polyP Synthesis Research Articles

How inositol pyrophosphates control cellular phosphate homeostasis?

Phosphorus in his phosphate PO(4)(3-) configuration is an essential constituent of all life forms. Phosphate diesters are at the core of nucleic acid structure, while phosphate monoester transmits information under the control of protein kinases and phosphatases. Due to these fundamental roles in biology it is not a surprise that phosphate cellular homeostasis is under tight control. Inositol pyrophosphates are organic molecules with the highest proportion of phosphate groups, and they are capable of regulating many biological processes, possibly by controlling energetic metabolism and adenosine triphosphate (ATP) production. Furthermore, inositol pyrophosphates influence inorganic polyphosphates (polyP) synthesis. The polymer polyP is solely constituted by phosphate groups and beside other known functions, it also plays a role in buffering cellular free phosphate [Pi] levels, an event that is ultimately necessary to generate ATP and inositol pyrophosphate. Although it is not yet clear how inositol pyrophosphates regulate cellular metabolism, understanding how inositol pyrophosphates influence phosphates homeostasis will help to clarify this important link. In this review I will describe the recent literature on this topic, with in the hope of inspiring further research in this fascinating area of biology.

Amino Acids Involved in Polyphosphate Synthesis and Its Mobilization Are Distinct in Polyphosphate Kinase-1 from Mycobacterium tuberculosis

BackgroundIn bacteria polyphosphates (poly-P) are involved in cellular metabolism and development especially during stress. The enzyme, principally involved in polyphosphate biosynthesis and its mobilization leading to generation of NTPs, is known as polyphosphate kinase (PPK).Principal FindingsAmong two genes of polyphosphate kinases present in Mycobacterium tuberculosis, we cloned and expressed PPK1 in Escherichia coli as histidine-tagged protein. This ∼86 kDa protein is capable of autophosphorylation and synthesis of poly-P as well as NTP. Among 22 conserved histidine residues, we found only His-491 is autophosphorylated and crucial for any enzymatic activity. Substitution of His-510 caused mPPK1 protein deficient but not defective in autophosphorylation, thereby contrary to earlier reports negating any role of this residue in the process. However, mutation of His-510 with either Ala or Gln affected ATP or poly-P synthesis depending on the substitution; while such effects were severe with H510A but mild with H510Q. Furthermore, mPPK1 also renders auxiliary nucleotide diphosphate kinase function by synthesizing virtually all NTPs/dNTPs from their cognate NDPs/dNDPs by utilizing poly-P as the phosphate donor albeit with varied efficiency. To assess the influence of other catalytic domain residues of mPPK1 towards its functionality, we designed mutations based on E. coli PPK1 crystal structure since it owes 68% amino acid sequence similarity with mPPK1. Interestingly, our results revealed that mutations in mPPK1 affecting poly-P synthesis always affected its ATP synthesizing ability; however, the reverse may not be true.Conclusions/SignificanceWe conclude that amino acid residues involved in poly-P and ATP synthesizing activities of mPPK1 are distinct. Considering conserved nature of PPK1, it seems our observations have broader implications and not solely restricted to M. tuberculosis only.

Aptamer-Mediated Inhibition of Mycobacterium tuberculosis Polyphosphate Kinase 2

Inorganic polyphosphate (polyP) plays a number of critical roles in bacterial persistence, stress, and virulence. PolyP intracellular metabolism is regulated by the polyphosphate kinase (PPK) protein families, and inhibition of PPK activity is a potential approach to disrupting polyP-dependent processes in pathogenic organisms. Here, we biochemically characterized Mycobacterium tuberculosis (MTB) PPK2 and developed DNA-based aptamers that inhibit the enzyme's catalytic activities. MTB PPK2 catalyzed polyP-dependent phosphorylation of ADP to ATP at a rate 838 times higher than the rate of polyP synthesis. Gel filtration chromatography suggested MTB PPK2 to be an octamer. DNA aptamers were isolated against MTB PPK2. Circular dichroism revealed that aptamers grouped into two distinct classes of secondary structure; G-quadruplex and non-G-quadruplex. A selected G-quadruplex aptamer was highly selective for binding to MTB PPK2 with a dissociation constant of 870 nM as determined by isothermal titration calorimetry. The binding between MTB PPK2 and the aptamer was exothermic yet primarily driven by entropy. This G-quadruplex aptamer inhibited MTB PPK2 with an IC(50) of 40 nM and exhibited noncompetitive inhibition kinetics. Mutational mechanistic analysis revealed an aptamer G-quadruplex motif is critical for enzyme inhibition. The aptamer was also tested against Vibrio cholerae PPK2, where it showed an IC(50) of 105 nM and insignificant inhibition against more distantly related Laribacter hongkongensis PPK2.

The early stage of polyphosphate accumulation in <i>saccharomyces cerevisiae</i>: comparative study by extraction and DAPI staining

Inorganic polyphosphate (PolyP), a bioactive polymer with multiple functions, plays a key role in biomineralizaion and phosphorus homeostasis in yeasts. After phosphate starvation, the cells of Saccharomyces cerevisiae restored their pool of PolyP during the first 30 min of incubation in the media containing phosphate and carbon sources. The cells of parent strain accumulated PolyP both in glucose and ethanol-containing media. In the medium with glucose, the strain with inactivated PPX1 and PPN1 genes (encoding two major yeast polyphosphatases) accumulated 2-fold more PolyP than the parent strain. The PolyP in the mutant cells had a greater average chain length compared to the parent strain. The strain with inactivated exopolyphosphatase genes РРХ1 and PPN1 was incapable of PolyP synthesis in the medium with ethanol. The in vivo staining of cells with DAPI show that in the cells of parent strain PolyP appeared first in cytoplasm and mitochondria under cultivation in glucose-containing medium and in cytoplasm and vacuoles in the medium with ethanol. In the ΔPPX1ΔPPN1double mutant PolyP accumulated in cytoplasm and vacuoles under cultivation in glucose-contained medium.

Effect of cycloheximide, iodoacetamide, and antimycin A on inorganic phosphate synthesis in Saccharomyces cerevisiae VKM Y-1173

The effect of inhibitors of protein synthesis (cycloheximide, CHI), glycolysis (iodoacetamide, IAA), and oxidative phosphorylation (antimycin A, ANM) on inorganic phosphate (polyP) synthesis during the first 0.5 h of their hypercompensation in Saccharomyces cerevisiae VKM Y-l173 grown on 2% glucose-containing media at low (hypoxia) or high aeration rates or in the presence of 1 vol % ethanol under high aeration conditions was studied. PolyP accumulation was highest in the medium with glucose under hypoxia; lower, with glucose at high aeration; and lowest, in the medium with ethanol. CHI had a small effect on the total polyP level but significantly stimulated ATP accumulation, irrespective of the culture growth conditions. The low-polymer acid-soluble polyP1 were synthesized most actively by the cells grown on glucose under hypoxia, alkali-soluble polyP3 were synthesized at en hanced aeration, and the most hig-molecular fraction, polyP5, was actively accumulated along with polyP3 at cultivation on ethanol. Regardless of the growth conditions, CHI inhibited accumulation of polyP4, the synthesis of which is associated with the synthesis of mannoproteins. IAA and ANM largely inhibited synthesis of all fractions at yeast growth under hypoxia and on ethanol, respectively. The results as a whole demonstrate the dependence of polyP formation on the main energy-generating cell processes and, at the same time, the absence of direct dependence of their synthesis on ATP concentration in Saccharomyces cerevisiae VKM Y-l 173.

Influence of nitrogen and phosphorus on polyphosphate accumulation in Gigaspora margarita during spore germination

Polyphosphate (polyP) is the form in which phosphorus (P) is transferred from extraradical hyphae into arbuscles in the symbiotic stage of arbuscular mycorrhizal fungi. However, polyP dynamics in the presymbiotic stage are less understood. In this study, we aimed to investigate polyP accumulation in Gigaspora margarita as influenced by nitrogen (N) and/or P supply during germination. Spores of G. margarita were incubated on medium with or without P or N addition. PolyP content in the fungal tissue was monitored using a polyP kinase/luciferase system, and polyP synthetic activity was determined with 32P labeling. The results showed that both N and P were necessary for polyP accumulation in germ tubes. Nitrate increased the polyP content in germ tubes, but ammonium did not. Along with germination, polyP content decreased in spores, but increased in germ tubes. 32P labeling indicated that polyP synthetic activity increased in germ tubes along with germination, but was negligible in spores. Our results suggest that, in the presymbiotic stage of G. margarita, uptake of environmental N and P increases polyP content in germ tubes, and that polyP synthesis occurs mainly therein, leading to polyP accumulation. The possible mechanism of transfer of polyP from spores to hyphae remains to be elucidated.

Importance of Polyphosphate Kinase 1 for Campylobacter jejuni Viable-but-Nonculturable Cell Formation, Natural Transformation, and Antimicrobial Resistance

Campylobacter jejuni, a gram-negative, microaerophilic bacterium, is a predominant cause of bacterial gastroenteritis in humans. Although considered fragile and fastidious and lacking many classical stress response mechanisms, C. jejuni exhibits a remarkable capacity for survival and adaptation, successfully infecting humans and persisting in the environment. Consequently, understanding the physiological and genetic properties that allow C. jejuni to survive and adapt to various stress conditions is crucial for therapeutic interventions. Of importance is polyphosphate (poly-P) kinase 1 (PPK1), which is a key enzyme mediating the synthesis of poly-P, an essential molecule for survival, mediating stress responses, host colonization, and virulence in many bacteria. Therefore, we investigated the role of PPK1 in C. jejuni pathogenesis, stress survival, and adaptation. Our findings demonstrate that a C. jejuni Deltappk1 mutant was deficient in poly-P accumulation, which was associated with a decreased ability to form viable-but-nonculturable cells under acid stress. The Deltappk1 mutant also showed a decreased frequency of natural transformation and an increased susceptibility to various antimicrobials. Furthermore, the Deltappk1 mutant was characterized by a dose-dependent deficiency in chicken colonization. Complementation of the Deltappk1 mutant with the wild-type copy of ppk1 restored the deficient phenotypes to levels similar to those of the wild type. Our results suggest that poly-P plays an important role in stress survival and adaptation and might contribute to genome plasticity and the spread and development of antimicrobial resistance in C. jejuni. These findings highlight the potential of PPK1 as a novel target for therapeutic interventions.

Polyphosphate Kinase Affects Oxidative Stress Response by Modulating cAMP Receptor Protein and rpoS Expression in Salmonella Typhimurium

Polyphosphate (polyP) plays diverse physiological functions in prokaryotes and eukaryotes, but most of their detailed mechanisms are still obscure. Here, we show that deletion of polyphosphate kinase (PPK), the principal enzyme responsible for synthesis of polyP, resulted in augmented expression of cAMP receptor protein (CRP) and rpoS and lowered H2O2 sensitivity in Salmonella Typhimurium ATCC14028. The binding of cAMP-CRP complex to rpoS promoter and further stimulation of its transcription were proved through electrophoretic mobility shift assay, lacZ fusion, and exogenous cAMP addition, respectively. The rpoS expression increased in cpdA (cAMP phosphodiesterase coding gene) mutant, further suggesting that cAMP-CRP upregulated rpoS expression. These results demonstrate that PPK affects oxidative stress response by modulating crp and rpoS expression in S. Typhimurium.

Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria

Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of phosphate residues linked by high-energy bonds. It is found in all organisms and has been proposed to serve as an energy source in a pre-ATP world. This ubiquitous and abundant biopolymer plays numerous and vital roles in metabolism and regulation in prokaryotes and eukaryotes, but the underlying molecular mechanisms for most activities of polyP remain unknown. In prokaryotes, the synthesis and utilization of polyP are catalyzed by 2 families of polyP kinases, PPK1 and PPK2, and polyphosphatases. Here, we present structural and functional characterization of the PPK2 family. Proteins with a single PPK2 domain catalyze polyP-dependent phosphorylation of ADP to ATP, whereas proteins containing 2 fused PPK2 domains phosphorylate AMP to ADP. Crystal structures of 2 representative proteins, SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomonas aeruginosa, revealed a 3-layer alpha/beta/alpha sandwich fold with an alpha-helical lid similar to the structures of microbial thymidylate kinases, suggesting that these proteins share a common evolutionary origin and catalytic mechanism. Alanine replacement mutagenesis identified 9 conserved residues, which are required for activity and include the residues from both Walker A and B motifs and the lid. Thus, the PPK2s represent a molecular mechanism, which potentially allow bacteria to use polyP as an intracellular energy reserve for the generation of ATP and survival.

Polyphosphate Kinase 1 Is a Pathogenesis Determinant in Campylobacter jejuni

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, relatively little is known about C. jejuni's precise pathogenesis mechanisms, particularly in comparison to other well-studied enteric organisms such as Escherichia coli and Salmonella spp. Altered expression of phosphate genes in a C. jejuni stringent response mutant, together with known correlations between the stringent response, polyphosphate (poly-P), and virulence in other bacteria, led us to investigate the role of poly-P in C. jejuni stress survival and pathogenesis. All sequenced C. jejuni strains harbor a conserved putative polyphosphate kinase 1 predicted to be principally responsible for poly-P synthesis. We generated a targeted ppk1 deletion mutant (Deltappk1) in C. jejuni strain 81-176 and found that Deltappk1, as well as the DeltaspoT stringent response mutant, exhibited low levels of poly-P at all growth stages. In contrast, wild-type C. jejuni poly-P levels increased significantly as the bacteria transitioned from log to stationary phase. Phenotypic analyses revealed that the Deltappk1 mutant was defective for survival during osmotic shock and low-nutrient stress. However, certain phenotypes associated with ppk1 deletion in other bacteria (i.e., motility and oxidative stress) were unaffected in the C. jejuni Deltappk1 mutant, which also displayed an unexpected increase in biofilm formation. The C. jejuni Deltappk1 mutant was also defective for the virulence-associated phenotype of intraepithelial cell survival in a tissue culture infection model and exhibited a striking, dose-dependent chick colonization defect. These results indicate that poly-P utilization and accumulation contribute significantly to C. jejuni pathogenesis and affect its ability to adapt to specific stresses and stringencies. Furthermore, our study demonstrates that poly-P likely plays both similar and unique roles in C. jejuni compared to its roles in other bacteria and that poly-P metabolism is linked to stringent response mechanisms in C. jejuni.

Inorganic polyphosphate and exopolyphosphatase in the nuclei of Saccharomyces cerevisiae: dependence on the growth phase and inactivation of the PPX1 and PPN1 genes

Nuclei of the yeast Saccharomyces cerevisiae possess inorganic polyphosphates (polyP) with chain lengths of ca. 10-200 phosphate residues. Subfractionation of the nuclei reveals that the most part of polyP is not associated with DNA. Transition of the yeast cells from stationary phase to active growth at orthophosphate (P(i)) excess in the medium is followed by the synthesis of the shortest polyP (<15 phosphate residues) and hydrolysis of the high-molecular polyP (>45 phosphate residues) in the nuclei. Nuclear exopolyphosphatase (exopolyPase) activity does not depend on the growth phase. The PPX1 gene encoding the major cytosolic exopolyPase does not encode the nuclear one and its inactivation has no effect on polyP metabolism in this compartment. Under inactivation of the PPN1 gene encoding another yeast exopolyPase, elimination of the nuclear exopolyPase is observed. The effect of PPN1 inactivation on the polyP level in the nuclei is insignificant in the stationary phase, while in the exponential phase this level increases 2.3-fold as compared with the parent strain of S. cerevisiae. In the active growth phase, no hydrolysis of high-molecular polyP is detected while the synthesis of short-chain polyP is retained. The data obtained indicate substantial changes in polyP metabolism in nuclei under the renewal of active growth, which only partially depends on the genes of polyP metabolism known to date.

Antibaterial effect of Scutellariae Radix on wild type and mutants of E. coli

Inorganic polyphosphate(poly P)is having been found in all microbes, fungi, plants and animals and Human. In E. coli, an enzyme responsible for polyP synthesis is the polyphosphate kinase(ppk), exopolyphospatase(ppx), which hydrolysed the terminal residues of polyP, was discovered at same operon. Natural herbal extract of Scutellariae Radix(Scutellaria baicalensis George) have been used as antibacterial substance against infected bacteria. Scutellariae Radix extract showed remarkable antimicrobial activity against S. typhimurium, but little attention has been paid to the selective antibacterial activity against pathogenic and nonpathogenic bacteria. This study suggests the possible mechanism(s) of Scutellariae Radix for the selectable antibacterial activities in terms of the relationship between ppk and other relating regulatory genes(sspA and hns)on E.coli. Wild type and ppk mutants strains of E. coli were grown up to 5 mg/ml of Scutellariae Radix containing media. It shows significant differences between deletion mutants and wild type strains on treatment of Scutellariae Radix. Additionally, the results showed not only increased growth rate but also the regulation of gene expression on sspA and hns. Dependent on activities of ppk, the transcriptional activities of sspA and hns altered significantly. So, it suggests that Scutellariae Radix stimulate the transcription(s) either directly or indirectly to ppk and other ppk-relating genes.

Crystal structure of a polyphosphate kinase and its implications for polyphosphate synthesis.

Polyphosphate (polyP), a linear polymer of hundreds of orthophosphate residues, exists in all tested cells in nature, from pathogenic bacteria to mammals. In bacteria, polyP has a crucial role in stress responses and stationary-phase survival. Polyphosphate kinase (PPK) is the principal enzyme that catalyses the synthesis of polyP in bacteria. It has been shown that PPK is required for bacterial motility, biofilm formation and the production of virulence factors. PPK inhibitors may thus provide a unique therapeutic opportunity against antibiotic-resistant pathogens. Here, we report crystal structures of full-length Escherichia coli PPK and its complex with AMPPNP (beta-gamma-imidoadenosine 5-phosphate). PPK forms an interlocked dimer, with each 80 kDa monomer containing four structural domains. The PPK active site is located in a tunnel, which contains a unique ATP-binding pocket and may accommodate the translocation of synthesized polyP. The PPK structure has laid the foundation for understanding the initiation of polyP synthesis by PPK.

BIOCHEMICAL MODEL AND MECHANISM FOR ACINETOBACTER NITRITE INHIBITION

Nitrite accumulation is not unusual in batch processes such as sequencing batch reactor (SBR) with high-strength of ammonium or nitrate wastewaters. A possible mechanism of nitrite inhibition on Acinetobacter was depicted in a biochemical model, which the protonated species, nitrous acid form of nitrite, affects proton relating transport at the proton-pumping site crossing the cell membrane under unlimited carbon and phosphorus conditions. This effect exerts inhibition of phosphorylation under aerobic condition and yields low APT/ADP ratio, consequently decrease poly-P synthesis and phosphorus uptake from outside the cell in the model.⠊礀Ѐ㔶〻ᨀ䙯獳楬猠☠灲敨楳瑯物挠汩晥

Phosphate pool dynamics in the arbuscular mycorrhizal fungus Glomus intraradices studied by in vivo31 P NMR spectroscopy.

• Polyphosphate (polyP) is presumably central to phosphate (P) metabolism of arbuscular mycorrhizal (AM) fungi, but its synthesis, location and chain lengths are poorly characterized. Here, we applied noninvasive and nondestructive nuclear magnetic resonance (NMR) spectroscopy to obtain novel information on AM fungal polyP. • In vivo 31 P NMR spectroscopy was used to characterize polyP and other P pools in external hyphae and in mycorrhizal roots of associations between Glomus intraradices and cucumber (Cucumis sativus). • A time-course study of P-starved external hyphae supplied with additional P showed that polyP appeared more rapidly than vacuolar inorganic P. These P metabolites also appeared in the roots, but later. PolyP considerably exceeded amounts of vacuolar inorganic P, where it was located in acidic, presumably vacuolar compartments, and had a short average chain length. • The rapid synthesis of polyP might be important for the maintenance of effective hyphal P uptake. Our data support the hypothesis that polyP is the major P species translocated in the tubular vacuolar network, the presence of which was previously demonstrated in AM fungi.

Intracellular phosphorus metabolism of Microcystis aeruginosa under various redox potential in darkness

Phosphorus metabolism of Microcystis aeruginosa was studied under gradient redox potential from 252 mV to -70 mV in darkness. The release of phosphorus occurred in all the treatments, and this process was accelerated in darkness when the redox potential was lowered. Low redox potential in darkness stimulated the accumulation of polyphosphate (PolyP) and the degradation of polyglucose. The synthesis of PolyP delayed the decrease of intracellular orthophosphate. The death of M. aeruginosa was slowered when the redox potential was low in darkness. The accumulation of PolyP under low redox potential in the dark was very important to M. aeruginosa for endurance through the unfavorable growth conditions for maintaining phosphorus concentration, energy storage, and other physiological functions. The ability to accumulate PolyP in the dark and negative redox potential may be of considerable advantage in the low-light, organically rich, and low-redox habitats.

The content and chain length of polyphosphates from vacuoles of Saccharomyces cerevisiae VKM Y-1173.

The content of inorganic polyphosphates (polyP) in vacuoles of the yeast Saccharomyces cerevisiae is approximately 15% of the total cellular polyP. Over 80% of the vacuole polyP are in an acid-soluble fraction. It was first established by (31)P-NMR spectroscopy that a polymeric degree (n) of two subfractions obtained by precipitation with Ba(2+) in succession at pH 4.5 and 8.2 was approximately 20 +/- 5 and 5 +/- 2 residues of ortho-phosphoric acid, respectively. Under a deficit of phosphate (P(i)) in the cultivation medium, the polyP content in vacuoles decreased approximately 7-fold with the same drastic reduction of their content in the cell. Unlike intact yeast cells, where polyP overcompensation is observed after their transfer from phosphate-free to phosphate-containing medium, the vacuoles do not show this effect. The data indicate the occurrence of special regulatory mechanisms of polyP synthesis in vacuoles differing from those in the whole cell.

Polyphosphate and phosphate pump.

In microbial cells, inorganic polyphosphate (polyP) plays a significant role in increasing cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. polyP is a polyfunctional compound. The most important of its functions are the following: phosphate and energy reservation, cation sequestration and storage, membrane channel formation, participation in phosphate transport, involvement in cell envelope formation and function, gene activity control, regulation of enzyme activities, and a vital role in stress response and stationary-phase adaptation. The functions of polyP have changed greatly during the evolution of living organisms. In prokaryotes, the most important functions are as an energy source and a phosphate reserve. In eukaryotic microorganisms, the regulatory functions predominate. Therefore, a great difference is observed between prokaryotes and eukaryotes in their polyP-metabolizing enzymes. Some key prokaryotic enzymes are not present in eukaryotes, and conversely, eukaryotes have developed new polyP-metabolizing enzymes that are not present in prokaryotes. The synthesis and degradation of polyP in each specialized organelle and compartment of eukaryotic cells are mediated by different sets of enzymes. This is consistent with the endosymbiotic hypothesis of eukaryotic cell origin.

The Multiple Activities of Polyphosphate Kinase ofEscherichia coli and Their Subunit Structure Determined by Radiation Target Analysis

Polyphosphate kinase (PPK), the principal enzyme required for the synthesis of inorganic polyphosphate (polyP) from ATP, also exhibits other enzymatic activities, which differ significantly in their biochemical optima and responses to chemical agents. These several activities include: polyP synthesis (forward reaction), nATP --> polyP(n) + nADP (Equation 1); ATP synthesis from polyP (reverse reaction), ADP + polyP(n) --> ATP + polyP(n - 1) (Equation 2); general nucleoside-diphosphate kinase, GDP + polyP(n) --> GTP + polyP(n - 1) (Equation 3); linear guanosine 5'-tetraphosphate (ppppG) synthesis, GDP + polyP(n) --> ppppG + polyP(n - 2) (Equation 4); and autophosphorylation, PPK + ATP --> PPK-P + ADP (Equation 5). The Mg(2+) optima are 5, 2, 1, and 0.2 mM, respectively, for the activities in Equations 1, 2, 3, and 4. Inorganic pyrophosphate inhibits the activities in Equations 1 and 3 but stimulates that in Equation 4. The kinetics of the activities in Equations 1, 2, and 3 are highly processive, whereas the transfer of a pyrophosphoryl group from polyP to GDP (Equation 4) is distributive and demonstrates a rapid equilibrium, random Bi-Bi catalytic mechanism. Radiation target analysis revealed that the principal functional unit of the homotetrameric PPK is a dimer. Exceptions are a trimer for the synthesis of ppppG (Equation 4) and a tetrameric state for the autophosphorylation of PPK (Equation 5) at low ATP concentrations. Thus, the diverse functions of this enzyme involve different subunit organizations and conformations. The highly conserved homology of PPK among 18 microorganisms was used to determine important residues and conserved regions by alanine substitution, by site-directed mutagenesis, and by deletion mutagenesis. Of 46 single-site mutants, seven exhibit none of the five enzymatic activities; in one mutant, ATP synthesis from polyP is reduced relative to GTP synthesis. Among deletion mutants, some lost all five PPK activities, but others retained partial activity for some reactions but not for others.

電極還元法による含ケイ素ポリマーの合成

The electroreduction of organochlorosilanes was carried out by using magnesium cathode and anode in a single compartment cell affording oligosilanes, polysilanes, and polycarbosilanes. The electroreductive coupling of organochlorosilanes gave the corresponding disilanes in high yield. The cross-coupling of chloroorganosilanes with dichloroorganosilanes under the same reaction conditions produced various types of oligosilanes such as trisilanes, tetrasilanes, and pentasilanes. This procedure was successfully applied to the stepwise elongation of silicon-silicon chains. The high efficiency of this electroreductive silicon-silicon bond formation reaction enabled the electroreductive synthesis of poly (alkylarylsilane) s having high molecular weight (Mn=5200-31000, Mw/Mn=1.4-1.8). The electroreductive polymerization of the dichlorosilanes bearing various types of functional groups was also carried out to afford the functionalized polysilanes. Moreover, the electroreductive polymerization of dichlorooligosilanes was successfully applied to the synthesis of sequence-ordered polysilanes. This electroreductive method was also applicable to the synthesis of poly-p- (disilanylene) phenylenes and silane-germane copolymers. The effects of electrode material, monomer concentration, amount of supplied electricity, and ultrasound were investigated in these reactions.