Function-Structure Studies and Identification of Three Enzyme Domains Involved in the Catalytic Activity in Rat Hepatic Squalene Synthase

Gu, Peide; Ishii, Yoshinori; Spencer, Thomas A.; Shechter, Ishaiahu

doi:10.1074/jbc.273.20.12515

Cited by 80 publications

(93 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Eukaryotic SQSs have four conserved regions (Fig. 2, I to IV), which kinetic studies with site-directed mutants and a crystal structure indicate are important for catalysis (10,32). Regions I, II, and III are involved in the first half-reaction, the condensation of two molecules of FPP to give PSPP.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Lee

Poulter

2008

J Bacteriol

View full text Add to dashboard Cite

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...

show abstract

Section: Resultsmentioning

confidence: 99%

“…Geranyl diphosphate (GPP) and FPP were synthesized by the procedure of Davisson et al (8). [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]isopentenyl diphosphate (IPP) and [1][2][3] H]FPP were purchased from GE Healthcare. General methods.…”

Section: Methodsmentioning

confidence: 99%

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

Lee

Poulter

2008

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…Thus, our results suggested that Zm-PSY1-NP was located in fibrillar plastoglobuli, which initiate from globular plastoglobuli in the presence of high concentrations of carotenoids. The presence of carotenoids in fibrils was supported by the use of the Spectral Dye Separation tool in LAS AF software (Leica), applied to the fluorescence intensity spectra of Zm-PSYs-GFP constructs expressed in protoplasts prepared from etiolated leaves of Zm-PSY1 knockout maize (see 168, Amino acid represented by Asn (N) in Zm-PSY1 and Ser (S) in all other PSYs; 257, amino acid represented by Thr (T) in Zm-PSY1 and Pro (P) in all other PSYs; 174 and 190, respective amino acids are shown to affect the activity of PSY in cassava and tomato (Gady et al, 2011); 285, mutation at this site inactivates SQS (Gu et al, 1998). Hyphens indicate putative cleavage site for chloroplast transit sequence, predicted by ChloroP.…”

Section: Single Amino Acid Variants Displayed Altered Psy1 Localizatimentioning

confidence: 99%

“…Predicted structural similarities between PSYs and SQS are presented in Figure 4. Mutagenesis of either of two highly conserved Asp residues 219 and 223 to Glu inactivates SQS (Gu et al, 1998). Thus, we mutagenized the corresponding Asp residue 285 to Glu and inactivated Zm-PSY1 (Table 1), BLAST alignment of Zm-PSY1 to all PSY sequences available from the National Center for Biotechnology Information.…”

Section: Single Amino Acid Variants Displayed Altered Psy1 Localizatimentioning

confidence: 99%

Plastid Localization of the Key Carotenoid Enzyme Phytoene Synthase Is Altered by Isozyme, Allelic Variation, and Activity

et al. 2012

View full text Add to dashboard Cite

Plant carotenoids have unique physiological roles related to specific plastid suborganellar locations. Carotenoid metabolic engineering could enhance plant adaptation to climate change and improve food security and nutritional value. However, lack of fundamental knowledge on carotenoid pathway localization limits targeted engineering. Phytoene synthase (PSY), a major rate-controlling carotenoid enzyme, is represented by multiple isozymes residing at unknown plastid sites. In maize (Zea mays), the three isozymes were transiently expressed and found either in plastoglobuli or in stroma and thylakoid membranes. PSY1, with one to two residue modifications of naturally occurring functional variants, exhibited altered localization, associated with distorted plastid shape and formation of a fibril phenotype. Mutating the active site of the enzyme reversed this phenotype. Discovery of differential PSY locations, linked with activity and isozyme type, advances the engineering potential for modifying carotenoid biosynthesis.

show abstract

“…Extensive investigations of squalene synthase including sitedirect mutagenesis (28) and structural elucidation of 3-dimensional structure (29) have focused on five highly conserved domains (domains I-V) thought associated with catalysis (30). Many studies have also utilized these highly conserved domains as a means for isolating the corresponding genes from a diverse range of organisms.…”

mentioning

confidence: 99%

Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii

Niehaus

Okada

Devarenne

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

134

152

View full text Add to dashboard Cite

Botryococcene biosynthesis is thought to resemble that of squalene, a metabolite essential for sterol metabolism in all eukaryotes. Squalene arises from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene. In principle, botryococcene could arise from an alternative rearrangement of the presqualene intermediate. Because of these proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence isolated squalene synthase-like genes from Botryococcus braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encodes for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when combinations of the synthase-like enzymes were mixed together, in vivo and in vitro, robust botryococcene (SSL-1+SSL-3) or squalene biosynthesis (SSL1+SSL-2) was observed. These findings were unexpected because squalene synthase, an ancient and likely progenitor to the other Botryococcus triterpene synthases, catalyzes a two-step reaction within a single enzyme unit without intermediate release, yet in B. braunii, these activities appear to have separated and evolved interdependently for specialized triterpene oil production greater than 500 MYA. Coexpression of the SSL-1 and SSL-3 genes in different configurations, as independent genes, as gene fusions, or targeted to intracellular membranes, also demonstrate the potential for engineering even greater efficiencies of botryococcene biosynthesis.algae | biofuels | terpene enzymology

show abstract

Function-Structure Studies and Identification of Three Enzyme Domains Involved in the Catalytic Activity in Rat Hepatic Squalene Synthase

Cited by 80 publications

References 48 publications

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

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

Plastid Localization of the Key Carotenoid Enzyme Phytoene Synthase Is Altered by Isozyme, Allelic Variation, and Activity

Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii

Contact Info

Product

Resources

About