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We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate, mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce [beta]-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 80 and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the 'universal' substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes. plant biochemistry | terpene synthases | cis-prenyltransferases | biochemical diversity | specialized metabolism

Liquid polyphosphate fertilizers have shown significant yield increases compared with granular orthophosphate fertilizer on highly P-fixing soils in Australian cropping trials. Polyphosphate fertilizers contain mixed P species (predominantly pyrophosphate and orthophosphate) and are therefore chemically different from most other inorganic P fertilizers, which contain only orthophosphate. To determine the mechanisms causing the yield responses to polyphosphate fertilizer, a method for investigating the isotopic exchangeability (lability) of P added as pyrophosphate was developed and tested. This method enabled the quantification of the hydrolysis reaction of pyrophosphate to orthophosphate in soil solution. As the rate of pyrophosphate applied increased, the proportion of pyrophosphate added that was isotopically exchangeable increased, and the percentage of pyrophosphate added that hydrolyzed to orthophosphate decreased. The method was further developed to test the hypothesis that pyrophosphate addition to soil may cause the mobilization of native orthophosphate reserves. The results suggested that there was no mobilization of native P after 7 d of incubation in two calcareous soils. Abbreviations: OP, orthophosphate; PP, pyrophosphate.

The thermodynamics of the conformational rearrangement step in the absence of magnesium is investigated for the wild type farnesyltransferase (FTase) and the Y300Fbeta mutant complexed with the peptide CVIM. The results show an increased transition barrier height for CVLM.

The article highlights that missense mutations in the gene cause a syndromic form of peripheral neuropathy associated with sensorineural hearing loss and optic neuropathy with the help of the cochlear expression database.

The article highlights that phosphoribosyl pyrophosphate synthetase 1 gene loss-of-function mutations cause arts syndrome.

A mutation in galU that causes the lack of O34-antigen lipopolysaccharide (LPS) in Aeromonas hydrophila strain AH-3 was identified. It was proved that A. hydrophila GalU is a UDP-glucose pyrophosphorylase responsible for synthesis of UDP-glucose from glucose 1-phosphate and UTP. The galU mutant from this strain showed two types of LPS structures, represented by two bands on LPS gels. The first one (slow-migrating band in gels) corresponds to a rough strain having the complete core, with two significant differences: it lacks the terminal galactose residue from the LPS-core and 4-amino-4-deoxyarabinose residues from phosphate groups in lipid A. The second one (fast-migrating band in gels) corresponds to a deeply truncated structure with the LPS-core restricted to one 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and three L-glycero-D-manno-heptose residues, galU mutants in several motile mesophilic Aeromonas strains from serotypes O1, O2, O11, O18, O21 and 044 were also devoid of the O-antigen LPS. The galU mutation reduced to less than 1% the survival of these Aeromonas strains in serum, decreased the ability of these strains to adhere and reduced by 1.5 or 2 log units the virulence of Aeromonas serotype O34 strains in a septicaemia model in either fish or mice. All the changes observed in the galU mutants were rescued by the introduction of the corresponding single wild-type gene.

Two newly isolated bacterial membrane-bound pyrophosphatase from the mesophile Methanosarcina mazei and the moderate thermophile Moorella thermoacetica that catalyze [Na.sup.} rather than [H.sup.} transport into Escherichia coli inner membrane vesicles (IMV) is demonstrated.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between [Leu.sup.102] and [Pro.sup.103] was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains.

The palatability and taste quality of pyrophosphates were evaluated in a series of behavioral and electrophysiological experiments. In two-bottle choice tests with water, rats strongly preferred some concentrations of [Na.sub.3]H[P.sub.2][O.sub.7] and [Na.sub.4][P.sub.2][O.sub.7], moderately preferred some concentrations of [K.sub.4][P.sub.2][O.sub.7] and [Fe.sub.4][([P.sub.2][O.sub.7]).sub.3], and were indifferent to or avoided all concentrations of [Ca.sub.2][P.sub.2][O.sub.7] and [Na.sub.2][H.sub.2][P.sub.2][O.sub.7]. The contribution of sodium to the preference for sodium pyrophosphates was ascertained: 1) Rats with a choice between [Na.sub.4][P.sub.2][O.sub.7] and NaCl preferred 1 mM [Na.sub.4][P.sub.2][O.sub.7] to 4 mM NaCl but preferred 40 or 150 mM NaCl to 10 mM [Na.sub.4][P.sub.2][O.sub.7], 2) blocking salt taste transduction by mixing [Na.sub.4][P.sub.2][O.sub.7] with amiloride reduced preferences but did not eliminate them, and 3) three mouse strains (FVB/J, C57BL/6J, and CBA/J) known to differ in sodium preference had the same rank order of preferences for [Na.sub.3]H[P.sub.2][O.sub.7] and NaCl, but peak preferences were higher for [Na.sub.3]H[P.sub.2][O.sub.7] than for NaCl. The taste qualities of pyrophosphates were determined by measuring taste-evoked responses of neurons in the nucleus of the solitary tract of rats. Across-neuron patterns of activity for sodium pyrophosphates were similar to that of NaCl but the pattern of [Na.sub.3]H[P.sub.2][O.sub.7] plus amiloride was unique from those of sweet, salty, sour, bitter, and umami stimuli. Taken together, the results indicate that the high palatability of some concentrations of [Na.sub.3]H[P.sub.2][O.sub.7] and [Na.sub.4][P.sub.2][O.sub.7] is due partially to their salty taste, but there must also be another cause, which may include a novel orosensory component distinct from the five major taste qualities. two-bottle preference test; gustatory electrophysiology; sodium; taste quality doi:10.1152/ajpregu.00886.2006

Coenzyme [Q.sub.10] (Co[Q.sub.10]) plays a pivotal role in oxidative phosphorylation (OXPHOS), as it distributes electrons among the various dehydrogenases and the cytochrome segments of the respiratory chain. We have identified [...]

Wheat yield is influenced by the efficacy of seed starch biosynthetic enzymes. ADP-glucose pyrophosphorylase (AGP) catalyzes a rate-limiting step in seed starch biosynthesis. We transformed the hard spring wheat (Triticum aestivum L.) cultivar Hi-Line with a modified maize AGP large subunit sequence (Sh2r6hs) to increase AGP activity. In previously described growth chamber studies, Sh2r6hs conditioned increased AGP activity, seed yield, and plant size. The primary objective of this study was to determine whether a similar yield enhancement could be detected under field conditions. Sh2r6hs transgenics were field tested over four growing years, in three locations, with varying planting density and irrigation. The results indicate that significant yield increases were more likely to occur in space-planted, irrigated environments than densely planted, rainfed environments, suggesting that limited abiotic resources may subsequently limit Sh2r6hs-associated yield enhancement. In elite lines, as in the [F.sub.2]--derived trials in which tissue culture derived mutations were reduced by out-crossing, Sh2r6hs appears to confer a yield advantage only when field conditions are nonlimiting.

Pyrophosphate is the main form of condensed P in the fluid fertilizer ammonium polyphosphate (APP). When APP is applied to soil, pyrophosphate is hydrolyzed to orthophosphate. Hydrolysis of pyrophosphate was investigated in a highly calcareous soil over 3 wk. Changes in P speciafion were measured using solid-state [sup.31]P nuclear magnetic resonance (NMR) spectroscopy for dried soil samples and ion chromatography for NaOH extracts. Both techniques showed a decrease in pyrophosphate and concomitant increase in orthophosphale concentration with time, but the rate of pyrophosphate hydrolysis calculated from the NaOH extract measured by ion chromatography was two times faster than the rate calculated from the NMR data indicating differences in P speciation between methods. The NMR technique employed spin counting to determine the proportion of P nuclei observable by NMR. Since this proportion (71-88%) was greater than the proportion of P extracted by NaOH and detected by ion chromatography (38-71%), we concluded that the NMR technique was able to quantify more of the pyrophosphate added than the extraction method. The results of the study suggest that solid-state [sup.31]P NMR is a powerful noninvasive technique for the investigation of pyrophosphate reactions in soil and that pyrophosphate has the ability to persist for several weeks in highly calcareous Australian soils in the presence of microbial activity. This has important implications for P availability processes where pyrophosphale is supplied as a fertilizer and is currently under investigation in Australian soil types.

In bacteria, thiamine pyrophosphate (TPP) is an essential cofactor that is synthesized de novo. Thiamine, however, is not an intermediate in the biosynthetic pathway but is salvaged from the environment and phosphorylated to TPP. We have isolated and characterized new mutants of Bacillus subtilis that deregulate thiamine biosynthesis and affect the export of thiamine products from the cell. Deletion of the ydiA gene, which shows significant similarity to the thiamine monophosphate kinase gene of Escherichia coli (thiL), did not generate the expected thiamine auxotroph but instead generated a thiamine bradytroph that grew to near-wild-type levels on minimal medium. From this [DELTA]thiL deletion mutant, two additional ethyl methanesulfonate-induced mutants that derepressed the expression of a thiC-lacZ transcriptional reporter were isolated. One mutant, Tx1, contained a nonsense mutation within the B. subtilis yloS (thiN) gene that encodes a thiamine pyrophosphokinase, a result which confirmed that B. subtilis contains a single-step, yeast-like thiamine-to-TPP pathway in addition to the bacterial TPP de novo pathway. A second mutant, strain Tx26, was shown to contain two lesions. Genetic mapping and DNA sequencing indicated that the first mutation affected yuaJ, which encodes a thiamine permease. The second mutation was located within the ykoD cistron of the ykoFEDC operon, which putatively encodes the ATPase component of a unique thiamine-related ABC transporter. Genetic and microarray studies indicated that both the mutant yuaJ and ykoD genes were required for the derepression of thiamine-regulated genes. Moreover, the combination of the four mutations (the [DELTA]thiL, thiN, yuaJ, and ykoD mutations) into a single strain significantly increased the production and excretion of thiamine products into the culture medium. These results are consistent with the proposed 'riboswitch' mechanism of thiamine gene regulation (W. C. Winkler, A. Nahvi, and R. R. Breaker, Nature 419:952-956, 2002).

Hexaprenyl pyrophosphate synthase (HexPPs) from Sulfolobus solfataricus catalyzes the synthesis of trans-[C.sub.30]-hexaprenyl pyrophosphate (HexPP) by reacting two isopentenyl pyrophosphate molecules with one geranyigeranyl pyrophosphate. The crystal structure of the homodimeric [C.sub.30]-HexPPs resembles those of other trans-prenyitransferases, 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 ([alpha]I, [alpha]J, [alpha]K, and part of [alpha]H) 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.

Human GTP-L-fucose pyrophosphorylase (GFPP) substrate discrimination that was dissected with a series of analogues is described. The combined substrate binding and enzyme analysis has revealed that the nature of the purine base is the major determinant in substrate specificity followed by the nature of the hexose-1-P and then by the ribose moiety.

The pnhA gene of Pasteurella muitocida encodes PnhA, which is a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases. PnhA hydrolyzes diadenosine tetra-, penta-, and hexaphosphates with a preference for diadenosine pentaphosphate, from which it forms ATP and ADP. PnhA requires a divalent metal cation, [Mg.sup.2+] or [Mn.sup.2+], and prefers an alkaline pH of 8 for optimal activity. A P. multocida strain that lacked a functional pnhA gene, ACP13, was constructed to further characterize the function of PnhA. The cellular size of ACP13 was found to be 60% less than that of wild-type P. multocida, but the growth rate of ACP13 and its sensitivity to heat shock conditions were similar to those of the wild type, and the wild-type cell size was restored in the presence of a functional pnhA gene. Wild-type and ACP13 strains were tested for virulence by using the chicken embryo lethality model, and ACP13 was found to be up to 1,000-fold less virulent than the wild-type strain. This is the first study to use an animal model in assessing the virulence of a bacterial strain that lacked a dinucleoside oligophosphate pyrophosphatase and suggests that the pyrophosphatase PnhA, catalyzing the hydrolysis of diadenosine pentaphosphates, may also play a role in facilitating P. multocida pathogenicity in the host.

Extracellular matrix (ECM) mineralization is a physiological process in bone and pathological one in soft tissues. A study reveals that coexpression in osteoblasts of otherwise broadly expressed genes is necessary and sufficient to induce bone mineralization and also provides evidence that pathological mineralization is prevented by modulating extracellular phosphate concentration.

Alignment of more than 20 deduced sequences for mevalonate diphosphate decarboxylase (MDD) indicates that the serines 34, 36, 120, 121, 153, and 155 are invariant residues that map within a proposed interdomain active site cleft. It is found that the catalyst consequences of mutating invariant series 36,120,153 and 155 are modest [

The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP). The pre-steady state and steady state methods were used to show that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond.

Two genes atmG and ggsA are cloned from Aspergillus flavus using degenerate primers for conserved domains of fungal geranylgeranyl diphosphate synthases. The study broadens ones understanding of indole-diterpene metabolite biosynthesis in filamentous fungi and provides the first genetic evidence for the biosynthetic pathway of aflatrem in Aspergillus flavus.

The physiological role of the membrane-bound pyrophosphatase of Rhodospirillum rubrum was investigated by the characterization of a mutant strain. Comparisons of growth levels between the wild type and the mutant under different low-potential conditions and during transitions between different metabolisms indicate that this enzyme provides R. rubrum with an alternative energy source that is important for growth in low-energy states.

Soluble and membrane-bound inorganic pyrophosphatases (sPPase and H+-PPase, respectively) of the purple nonsulfur bacterium Rhodospirillum rubrum are differentially regulated by environmental growth conditions. Both proteins and their transcripts were found in cells of anaerobic phototrophic batch cultures along all growth phases, although they displayed different time patterns. However, in aerobic cells that grow in the dark, which exhibited the highest growth rates, Northern and Western blot analyses as well as activity assays demonstrated high sPPase levels but no H+-PPase. It is noteworthy that H+-PPase is highly expressed in aerobic cells under acute salt stress (1 M NaCl). H+-PPase was also present in anaerobic cells growing at reduced rates in the dark under either fermentative or anaerobic respiratory conditions. Since H+-PPase was detected not only under all anaerobic growth conditions but also under salt stress in aerobiosis, the corresponding gene is not invariably repressed by oxygen. Primer extension analyses showed that, under all anaerobic conditions tested, the R. rubrum H+-PPase gene utilizes two activator-dependent tandem promoters, one with an FNR-like sequence motif and the other with a RegA motif, whereas in aerobiosis under salt stress, the H+-PPase gene is transcribed from two further tandem promoters involving other transcription factors. These results demonstrate a tight transcriptional regulation of the H+-PPase gene, which appears to be induced in response to a variety of environmental conditions, all of which constrain cell energetics.

The thermophilic properties of the choline-binding tag (ChB tag) is used by fusing it to the pyrophosphatase (Pyr) from Thermus thermophilus in order to facilitate its purification and immobilization. This fusion has revealed novel and interesting biotechnological applications for a thermostable tag.

Stringent control mediated by the bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp) is a key regulatory process governing bacterial gene expression. By devising a system to measure ppGpp in plants, we have been able to identify ppGpp in the chloroplasts of plant cells. Levels of ppGpp increased markedly when plants were subjected to such biotic and abiotic stresses as wounding, heat shock, high salinity, acidity, heavy metal, drought, and UV irradiation. Abrupt changes from light to dark also caused a substantial elevation in ppGpp levels. In vitro, chloroplast RNA polymerase activity was inhibited in the presence of ppGpp, demonstrating the existence of a bacteria-type stringent response in plants. Elevation of ppGpp levels was elicited also by treatment with plant hormones jasmonic acid, abscisic acid, and ethylene, but these effects were blocked completely by another plant hormone, indole-3-acetic acid. On the basis of these findings, we propose that ppGpp plays a critical role in systemic plant signaling in response to environmental stresses, contributing to the adaptation of plants to environmental changes.

Stable and regular shaped colloidal nanocrystals are seen in a dilute electrostatically stabilized aqueous solution of lipid A diphosphate. It is reported that they are fragile and easily damaged when exposed to the electron beam.

A study is conducted to demonstrate that significant amounts of germacrene A are indeed released from wild-type aristolochene synthase (AS) from pencillium roqueforti. Germacrene Awas is identified through direct GC-MS comparison to an authentic sample and through production of beta-elemene in thermal Cope rearrangements.

The two hydrogen bonding motifs for the title module, a C4h eclipsed and a D4 staggered structure is identified by the density functional calculations at the B3LYP/LanL2DZ+(f,d,p) level. The two structures can be distinguished in an X-ray crystallographic analysis on the basis of the Pt-P-O-P dihedral angle without the need to explicitly locate hydrogen atoms in the structure.

Free diffusive transfer of (+)-copalyl diposphate between two active sites is investigated with regard to the bifunctional abietadiene synthase.

Research is presented concerning the investigation of the formation of diphosphate from 2-hydroxy-2-oxo-5,5-dimethyl-4-(R)-phenyl-1,3,2-dioxaphosphorninane and (R)-2-chloro-2-oxo-5,5-dimethyl-4-(R)-phenyl-1,3,2-dioxaphosphorinane.

The synthesis, characterization and conformational analysis of a sterically congested pyrophosphite are described. A specific parameterization of the pyrophosphite structure, within the AMBER force field is determined by a fit to ab initio data at the MP2/6-31G(d) level.

Research has been conducted on the medium-chain (E)-prenyl diphosphate synthases and their heterodimeric structures. The substitution of some amino acid residues in hexaprenyl diphosphate synthase subunits by site-selected mutagenesis has been carried out to examine the product chain length determination mechanism of these enzymes.

Two enzyme-pyrophosphate intermediates are formed during the reaction of cytoplasmic pyrophosphatase on pyrophosphate. The conversion of the intermediates is the rate-limiting step during synthesis and a partially rate-limiting step during hydrolysis.

Research has been conducted on the nucleoside diphosphate kinase (Ndk). The localization of the Ndk secretion motif has been carried out.

Research into the structural and conformational behaviour of the sugar-diphosphate linkage is presented. It was established that interactions of the diphosphate group with the Mg2+ cation alter the conformational preferences around the diphosphate linkage.

Research is presented describing the study of diphosphate nucleotides and thymidine mono in a water solution.

Research has been conducted on the N-terminal fragment of adenosine diphosphate ribosylation factor 1. Results indicate that there may be association of the bilayer disk peptides through the surface and edge interactions combination.

Research has been conducted on the squalene synthase. The catalysis of two reactions in sterol biosynthesis to form squalene and cyclopropylcarbinyl intermediate presqualene diphosphate is described.

Research has been conducted on the Streptomyces sp. strain CL190. Results indicate that this strain utilizes mevalonate and nonmevalonate pathways for the isopentenyl diphosphate biosynthesis.

A study concerning independent active site of farnesyl diphosphate synthase subunits is presented. Construction of expression plasmids overproducing hybrid-type heterodimers of Bacillus stearothermophilus is discussed.

Research has been conducted on the O,O-alkylene dithiophosphate derivatives of halodimethyltellurium(IV). The synthesis of the compound by the reaction in dichloromehtane is described.

Cell cultures of Catharanthus roseus were supplied with [2-13C,3-2H]-deoxyxylulose or [2-13C,4-2H]1-deoxyxylulose. Lutein and chlorophylls were isolated from the cell mass, and hydrolysis of the chlorophyll mixtures afforded phytol. Isotope labeling patterns of phytol and lutein were determined by 2H NMR and 1H,2H-decoupled 13C NMR. From the data it must be concluded that the deuterium atom in position 3 of deoxyxylulose was incorporated into both isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate with a rate of 75% (with respect to the internal 13C label). The detected stereochemical signature implies that the label is located preferentially in the (E)-hydrogen atom of IPP. This preferential labeling, in turn, rules out dimethylallyl pyrophosphate as the compulsory precursor of IPP. In the experiment with [2-13C,4-2H]1-deoxyxylulose, the 13C label was efficiently transferred to the terpenoids whereas the 2H label was completely washed out, most probably after IPP formation as a consequence of the isomerization and elongation process. In addition, the data cast light on the stereochemical course of the dehydrogenation and cyclization steps involved in the biosynthesis of lutein.

Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: [Alpha]-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield. Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP. Escherichia coli cells expressing wild type (WT) Shrunken2 (Sh2), and Brittle2 (Bt2) exhibit a reduced capacity to produce glycogen when grown at 42 [degrees] C. Mutagenesis of Sh2 and coexpression with WT Bt2 led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature. An increase in AGP stability was found in each of four mutants examined. Initial characterization revealed that the BT2 protein was elevated in two of these mutants. Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly. These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits. In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the routants, Sh2hs33, identified a greater propensity for heterotetramer dissociation in WT AGP. Sequencing of Sh2hs33 and several other mutants identified a His-to-Tyr mutation at amino acid position 333. Hence, a single point mutation in Sh2 can increase the stability of maize endosperm AGP through enhanced subunit interactions.

The biosyntheses of inositol diphosphates, diphosphoinositol pentakisphosphate (PP-IP5) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4), were characterized through the purification and identification of the inositol hexakisphosphate kinase (IP6-K) from rat brain. It was established that IP6-K can move the terminal phosphate of the PP-IP5 diphosphate to adenosine diphosphate to form adenosine triphosphate (ATP). It was discovered that PP-IP5-K contributed to the formation of bis-PP-IP4 and that purified PP-IP5-K was capable of the ATP synthase function.

Several eubacteria including Esherichia coil use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-D-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-D-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-D-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 rain on the chromosomal map of E. coil. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E. coli and found to catalyze the formation of 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-o-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required [Mn.sup.2+], [Co.sup.2+], or [Mg.sup.2+] as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-o-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-D-erythritol 4-phosphate from l-deoxy-o-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E. coli.