Cloning, Solubilization, and Characterization of Squalene Synthase from Thermosynechococcus elongatus BP-1

Abstract

Squalene synthase (SQS) is a bifunctional enzyme that catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the subsequent rearrangement of PSPP to squalene. These reactions constitute the first pathway-specific steps in hopane biosynthesis in Bacteria and sterol biosynthesis in Eukarya. The genes encoding SQS were isolated from the hopane-producing bacteria Thermosynechococcus elongatus BP-1, Bradyrhizobium japonicum, and Zymomonas mobilis and cloned into an Escherichia coli expression system. The expressed proteins with a His 6 tag were found exclusively in inclusion bodies when no additives were used in the buffer. After extensive optimization, soluble recombinant T. elongatus BP-1 SQS was obtained when cells were disrupted and purified in buffers containing glycerol. The recombinant B. japonicum and Z. mobilis SQSs could not be solubilized under any of the expression and purification conditions used. Purified T. elongatus His 6 -SQS gave a single band at 42 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and molecular ion at m/z 41886 by electrospray mass spectrometry. Incubation with FPP and NADPH gave squalene as the sole product. Incubation of the enzyme with [14 C]FPP in the absence of NADPH gave PSPP. The enzyme requires Mg 2؉ for activity, has an optimum pH of 7.6, and is strongly stimulated by detergent. Under optimal conditions, the K m of FPP is 0.97 ؎ 0.10 M and the k cat is 1.74 ؎ 0.04 s ؊1 . Zaragozic acid A, a potent inhibitor of mammalian, fungal, and Saccharomyces cerevisiae SQSs, also inhibited recombinant T. elongatus BP-1 SQS, with a 50% inhibitory concentration of 95.5 ؎ 13.6 nM.Squalene synthase ([SQS] farnesyl diphosphate:farnesyl diphosphate transferase; EC 2.5.1.21) catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to form a c1Ј-2-3-linked triterpene intermediate, presqualene diphosphate (PSPP), and the subsequent NADPH-dependent rearrangement and reduction of PSPP to produce squalene (SQ), as outlined in Fig. 1 (36, 37). These transformations are the first pathway-specific reactions in the hopanoid biosynthetic pathway in Bacteria (7,33,42,48) and the sterol pathway in Eukarya (37). Studies of yeast SQS show that substrate addition is ordered with two molecules of FPP added first, followed by NADPH (24, 39). When NADPH is present in the buffer, PSPP is converted directly to SQ without dissociating from the active site. Formation of PSPP or a prior conformational change in SQS is the rate-limiting step in the overall conversion of FPP to SQ. PSPP is formed and released when NADPH is absent. Under these conditions, SQS catalyzes a slow "solvolysis" of PSPP to give triterpene alcohols and hydrocarbons with irregular isoprenoid skeletons (14).SQS has been cloned from a variety of eukaryotes, including fungal (15, 21, 57), protozoan (45), rat (22), mouse (12), human (41, 50), and plant sources (27). The eukaryotic enzyme is associated with membranes, where it appears to be anchored by a h...

Functional microdomains in bacterial membranes

“…As expected from its predicted amino acid sequence, purified recombinant YisP had the enzymatic features of a phytoene or squalene synthase, and preferentially used farnesyl pyrophosphate (FPP) as a substrate (Supplemental Fig. S3; Lee and Poulter 2008). In addition, YisP enzymatic activity was dependent on NADH and was blocked by nanomolar concentrations of the competitive inhibitor of squalene synthases, zaragozic acid ( Fig.…”

Molecular Cloning, Expression and Characterization of a Squalene Synthase Gene from Grain Amaranth (<i>Amaranthus cruentus</i> L.)

“…Isolation and characterization of a SQS gene from grain amaranth is the first step towards understanding regulation of the squalene biosynthetic pathway. Genes encoding SQS have been isolated from many sources, such as fungi (Jennings et al 1991), bacteria (Lee & Poulter 2008), animals (McKenzie et al 1992), and plants (Nakashima et al 1995, Hata et al 1997, Devarenne et al 1998, Hayashi et al 1999, Akamine et al 2003, Huang et al 2007, Uchida et al 2009, Kim et al 2011). To our knowledge, this is the first report on the molecular cloning and expression analysis of an SQS gene from grain amaranth.…”

Directed optimization of a newly identified squalene synthase from Mortierella alpine based on sequence truncation and site-directed mutagenesis

“…SQS was assayed by modification of the procedure reported by Lee, Poulter [21]. The assay was performed in 50 mM MOPS buffer (pH 7.0), containing 40 μM FPP, 30 μg purified SQS, 10 mM MgCl 2 , 1 mM NADPH, 1 mM dithiothreitol (DTT), 0.2 mg BSA, in a final volume of 200 μL.…”