Molecules Of Isopentenyl Pyrophosphate Research Articles

A fat story—antigen presentation by butyrophilin 3A1 to γδ T cells

A fat story—antigen presentation by butyrophilin 3A1 to γδ T cells

Molecular characterization of farnesyl pyrophosphate synthase from Bacopa monniera by comparative modeling and docking studies.

Farnesyl pyrophosphate synthase (FPS; EC 2.5.1.10) is a key enzyme in isoprenoid biosynthetic pathway and provides precursors for the biosynthesis of various pharmaceutically important metabolites. It catalyzes head to tail condensation of two isopentenyl pyrophosphate molecules with dimethylallyl pyrophosphate to form C15 compound farnesyl pyrophosphate. Recent studies have confirmed FPS as a molecular target of bisphosphonates for drug development against bone diseases as well as pathogens. Although large numbers of FPSs from different sources are known, very few protein structures have been reported till date. In the present study, FPS gene from medicinal plant Bacopa monniera (BmFPS) was characterized by comparative modeling and docking. Multiple sequence alignment showed two highly conserved aspartate rich motifs FARM and SARM (DDXXD). The 3-D model of BmFPS was generated based on structurally resolved FPS crystal information of Gallus gallus. The generated models were validated by various bioinformatics tools and the final model contained only α-helices and coils. Further, docking studies of modeled BmFPS with substrates and inhibitors were performed to understand the protein ligand interactions. The two Asp residues from FARM (Asp100 and Asp104) as well as Asp171, Lys197 and Lys262 were found to be important for catalytic activity. Interaction of nitrogen containing bisphosphonates (risedronate, alendronate, zoledronate and pamidronate) with modeled BmFPS showed competitive inhibition; where, apart from Asp (100, 104 and 171), Thr175 played an important role. The results presented here could be useful for designing of mutants for isoprenoid biosynthetic pathway engineering well as more effective drugs against osteoporosis and human pathogens.AbbreviationsIPP - Isopentenyl Pyrophosphate, DMAPP - Dimethylallyl Pyrophosphate, GPP - Geranyl Pyrophosphate, FPP - FPPFarnesyl Pyrophosphate, DOPE - Discrete Optimized Protein Energy, BmFPS - Bacopa monniera Farnesyl Pyrophosphate Synthase, RMSD - Root Mean square Deviation, OPLS-AA - Optimized Potentials for Liquid Simulations- All Atom, FARM - First Aspartate Rich Motif, SARM - Second Aspartate Rich Motif.

Biophysical Investigation of the Mode of Inhibition of Tetramic Acids, the Allosteric Inhibitors of Undecaprenyl Pyrophosphate Synthase

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate the C55 undecaprenyl pyrophosphate (UPP). It has been demonstrated that tetramic acids (TAs) are selective and potent inhibitors of UPPS, but the mode of inhibition was unclear. In this work, we used a fluorescent FPP probe to study possible TA binding at the FPP binding site. A photosensitive TA analogue was designed and synthesized for the study of the site of interaction of TA with UPPS using photo-cross-linking and mass spectrometry. The interaction of substrates with UPPS and with the UPPS·TA complex was investigated by protein fluorescence spectroscopy. Our results suggested that tetramic acid binds to UPPS at an allosteric site adjacent to the FPP binding site. TA binds to free UPPS enzyme but not to substrate-bound UPPS. Unlike Escherichia coli UPPS which follows an ordered substrate binding mechanism, Streptococcus pneumoniae UPPS appears to follow a random-sequential substrate binding mechanism. Only one substrate, FPP or IPP, is able to bind to the UPPS·TA complex, but the quaternary complex, UPPS·TA·FPP·IPP, cannot be formed. We propose that binding of TA to UPPS significantly alters the conformation of UPPS needed for proper substrate binding. As the result, substrate turnover is prevented, leading to the inhibition of UPPS catalytic activity. These probe compounds and biophysical assays also allowed us to quickly study the mode of inhibition of other UPPS inhibitors identified from a high-throughput screening and inhibitors produced from a medicinal chemistry program.

Homodimeric hexaprenyl pyrophosphate synthase from the thermoacidophilic crenarchaeon Sulfolobus solfataricus displays asymmetric subunit structures.

Hexaprenyl pyrophosphate synthase (HexPPs) from Sulfolobus solfataricus catalyzes the synthesis of trans-C(30)-hexaprenyl pyrophosphate (HexPP) by reacting two isopentenyl pyrophosphate molecules with one geranylgeranyl pyrophosphate. The crystal structure of the homodimeric C(30)-HexPPs resembles those of other trans-prenyltransferases, including farnesyl pyrophosphate synthase (FPPs) and octaprenyl pyrophosphate synthase (OPPs). In both subunits, 10 core helices are arranged about a central active site cavity. Leu164 in the middle of the cavity controls the product chain length. Two protein conformers are observed in the S. solfataricus HexPPs structure, and the major difference between them occurs in the flexible region of residues 84 to 100. Several helices (alphaI, alphaJ, alphaK, and part of alphaH) and the associated loops have high-temperature factors in one monomer, which may be related to the domain motion that controls the entrance to the active site. Different side chain conformations of Trp136 in two HexPPs subunits result in weaker hydrophobic interactions at the dimer interface, in contrast to the symmetric pi-pi stacking interactions of aromatic side chains found in FPPs and OPPs. Finally, the three-conformer switched model may explain the catalytic process for HexPPs.

A molecular ruler for chain elongation catalyzed by octaprenyl pyrophosphate synthase and its structure-based engineering to produce unprecedented long chain trans-prenyl products.

Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of farnesyl pyrophosphate (FPP) with five molecules of isopentenyl pyrophosphate (IPP) to generate C(40) octaprenyl pyrophosphate (OPP) which constitutes the side chain of menaquinone. We have previously reported the X-ray structure of OPPs from Thermotoga maritima, which is composed entirely of alpha-helices joined by connecting loops and is arranged with nine core helices around a large central cavity [Guo, R. T., Kuo, C. J., Ko, T. P., Chou, C. C., Shr, R. L., Liang, P. H., and Wang, A. H.-J. (2004) J. Biol. Chem. 279, 4903-4912]. A76 and S77 are located on top of the active site close to where FPP is bound. A76Y and A76Y/S77F OPPs mutants produce C(20), indicating that the substituted larger residues interfere with the substrate chain elongation. Surprisingly, the A76Y/S77F mutant synthesizes a larger amount of C(20) than the A76Y mutant. In the crystal structure of the A76Y/S77F mutant, F77 is pushed away by Y76, thereby creating more space between those two large amino acids to accommodate the C(20) product. A large F132 residue at the bottom of the tunnel-shaped active site serves as the "floor" and determines the final product chain length. The substitution of F132 with a small Ala, thereby removing the blockade, led to the synthesis of a C(50) product larger than that produced by the wild-type enzyme. On the basis of the structure, we have sequentially mutated the large amino acids, including F132, L128, I123, and D62, to Ala underneath the tunnel. The products of the F132A/L128A/I123A/D62A mutant reach C(95), beyond the largest chain length generated by all known trans-prenyltransferases. Further modifications of the enzyme reaction conditions, including new IPP derivatives, may allow the preparation of high-molecular weight polyprenyl products resembling the rubber molecule.

Reaction kinetic pathway of the recombinant octaprenyl pyrophosphate synthase from Thermotoga maritima: how is it different from that of the mesophilic enzyme

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the chain elongation of farnesyl pyrophosphate (FPP) via consecutive condensation reactions with five molecules of isopentenyl pyrophosphate (IPP) to generate all- trans C 40-octaprenyl pyrophosphate. The polymer forms the side chain of ubiquinone that is involved in electron transport system to produce ATP. Our previous study has demonstrated that Escherichia coli OPPs catalyzes IPP condensation with a rate of 2 s −1 but product release limits the steady-state rate at 0.02 s −1 [Biochim. Biophys. Acta 1594 (2002) 64]. In the present studies, a putative gene encoding for OPPs from Thermotoga maritima, an anaerobic and thermophilic bacterium, was expressed, purified, and its kinetic pathway was determined. The enzyme activity at 25 °C was 0.005 s −1 under steady-state condition and was exponentially increased with elevated temperature. In contrast to E. coli OPPs, IPP condensation rather than product release was rate limiting in enzyme reaction. The product of chain elongation catalyzed by T. maritima OPPs was C 40 and the rate of its conversion to C 45 was negligible. Under single-turnover condition with 10 μM OPPs·FPP complex and 1 μM IPP, only the C 20 was formed rather than C 20–C 40 observed for E. coli enzyme. Together, our data suggest that the thermophilic OPPs from T. maritima has lower enzyme activity at 25 °C, higher product specificity, higher thermal stability and lower structural flexibility than its mesophilic counterpart from E. coli.

Insight into the activation mechanism of Escherichia coli octaprenyl pyrophosphate synthase derived from pre-steady-state kinetic analysis

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the sequential condensation of five molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to generate all- trans C 40-octaprenyl pyrophosphate, which constitutes the side chain of ubiquinone. Due to the slow product release, a long-chain polyprenyl pyrophosphate synthase often requires detergent or another factor for optimal activity. Our previous studies in examining the activity enhancement of Escherichia coli undecaprenyl pyrophosphate synthase have demonstrated a switch of the rate-determining step from product release to isopentenyl pyrophosphate (IPP) condensation reaction in the presence of Triton [12]. In order to understand the mechanism of enzyme activation for E. coli OPPs, a single-turnover reaction was performed and the measured IPP condensation rate (2 s −1) was 100 times larger than the steady-state rate (0.02 s −1). The high molecular weight fractions and Triton could accelerate the steady-state rate by 3-fold (0.06 s −1) but insufficient to cause full activation (100-fold). A burst product formation was observed in enzyme multiple turnovers indicating a slow product release.

Effect of site-directed mutagenesis of the conserved aspartate and glutamate on E. coli undecaprenyl pyrophosphate synthase catalysis.

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes condensation of eight molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to yield C(55)-undecaprenyl pyrophosphate. We have mutated the aspartates and glutamates in the five conserved regions (I to V) of UPPs protein sequence to evaluate their effects on substrate binding and catalysis. The mutant enzymes including D26A, E73A, D150A, D190A, E198A, E213A, D218A, and D223A were expressed and purified to great homogeneity. Kinetic analyses of these mutant enzymes indicated that the substitution of D26 in region I with alanine resulted in a 10(3)-fold decrease of k(cat) value compared to wild-type UPPs. Its IPP K(m) value has only minor change. The mutagenesis of D150A has caused a much lower IPP affinity with IPP K(m) value 50-fold larger than that of wild-type UPPs but did not affect the FPP K(m) and the k(cat). The E213A mutant UPPs has a 70-fold increased IPP K(m) value and has a 100-fold decreased k(cat) value compared to wild-type. These results suggest that D26 of region I is critical for catalysis and D150 in region IV plays a significant role of IPP binding. The E213 residue in region V is also important in IPP binding as well as catalysis. Other mutant UPPs enzymes in this study have shown no significant change (<5-fold) of k(cat) with exception of E73A and D218A. Both enzymes have 10-fold lower k(cat) value relative to wild-type UPPs.

Product distribution and pre-steady-state kinetic analysis of Escherichia coli undecaprenyl pyrophosphate synthase reaction.

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate. We investigated the kinetics and mechanism of this reaction pathway using Escherichia coli UPPs. With a variety of different ratios of enzyme to substrate and FPP to IPP in the presence or absence of Triton, different product distributions were found. In the presence of excess FPP, the intermediates (C(25)-C(50)) accumulated. Under a condition with enzyme and FPP in excess of IPP, instead of C(20)-geranylgeranyl pyrophosphate, C(20), C(25), and C(30) were the major products. The UPPs steady-state k(cat) value (2.5 s(-1)) in the presence of 0.1% Triton was 190-fold larger than in the absence of Triton (0.013 s(-1)). The k(cat) value matched the rate constant of each IPP condensation obtained from the enzyme single-turnover experiments. This suggested that the IPP condensation rather than product release was the rate-limiting step in the presence of Triton. In the absence of Triton, the intermediates formed and disappeared in a similar manner under enzyme single turnover in contrast to the slow steady-state rate, which indicated a step after product generation was rate limiting. This was further supported by a burst product formation. Judging from the accumulation level of C(55), C(60), and C(65), their dissociation from the enzyme cannot be too slow and an even slower enzyme conformational change with a rate of 0.001 s(-1) might govern the UPPs reaction rate under the steady-state condition in the absence of Triton.

24] Heptaprenylpyrophosphate synthetase from Bacillus subtilis

This chapter describes the isolation and purification of the heptaprenylpyrophosphate synthetase from Bacillus subtilis. Heptaprenylpyrophosphate synthetase is obtained from Bacillus subtilis, which produces menaquinone. This enzyme catalyzes the synthesis of all-trans-heptaprenyl pyrophosphate by successive condensation of four molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate as a primer all-trans-Geranylgeranyl pyrophosphate can also be a primer, but neither dimethylallyl nor geranyl pyrophosphate is accepted as a primer. This enzyme consists of two essential protein components, which are separated from each other by ion exchange chromatography. Principle and procedure of the assay method are discussed in the chapter. Heptaprenylpyrophosphate synthetase resembles geranylgeranylpyrophosphate synthetase in that neither enzyme is susceptible to monovalent cations or detergents. These properties suggest that the former two operate in intracellular environments closely related to each other, probably being peripherally associated with membrane so that undecaprenyi pyrophosphate of cis, trans-mixed stereochemistry can be efficiently synthesized, and that the latter two cooperate in the cytosol to produce alltrans-C 35 -prenyl pyrophosphate, which is the precursor for the side chain of menaquinone occurring in B. subtilis.

20] Purification of farnesylpyrophosphate synthetase by affinity chromatography

Publisher Summary This chapter describes the synthesis of an affinity column for farnesylpyrophosphate synthetase based on the geranyl moiety and a rapid purification of the enzyme from avian liver and yeast. Farnesylpyrophosphate synthetase is a 1'-4 prenyltransferase that produces a key intermediate in the isoprenoid pathway, which is the precursor for a variety of essential metabolites, including sterols, ubiquinones, dolichols, and some hemes. The enzyme synthesizes (E,E)-farnesyl pyrophosphate from dimethylallyl pyrophosphate and two molecules of isopentenyl pyrophosphate in two steps. Beginning with dimethylallyl pyrophosphate, a variety of products that differ in the length of the isoprenoid chain and the stereochemistry of the double bonds can be formed. A family of enzymes catalyzes 1'-4 prenyl transfers. Individual members present different substrate specificities based on chain length and double bond stereochemistry of the allylic substrate and produce five carbon homologs with exclusively E or Z trisubstituted double bonds. In principle, it should be possible to use the different substrate specificities to purify selectively individual members of the family from a crude homogenate by affinity chromatography. The product of the first step, geranyl pyrophosphate, binds to the enzyme more tightly than the other substrates or the final product and is therefore a logical candidate for the ligand in an affinity column to purify the enzyme.

Stereospecific studies on carotenoid biosynthesis.

Abstract

Studies on the biosynthesis of cholesterol XIX. Steric course of hydrogen eliminations and of C-C bond formations in squalene biosynthesis.

1. Three molecules of isopentenyl pyrophosphate are used in the biosynthesis of one molecule of farnesyl pyrophosphate. In this process, one hydrogen atom is added and three are eliminated : these three were all originally attached to C-4 of an earlier precursor, mevalonate. It is demonstrated that these eliminations are stereospecific and that all three occur in the same stereochemical sense. The absolute configuration of the eliminated hydrogen is defined. 2. In the association of three isopentenyl pyrophosphate molecules to form farnesyl pyrophosphate, two new C—C bonds are formed with expulsion of two pyrophosphate ions. It is demonstrated that both these processes occur with inversion of configuration at the pyrophosphate-bearing carbon atom. 3. In the biosynthesis of squalene from two molecules of farnesyl pyrophosphate, one farnesyl residue suffers exchange of one hydrogen atom and one new C—C bond is formed. It is demonstrated that this bond is formed with inversion of configuration at that farnesyl residue which does not exchange hydrogen.