Expression, Purification, and Characterization of the Human Squalene Synthase: Use of Yeast and Baculoviral Systems

Soltis, D A; McMahon, Gerald; Caplan, S.L.; Dudas, Debra; Chamberlin, Helena; Vattay, Anthony; Dottavio, Diane; Rucker, M.L.; Engstrom, Robert; Cornell-Kennon, Susan; Boettcher, Brian R.

doi:10.1006/abbi.1995.1095

Cited by 23 publications

(9 citation statements)

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“…SQS is located at the entry point to the sterol and hopanoid pathways, and it synthesizes the common precursor SQ with remarkable specificity. All three SQSs tested in this study have K m values ranging 0.2–0.5 mM for NADPH [23,25,26], which is comparable to the reported NADPH concentration values in E. coli (0.56 mM) [27]. Given these data, a non‐negligible fraction of SQSs should be NADPH‐free in E. coli cells.…”

Section: Discussionsupporting

confidence: 82%

Directed evolution of squalene synthase for dehydrosqualene biosynthesis

Furubayashi

Katabami

et al. 2014

FEBS Letters

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a b s t r a c tSqualene synthase (SQS) catalyzes the first step of sterol/hopanoid biosynthesis in various organisms. It has been long recognized that SQSs share a common ancestor with carotenoid synthases, but it is not known how these enzymes selectively produce their own product. In this study, SQSs from yeast, human, and bacteria were independently subjected to directed evolution for the production of the C 30 carotenoid backbone, dehydrosqualene. This was accomplished via high-throughput screening with Pantoea ananatis phytoene desaturase, which can selectively convert dehydrosqualene into yellow carotenoid pigments. Genetic analysis of the resultant mutants revealed various mutations that could effectively convert SQS into a ''dehydrosqualene synthase.'' All of these mutations are clustered around the residues that have been proposed to be important for NADPH binding.

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Section: Discussionsupporting

confidence: 82%

Directed evolution of squalene synthase for dehydrosqualene biosynthesis

Furubayashi

Katabami

et al. 2014

FEBS Letters

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“…successfully expressed in E. coli and substantial In the presence of an exogenous source of sterol, rat (4) amounts of SS enzyme purified from SF-9 insect cells and human (5) SS mRNA levels decrease 60 to 95%, infected with a recombinant baculovirus containing the respectively. The HMGR enzyme activity inhibitor lo-HSS cDNA (21). Purification of SS from plants has vastatin has also been used to demonstrate an increase shown difficulties similar to those of mammalian SS.…”

mentioning

confidence: 99%

Molecular Characterization of Tobacco Squalene Synthase and Regulation in Response to Fungal Elicitor

Devarenne

Shin

Back

et al. 1998

Archives of Biochemistry and Biophysics

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“…As a key precursor of isoprenoids, SQ is synthesized from FPP by SQS (Fig. 1), which has been identified in human beings [36,38], animals [14,24,34], plants [11,12,18,26,42,50], fungi [16,20,51,52], bacteria [21]. SQS is commonly considered to be a crucial enzyme and a potential regulatory branch node that controls carbon flux towards the triterpenes and sterols biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

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

Huang

Yao

Zhang

et al. 2015

Journal of Industrial Microbiology and Biotechnology

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Terpenoids, a class of isoprenoids usually isolated from plants, are always used as commercial flavor and anticancer drugs. As a key precursor for triterpenes and sterols, biosynthesis of squalene (SQ) can be catalyzed by squalene synthase (SQS) from two farnesyl diphosphate molecules. In this work, the key SQS gene involved in sterols synthesis by Mortierella alpine, an industrial strain often used to produce unsaturated fatty acid such as γ-linolenic acid and arachidonic acid, was identified and characterized. Bioinformatic analysis indicated that MaSQS contained 416 amino acid residues involved in four highly conserved regions. Phylogenetic analysis revealed the closest relationship of MaSQS with Ganoderma lucidum and Aspergillus, which also belonged to the member of the fungus. Subsequently, the recombinant protein was expressed in Escherichia coli BL21(DE3) and detected by SDS-PAGE. To improve the expression and solubility of protein, 17 or 27 amino acids in the C-terminal were deleted. In vitro activity investigation based on gas chromatography-mass spectrometry revealed that both the truncated enzymes could functionally catalyze the reaction from FPP to SQ and the enzymatic activity was optimal at 37 °C, pH 7.2. Moreover, based on the site-directed mutagenesis, the mutant enzyme mMaSQSΔC17 (E186K) displayed a 3.4-fold improvement in catalytic efficiency (k(cat)/K(m)) compared to the control. It was the first report of characterization and modification of SQS from M. alpine, which facilitated the investigation of isoprenoid biosynthesis in the fungus. The engineered mMaSQSΔC17 (E186K) can be a potential candidate of the terpenes and steroids synthesis employed for synthetic biology.

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Expression, Purification, and Characterization of the Human Squalene Synthase: Use of Yeast and Baculoviral Systems

Cited by 23 publications

References 0 publications

Directed evolution of squalene synthase for dehydrosqualene biosynthesis

Directed evolution of squalene synthase for dehydrosqualene biosynthesis

Molecular Characterization of Tobacco Squalene Synthase and Regulation in Response to Fungal Elicitor

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

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