The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment

We obtained detailed kinetic characteristics–stoichiometry, reaction rates, substrate affinities and equilibrium conditions–of human PPIP5K2 (diphosphoinositol pentakisphosphate kinase 2). This enzyme synthesizes ‘high-energy’ PP-InsPs (diphosphoinositol polyphosphates) by metabolizing InsP₆ (inositol hexakisphosphate) and 5-InsP₇ (5-diphosphoinositol 1,2,3,4,6-pentakisphosphate) to 1-InsP₇ (1-diphosphoinositol 2,3,4,5,6-pentakisphosphate) and InsP₈ (1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate), respectively. These data increase our insight into the PPIP5K2 reaction mechanism and clarify the interface between PPIP5K catalytic activities and cellular bioenergetic status. For example, stochiometric analysis uncovered non-productive, substrate-stimulated ATPase activity (thus, approximately 2 and 1.2 ATP molecules are utilized to synthesize each molecule of 1-InsP₇ and InsP₈, respectively). Impaired ATPase activity of a PPIP5K2-K248A mutant increased atomic-level insight into the enzyme''s reaction mechanism. We found PPIP5K2 to be fully reversible as an ATP-synthase in vitro, but our new data contradict previous perceptions that significant ‘reversibility’ occurs in vivo. PPIP5K2 was insensitive to physiological changes in either [AMP] or [ATP]/[ADP] ratios. Those data, together with adenine nucleotide kinetics (ATP K_m=20–40 μM), reveal how insulated PPIP5K2 is from cellular bioenergetic challenges. Finally, the specificity constants for PPIP5K2 revise upwards by one-to-two orders of magnitude the inherent catalytic activities of this enzyme, and we show its equilibrium point favours 80–90% depletion of InsP₆/5-InsP₇.     

Methylerythritol phosphate pathway to isoprenoids: Kinetic modeling and in silico enzyme inhibitions in Plasmodium falciparum

The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system.     

Chlorophyll a phytylation is required for the stability of photosystems I and II in the cyanobacterium Synechocystis sp. PCC 6803

In oxygenic phototrophic organisms, the phytyl ‘tail’ of chlorophyll a is formed from a geranylgeranyl residue by the enzyme geranylgeranyl reductase. Additionally, in oxygenic phototrophs, phytyl residues are the tail moieties of tocopherols and phylloquinone. A mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking geranylgeranyl reductase, ΔchlP, was compared to strains with specific deficiencies in either tocopherols or phylloquinone to assess the role of chlorophyll a phytylatation (versus geranylgeranylation). The tocopherol‐less Δhpt strain grows indistinguishably from the wild‐type under ‘standard’ light photoautotrophic conditions, and exhibited only a slightly enhanced rate of photosystem I degradation under strong irradiation. The phylloquinone‐less ΔmenA mutant also grows photoautotrophically, albeit rather slowly and only at low light intensities. Under strong irradiation, ΔmenA retained its chlorophyll content, indicative of stable photosystems. ΔchlP may only be cultured photomixotrophically (due to the instability of both photosystems I and II). The increased accumulation of myxoxanthophyll in ΔchlP cells indicates photo‐oxidative stress even under moderate illumination. Under high‐light conditions, ΔchlP exhibited rapid degradation of photosystems I and II. In conclusion, the results demonstrate that chlorophyll a phytylation is important for the (photo)stability of photosystems I and II, which, in turn, is necessary for photoautotrophic growth and tolerance of high light in an oxygenic environment.     

Synthesis of Polyoxamic Acid (2-Amino-2-deoxy-l-xylonic Acid

The rate of precipitation of the retrograded amylose product from a dil. amylose solution was determined by the centrifugal method. The results showed that the relation of the quantity of precipitate vs. time did not fit the typical second order reaction for the coalescence of colloidal particles but fitted the crystallization formula, in appearance.

The rate of precipitation was in proportion to (c-c_a)^1.5, where c is the amylose concentration and c_a the concentration of the dil. solution phase in the phase-separated solution. When the temperature dependence of the rate was treated according to the crystallization of polymers, it was found that the rate was in proportion to T_m²/T(ΔT)², where T_m is the melting point of the polymer in solution and ΔT is (T_m?T). The T_m thus obtained was 120°C for an amylose solution. These results suggested a certain correlation between the amylose retrogradation and the crystallization.     

High-resolution nuclear magnetic resonance spectroscopy studies of polysaccharides crosslinked by sodium trimetaphosphate: a proposal for the reaction mechanism

An NMR spectroscopy study ((31)P, (1)H, (13)C) of the postulated crosslinking mechanism of sodium trimetaphosphate (STMP) on polysaccharides is reported using methyl alpha-D-glucopyranoside as a model. In a first step, reaction of STMP with Glc-OMe gives grafted sodium tripolyphosphate (STPP(g)). On the one hand, STTP(g) can react with a second alcohol functionality to give a crosslinked monophosphate. On the other hand, a monophosphate (grafted phosphate) could be obtained by alkaline degradation of STPP(g). NMR spectroscopy allows to detect the various species formed and to obtain the crosslinking density of STMP-polysaccharides hydrogels.     

Hepatocyte susceptibility to glyoxal is dependent on cell thiamin content

Glyoxal, a reactive dicarbonyl, is detoxified primarily by the glyoxalase system utilizing glutathione (GSH) and by the aldo-keto reductase enzymes which utilizes NAD[P]H as the co-factor. Thiamin (Vitamin B(1)) is an essential coenzyme for transketolase (TK) that is part of the pentose phosphate pathway which helps maintain cellular NADPH levels. NADPH plays an intracellular role in regenerating glutathione (GSH) from oxidized GSH (GSSG), thereby increasing the antioxidant defenses of the cell. In this study we have focused on the prevention of glyoxal toxicity by supplementation with thiamin (3mM). Thiamin was cytoprotective and restored NADPH levels, glyoxal detoxification and mitochondrial membrane potential. Hepatocyte reactive oxygen species (ROS) formation, lipid peroxidation and GSH oxidation were decreased. Furthermore, hepatocytes were made thiamin deficient with oxythiamin (3mM) as measured by the decreased hepatocyte TK activity. Under thiamin deficient conditions a non-toxic dose of glyoxal (2mM) became cytotoxic and glyoxal metabolism decreased; while ROS formation, lipid peroxidation and GSH oxidation was increased.     

Identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases

A combination of sequence homology analyses of mevalonate diphosphate decarboxylase (MDD) proteins and structural information for MDD leads to the hypothesis that Asp 302 and Lys 18 are active site residues in MDD. These residues were mutated to replace acidic/basic side chains and the mutant proteins were isolated and characterized. Binding and competitive displacement studies using trinitrophenyl-ATP, a fluorescent analog of substrate ATP, indicate that these mutant enzymes (D302A, D302N, K18M) retain the ability to stoichiometrically bind nucleotide triphosphates at the active site. These observations suggest the structural integrity of the mutant MDD proteins. The functional importance of mutated residues was evaluated by kinetic analysis. The 10(3) and 10(5)-fold decreases in k(cat) observed for the Asp 302 mutants (D302N and D302A, respectively) support assignment of a crucial catalytic role to Asp 302. A 30-fold decrease in activity and a 16-fold inflation of the K(m) for ATP is documented for the K18M mutant, indicating that Lys 18 influences the active site but is not crucial for reaction chemistry. Demonstration of the influence of conserved aspartate 302 appears to represent the first documentation of the functional importance of a residue in the MDD catalytic site and affords insight into phosphotransferase reactions catalyzed by a variety of enzymes in the galactokinase, homoserine kinase, mevalonate kinase, phosphom-evalonate kinase (GHMP kinase) family.     

Pyruvate:NADP+ oxidoreductase is stabilized by its cofactor,thiamin pyrophosphate,in mitochondria of Euglena gracilis

Pyruvate:NADP(+) oxidoreductase (PNO) is a thiamin pyrophosphate (TPP)-dependent enzyme that plays a central role in the respiratory metabolism of Euglena gracilis, which requires thiamin for growth. When thiamin was depleted in Euglena cells, PNO protein level was greatly reduced, but its mRNA level was barely changed. In addition, a large part of PNO occurred as an apoenzyme lacking TPP in the deficient cells. The PNO protein level increased rapidly, without changes in the mRNA level, after supplementation of thiamin into its deficient cells. In the deficient cells, in contrast to the sufficient ones, a steep decrease in the PNO protein level was induced when the cells were incubated with cycloheximide. Immunofluorescence microscopy indicated that most of the PNO localized in the mitochondria in either the sufficient or the deficient cells. These findings suggest that PNO is readily degraded when TPP is not provided in mitochondria, and consequently the PNO protein level is greatly reduced by thiamin deficiency in E. gracilis.     

Organization of the Rab-GDI/CHM superfamily: the functional basis for choroideremia disease

Choroideremia is an X-chromosome-linked disease that leads to the degeneration of the choriocapillaris, the retinal pigment epithelium and the photoreceptor layer in the eye. The gene product defective in choroideremia, CHM, is identical to Rab escort protein 1 (REP1). CHM/REP1 is an essential component of the catalytic geranylgeranyltransferase II complex (GGTrII) that delivers newly synthesized small GTPases belonging to the RAB gene family to the catalytic complex for post-translational modification. CHM/REP family members are evolutionarily related to members of the guanine nucleotide dissociation inhibitor (GDI) family, proteins involved in the recycling of Rab proteins required for vesicular membrane trafficking through the exocytic and endocytic pathways, forming the GDI/CHM superfamily. Biochemical and structural analyses have now revealed a striking parallel in the organization and function of these two families allowing us to generate a general model for GDI/CHM superfamily function in health and disease.