By comparing the SQase-catalyzed solvolysis of <b>PSPP</b> in the absence of NADPH<sub>3</sub> to the reaction in the presence of NADPH<sub>3</sub>, it is apparent that the binding of the cofactor analogue substantially enhances the ability of SQase to control the regio- and stereochemistry of the rearrangements of <b>PSPP</b>.