title: Structural insights into the catalytic mechanism of human squalene synthase.
abstract: Squalene synthase (SQS) is a divalent metal-ion-dependent enzyme that catalyzes the two-step reductive `head-to-head' condensation of two molecules of farnesyl pyrophosphate to form squalene using presqualene diphosphate (PSPP) as an intermediate. In this paper, the structures of human SQS and its mutants in complex with several substrate analogues and intermediates coordinated with Mg2+ or Mn2+ are presented, which stepwise delineate the biosynthetic pathway. Extensive study of the SQS active site has identified several critical residues that are involved in binding reduced nicotinamide dinucleotide phosphate (NADPH). Based on mutagenesis data and a locally closed (JK loop-in) structure observed in the hSQS-(F288L)-PSPP complex, an NADPH-binding model is proposed for SQS. The results identified four major steps (substrate binding, condensation, intermediate formation and translocation) of the ordered sequential mechanisms involved in the `1'-1' isoprenoid biosynthetic pathway. These new findings clarify previous hypotheses based on site-directed mutagenesis and biochemical analysis.
authors: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors: Chia-I
authors: CI
authors: Liu
authors: University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan.
authors: Wen-Yih
authors: WY
authors: Jeng
authors: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors: Wei-Jung
authors: WJ
authors: Chang
authors: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors: Min-Fang
authors: MF
authors: Shih
authors: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors: Tzu-Ping
authors: TP
authors: Ko
authors: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
authors: Andrew H-J
authors: AH
authors: Wang
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D002413:Cations, Divalent; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012717:Sesquiterpenes / Q000378:metabolism; D013185:Squalene / Q000737:chemistry* / Q000378:metabolism; D055672:Static Electricity
chemical_list: D002413:Cations, Divalent; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C017672:presqualene pyrophosphate; D008345:Manganese; D009249:NADP; C004808:farnesyl pyrophosphate; D013185:Squalene; D013186:Farnesyl-Diphosphate Farnesyltransferase; D008274:Magnesium
keywords: cholesterol synthesis; farnesyl pyrophosphate; nicotinamide adenine dinucleotide; squalene synthase; terpenoids