Thermostable Farnesyl Diphosphate Synthase of Bacillus stearothermophilus: Molecular Cloning, Sequence Determination, Overproduction, and Purification1

Koyama, Tanetoshi; Obata, Seiji; Osabe, Masami; Takeshita, Ayumi; Yokoyama, Ken; Uchida, Masatoshi; Nishino, Tokuzo; Ogura, Kyozo

doi:10.1093/oxfordjournals.jbchem.a124051

Cited by 125 publications

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“…Restriction enzyme digestions, transformations, and other standard molecular biology techniques were carried out as described by Sambrook et al (24). Bacteria were cultured in Luria-Bertani (LB) or M9YG medium (3). All other chemicals were of analytical grade.…”

Section: Materials and General Procedures-[1-mentioning

confidence: 99%

“…To date, the structural genes for many types of trans-prenyltransferases have been cloned and characterized. Multiple alignments of the deduced amino acid sequences of these transprenyltransferases showed the presence of several conserved regions in the primary structures (3,4). The conserved regions include two characteristic aspartate-rich motifs (DDXXD), which have been shown to be essential for the catalytic function as well as substrate binding by site-directed mutational analysis (5)(6)(7)(8).…”

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confidence: 99%

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Identification of Significant Residues for Homoallylic Substrate Binding of Micrococcus luteus B-P 26 Undecaprenyl Diphosphate Synthase

Kharel

Zhang

Fujihashi

et al. 2001

Journal of Biological Chemistry

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The primary structure of cis-prenyltransferase is totally different from those of trans-prenyltransferases (Shimizu, N., Koyama, T., and Ogura, K. (1998) J. Biol. Chem. 272, 19476 -19481). To better understand the molecular mechanism of enzymatic cis-prenyl chain elongation, we selected seven charged residues in the con- Prenyltransferases, also referred to as prenyl diphosphate synthases, are enzymes catalyzing the sequential condensation of isopentenyl diphosphate (IPP) 1 with allylic diphosphates to produce linear prenyl diphosphates in the biosynthetic pathway of isoprenoid compounds, most of which are essential components of the cellular machinery, such as cholesterol, carotenoids, prenyl quinones, prenyl proteins, and dolichols. Although these condensation reactions are similar in terms of chemical mechanism, there are a number of enzymes having different specificities with respect to the chain length and double-bond stereochemistry of its final product. These enzymes can be classified into two major subgroups (cis and trans types) according to the geometry of the products and are extremely interesting from an enzymological viewpoint in which the reactions are regulated to proceed consecutively and terminate precisely at definite chain lengths depending on the specificities of individual enzymes (1, 2). To date, the structural genes for many types of trans-prenyltransferases have been cloned and characterized. Multiple alignments of the deduced amino acid sequences of these transprenyltransferases showed the presence of several conserved regions in the primary structures (3, 4). The conserved regions include two characteristic aspartate-rich motifs (DDXXD), which have been shown to be essential for the catalytic function as well as substrate binding by site-directed mutational analysis (5-8). Tarshis et al. (9) determined the crystal structure of chicken FPP synthase, in which most of the conserved regions are found in a large central cavity. and Tarshis et al. (15) have intensively investigated the product chain length determination mechanisms of short chain transprenyltransferases such as FPP synthase from Bacillus stearothermophilus or chicken and geranylgeranyl diphosphate (GGPP) synthase from Sulfolobus acidocaldarius.On the other hand, cis-prenyltransferases catalyze cis-prenyl chain elongation to produce prenyl diphosphates with E,Zmixed stereochemistry. In bacteria, sequential cis addition of IPP onto FPP as an allylic primer to give undecaprenyl diphosphate (UPP, C 55 ) is catalyzed by UPP synthase. UPP is the direct precursor of glycosyl carrier lipid in the biosynthesis of bacterial cell wall polysaccharide components such as peptidoglycan and lipopolysaccharide. In eukaryotic cells, dehydrodolichyl diphosphate synthase responsible for cis-prenyl chain elongation catalyzes much longer chain elongation than does the bacterial enzyme. Dehydrodolichyl diphosphate is an essential precursor of carbohydrate carrier lipid in the biosynthesis of N-linked glycoprotein or glycosylphosphatidylinositolan...

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Section: Materials and General Procedures-[1-mentioning

confidence: 99%

mentioning

confidence: 99%

Identification of Significant Residues for Homoallylic Substrate Binding of Micrococcus luteus B-P 26 Undecaprenyl Diphosphate Synthase

Kharel

Zhang

Fujihashi

et al. 2001

Journal of Biological Chemistry

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“…1C). Since the E. coli enzyme shares conserved active site residues with human FPPS (19), these complexes provide a structural framework for the development of novel FPPS inhibitors.…”

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Structural Basis for Bisphosphonate-mediated Inhibition of Isoprenoid Biosynthesis

Hosfield¹,

Zhang²,

Dougan³

et al. 2004

Journal of Biological Chemistry

252

351

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Farnesyl pyrophosphate synthetase (FPPS) synthesizes farnesyl pyrophosphate through successive condensations of isopentyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. Nitrogen-containing bisphosphonate drugs used to treat osteoclast-mediated bone resorption and tumor-induced hypercalcemia are potent inhibitors of the enzyme. Here we present crystal structures of substrate and bisphosphonate complexes of FPPS. The structures reveal how enzyme conformational changes organize conserved active site residues to exploit metal-induced ionization and substrate positioning for catalysis. The structures further demonstrate how nitrogen-containing bisphosphonates mimic a carbocation intermediate to inhibit the enzyme. Together, these FPPS complexes provide a structural template for the design of novel inhibitors that may prove useful for the treatment of osteoporosis and other clinical indications including cancer.Post-translational modification of C-terminal CAAX sequences by covalent attachment of isoprenyl chains is crucial for intracellular localization and proper function of small GTPases such as Ras, Rac, Rho, and CDC42 (1, 2). The substrates for these modifications are the 15-carbon isoprenoid farnesyl pyrophosphate (FPP) 1 or the 20-carbon isoprenoid geranyl-geranyl pyrophosphate synthesized by enzymes of the mevalonate pathway (3) (Fig. 1A). A key branch point enzyme of the mevalonate pathway is farnesyl pyrophosphate synthetase (FPPS), a ϳ30-kDa Mg 2ϩ -dependent homodimeric enzyme that synthesizes (E,E)-FPP from isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) (4, 5) (Fig. 1B). Interest in understanding FPPS activity stems from the recent discovery that FPPS is the molecular target of nitrogencontaining bisphosphonates (6,7,31,32). Bisphophonates are non-cleavable pyrophosphate (P-O-P) analogues in which the central oxygen is replaced by a carbon (P-C-P) with various side chains (Fig. 1C). Against parasitic organisms (8, 9) these agents have been shown in vitro to disrupt cell growth through FPPS inhibition. In people, bisphosphonates are targeted to bone tissue (10) where FPPS inhibition in bone-resorbing osteoclasts is a current therapeutic approach for treating postmenopausal osteoporosis (11,12). Because of their bone-targeting properties, bisphosphonates have also found use as agents to treat tumor-induced hypercalcemia (13), Paget's disease (14), and osteolytic metastases (15).Although structures of apo-and ligand-bound avian FPPS have been solved (16,17), the active sites are unassembled and do not provide substantial information concerning catalysis. Thus, to resolve the molecular basis of catalysis, and also to understand the structural features governing bisphosphonate recognition, we determined the structures of unliganded Staphylococcus aureus FPPS (FPPS-Sa), as well as two Escherichia coli FPPS (FPPS-Ec) ternary complexes. These ternary complexes include a 2.4-Å "substrate-bound" structure containing IPP and the noncleavable DMAPP analogue dimethyla...

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“…The amino acid sequence alignments of these enzymes have shown the presence of two characteristic aspartate-rich DDXXD motifs, which have been shown to be essential for the catalytic function as well as the substrate binding (2)(3)(4)(5)(6). The three-dimensional (3D) structure of farnesyl diphosphate synthase (FPS), a trans-type prenyltransferase, has been determined as the only structure in all prenyl chain elongating enzymes (7).…”

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Crystal structure of cis -prenyl chain elongating enzyme, undecaprenyl diphosphate synthase

Fujihashi

Zhang

Higuchi

et al. 2001

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

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Undecaprenyl diphosphate synthase (UPS) catalyzes the cis-prenyl chain elongation onto trans, trans-farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of bacterial cell walls. We report here the crystal structure of UPS as the only three-dimensional structure among cis-prenyl chain elongating enzymes. The structure is classified into a protein fold family and is completely different from the so-called ''isoprenoid synthase fold'' that is believed to be a common structure for the enzymes relating to isoprenoid biosynthesis. Conserved amino acid residues among cis-prenyl chain elongating enzymes are located around a large hydrophobic cleft in the UPS structure. A structural P-loop motif, which frequently appears in the various kinds of phosphate binding site, is found at the entrance of this cleft. The catalytic site is determined on the basis of these structural features, from which a possible reaction mechanism is proposed.

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Thermostable Farnesyl Diphosphate Synthase of Bacillus stearothermophilus: Molecular Cloning, Sequence Determination, Overproduction, and Purification1

Cited by 125 publications

References 0 publications

Identification of Significant Residues for Homoallylic Substrate Binding of Micrococcus luteus B-P 26 Undecaprenyl Diphosphate Synthase

Identification of Significant Residues for Homoallylic Substrate Binding of Micrococcus luteus B-P 26 Undecaprenyl Diphosphate Synthase

Structural Basis for Bisphosphonate-mediated Inhibition of Isoprenoid Biosynthesis

Crystal structure of cis -prenyl chain elongating enzyme, undecaprenyl diphosphate synthase

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