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

Abstract

Squalene synthase (SQase) catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP) and the subsequent rearrangement and NADPH-dependent reduction of PSPP to squalene (SQ). These reactions are the first committed steps in cholesterol biosynthesis. When recombinant SQase was incubated with FPP in the presence of dihydroNADPH (NADPH3, an unreactive analogue lacking the 5,6-double bond in the nicotinamide ring), three products were formed: dehydrosqualene (DSQ), a C30 analogue of phytoene; 10(S)-hydroxysqualene (HSQ), a hydroxy analogue of squalene; and rillingol (ROH), a cyclopropylcarbinyl alcohol formed by addition of water to the tertiary cyclopropylcarbinyl cation previously proposed as an intermediate in the rearrangement of PSPP to SQ (Poulter, C. D. Acc. Chem. Res. 1990, 23, 70-77). The structure and absolute stereochemistry of the tertiary cyclopropylcarbinyl alcohol were established by synthesis using two independent routes. Isolation of ROH 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 PSPP in the absence of NADPH3 to the reaction in the presence of NADPH3, 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 PSPP.

Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii

“…The initial reaction step consists of a head-to-head condensation of two farnesyl diphosphate (FPP) molecules to form a stable cyclopropyl intermediate, presqualene diphosphate (PSPP) (24,25). In the second reaction step, PSPP undergoes a reductive rearrangement in the presence of NADPH to yield squalene possessing a C1-C1′ linkage between the two farnesyl substituents (26,27) (Fig. 1B).…”

Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

“…Unable to convert into phytoene in wild-type CrtM, prephytoene diphosphate either remains stuck in the enzyme, departs, or undergoes other types of rearrangement to yield noncarotenoid products. (A similar phenomenon is well known for SqS; in the absence of NADPH, which is required to convert presqualene diphosphate to squalene, SqS produces a complex mixture of nonsqualene compounds including rillingol, 10-hydroxybotryococcene, and 12-hydroxysqualene [22,89].) However, in CrtM mutants where F26 or W38 is replaced with a smaller or more flexible amino acid, the prephytoene diphosphate formed from two molecules of C 20 PP is efficiently rearranged to form phytoene.…”

“…The biosynthesis of carotenoid backbones (and squalene) has proven to be a complex process (22,88,89) (Fig. 5a).…”

Evolution of a Pathway to Novel Long-Chain Carotenoids

“…This mechanism is virtually identical to that of squalene synthase (SqS), the enzyme that catalyzes the first step in cholesterol biosynthesis. Indeed, when deprived of NADPH, SqS produces product 1 as the main product (3,11). Carotene synthases are similar to SqS in sequence and predicted secondary structure; they probably share a common ancestor and have virtually identical folds.…”

“…Carotene synthases are similar to SqS in sequence and predicted secondary structure; they probably share a common ancestor and have virtually identical folds. Although detailed biochemical information on SqS is available (3,11), the basis of its specificity is also poorly understood. Mapped onto the crystal structure of human SqS (hSqS) (19), F26 and W38 appear in helices B and C. Both side chains point into the pocket that accommodates the second half-reaction (Fig.…”