Multisubstrate analogs for deoxynucleoside kinases. Triphosphate end products and synthetic bisubstrate analogs exhibit identical modes of binding and are useful probes for distinguishing kinetic mechanisms

Comparative inhibition kinetics with natural dNTP end products (dNp3) and new synthetic bisubstrate-type analogs, dNp4A (deoxynucleoside 5'-adenosine 5'-P1,P4-tetraphosphate), have been studied with their target deoxynucleoside kinases from Lactobacillus acidophilus. Analysis of inhibition specificity, inhibition patterns, and Ki(app) under various conditions has revealed the following conclusions. Both dNTP and dNp4A bind to the active site of the corresponding kinase through multiple binding determinants. The deoxynucleoside moiety of dNTP fits optimally at the deoxynucleoside binding site and provides the basis for its inhibition specificity, whereas the triphosphate group interacts with the ATP binding site, reinforcing the affinity of the molecule as a potent end product inhibitor (Ki = 0.4-3 microM). The adenosine moiety of dNp4A does not contribute to the binding of this compound, whereas the tetraphosphate portion is the second binding determinant, just as in the model developed for dNTP. dNTP and dNp4A proved to be useful tools for distinguishing the kinetic mechanisms of kinases which follow sequential pathways, i.e. the rapid equilibrium Random Bi Bi for dCyd and dGuo kinases and the steady state Ordered Bi Bi mechanism for two dAdo kinases associated either with dCyd kinase or with dGuo kinase on different multifunctional proteins.     

Initial rate and isotope exchange studies of rat skeletal muscle hexokinase

The kinetic mechanism of rat skeletal muscle hexokinase (hexokinase II) was investigated in light of a proposal by Cornish-Bowden and his co-workers (Gregoriou, M., Trayer, I. P., and Cornish-Bowden, A. (1983) Eur. J. Biochem. 134, 283-288). These investigators reported that the kinetic mechanism is ordered, with glucose adding before ATP and ADP dissociating from hexokinase before glucose-6-P. In addition, these workers suggest that glucose-6-P and ATP add to allosteric sites on hexokinase. We investigated the mechanism of action of hexokinase II by studying initial rate kinetics in the nonphysiological direction and by isotope exchange at chemical equilibrium. The former experiments were carried out in the absence of inhibitors and then with AMP, which is a competitive inhibitor of ADP, and with glucose 1,6-bisphosphate, a competitive inhibitor of glucose-6-P. The findings from these experiments suggest that the kinetic mechanism is rapid equilibrium Random Bi Bi. Isotope exchange at equilibrium studies also supports the random nature of the muscle hexokinase reaction; however, they also suggest that the mechanism is partially ordered, i.e. there is a preferred pathway associated with the branched mechanism. Approximately two-thirds of the flux through the hexokinase reaction involves the glucose on first glucose-6-P off last branch of the Random Bi Bi mechanism. These results imply that the kinetic mechanism is steady state Random Bi Bi. There is some evidence to suggest that glucose-6-P binds to an allosteric site on muscle hexokinase, but none to suppose that ATP binds allosterically. Analysis of the mechanism of Gregoriou et al. suggests that it is at variance with the findings of this report as well as with data available from other laboratories.     

Kinetic model of functioning and regulation of <Emphasis Type="Italic">Escherichia coli</Emphasis> isocitrate dehydrogenase

To describe published experimental data on the functioning of E. coli isocitrate dehydrogenase (IDH), a Rapid Equilibrium Random Bi Ter mechanism involving the formation of two dead-end enzyme complexes is proposed and a kinetic model of enzyme functioning is constructed. The enzyme is shown to be regulated through reversible phosphorylation by IDH kinase/phosphatase; the latter, in its turn, is controlled by IDH substrates and also by a number of central metabolites—pyruvate, 3-phosphoglycerate, and AMP—reflecting the energy demand of the cell. Using the model, it is shown that an increase in the concentration of the above effectors raises the fraction of active IDH and thus enhances the Krebs cycle flux. The ratio between the free and the phosphorylated forms of IDH is more sensitive to AMP, NADP, and isocitrate than to pyruvate and 3-phosphoglycerate. The model also predicts changes in the ratio between phosphorylated and active forms of IDH in the Krebs cycle that occur with a change in the energy and biosynthetic loads on E. coli cells.     

Killing of Bacteria by Copper Surfaces Involves Dissolved Copper

Bacteria are rapidly killed on copper surfaces. However, the mechanism of this process remains unclear. Using Enterococcus hirae, the effect of inactivation of copper homeostatic genes and of medium compositions on survival and copper dissolution was tested. The results support a role for dissolved copper ions in killing.The rapid killing of bacteria by solid copper surfaces is receiving rapidly growing attention. In laboratory experiments, it has been shown that many bacterial species, such as Escherichia coli O157, Staphylococcus aureus, Salmonella enterica, Campylobacter jejuni, Clostridium difficile, and Mycobacterium tuberculosis, are efficiently killed on copper or copper alloy surfaces (1, 3, 4, 6, 7, 12-14). In contrast, on stainless steel, living cells could be recovered even after 28 days. The antimicrobial activity of copper and copper alloys is now well established, and copper has recently been registered at the U.S. Environmental Protection Agency as the first solid antimicrobial material. A key focus is the use of copper in health care facilities, food processing plants, and other areas where clean or aseptic working procedures are required (2). In this connection, it has become important to understand the mechanism of bacterial killing, as it may bear on the possibility of the emergence of resistant organisms, cleaning procedures, and material and object engineering. We here used wild-type and mutant strains of Enterococcus hirae to investigate the influence of copper resistance genes on killing rates. We also evaluated copper dissolution by various media and its relation to killing efficiency. Our findings provide support for a prominent role for dissolved copper in the killing process.     

Comparing the efficiency of pollination mechanisms in Papilionoideae

The pump pollination mechanism is typical of basal clades within Papilionoideae and might be associated with less efficient pollen transfer systems, while the explosive tripping mechanism is considered more advanced and might represent the highest expression of the trend in pollen economy. Crotalaria pumila, C. stipularia, Desmodium incanum and D. subsericeum present secondary pollen presentation with pump and explosive pollination mechanisms, respectively. In the present study, we evaluate and compare (1) pollen removal, (2) pollen deposition and (3) pollen transfer efficiency of both mechanisms, considering single visits by Megachile spp., common pollinators of the four plant species in Salta Province, Argentina. Comparisons of visit durations are made in relation to the type of mechanism and rewards offered. We detected significant differences between both mechanisms in the proportion of pollen grains removed and deposited in a flower after a single visit of Megachile. We found that efficiency in pollen transfer was significantly higher for explosive mechanism (2.13?±?0.42 pollen grains deposited per 100 removed) than for pump mechanism (0.33?±?0.17 pollen grains deposited per 100 removed). This is the first study that compares efficiency between pollination mechanisms in this group of plants.     

Metabolomics Study of Immune Responses of New Zealand Greenshell™ Mussels (<Emphasis Type="Italic">Perna canaliculus</Emphasis>) Infected with Pathogenic <Emphasis Type="Italic">Vibrio</Emphasis> sp.

Vibrio coralliilyticus is a bacterial pathogen which can affect a range of marine organisms, such as corals, fish and shellfish, with sometimes devastating consequences. However, little is known about the mechanisms involved in the host-pathogen interaction, especially within molluscan models. We applied gas chromatography-mass spectrometry (GC-MS)-based metabolomics to characterize the physiological responses in haemolymph of New Zealand Greenshell? mussels (Perna canaliculus) injected with Vibrio sp. DO1 (V. coralliilyticus/neptunius-like isolate). Univariate data analyses of metabolite profiles in Vibrio-exposed mussels revealed significant changes in 22 metabolites at 6 h post-infection, compared to non-exposed mussels. Among them, 10 metabolites were up-regulated, while 12 metabolites were down-regulated in infected mussels. Multivariate analyses showed a clear distinction between infected and non-infected mussels. In addition, secondary pathway analyses indicated perturbations of the host innate immune system following infection, including oxidative stress, inflammation and disruption of the TCA cycle, change in amino acid metabolism and protein synthesis. These findings provide new insights into the pathogenic mechanisms of Vibrio infection of mussels and demonstrate our ability to detect detailed and rapid host responses from haemolymph samples using a metabolomics approach.     

Various <Emphasis Type="Italic">Wolbachia</Emphasis> genotypes differently influence host <Emphasis Type="Italic">Drosophila</Emphasis> dopamine metabolism and survival under heat stress conditions

Background

One of the most widespread prokaryotic symbionts of invertebrates is the intracellular bacteria of Wolbachia genus which can be found in about 50% of insect species. Wolbachia causes both parasitic and mutualistic effects on its host that include manipulating the host reproductive systems in order to increase their transmission through the female germline, and increasing the host fitness. One of the mechanisms, promoting adaptation in biological organisms, is a non-specific neuroendocrine stress reaction. In insects, this reaction includes catecholamines, dopamine, serotonin and octopamine, which act as neurotransmitters, neuromodulators and neurohormones. The level of dopamine metabolism correlates with heat stress resistance in Drosophila adults.

Results

To examine Wolbachia effect on Drosophila survival under heat stress and dopamine metabolism we used five strains carrying the nuclear background of interbred Bi90 strain and cytoplasmic backgrounds with different genotype variants of Wolbachia (produced by 20 backcrosses of Bi90 males with appropriate source of Wolbachia). Non-infected Bi90 strain (treated with tetracycline for 3 generations) was used as a control group. We demonstrated that two of five investigated Wolbachia variants promote changes in Drosophila heat stress resistance and activity of enzymes that produce and degrade dopamine, alkaline phosphatase and dopamine-dependent arylalkylamine N-acetyltransferase. What is especially interesting, wMelCS genotype of Wolbachia increases stress resistance and the intensity of dopamine metabolism, whereas wMelPop strain decreases them. wMel, wMel2 and wMel4 genotypes of Wolbachia do not show any effect on the survival under heat stress or dopamine metabolism. L-DOPA treatment, known to increase the dopamine content in Drosophila, levels the difference in survival under heat stress between all studied groups.

Conclusions

The genotype of symbiont determines the effect that the symbiont has on the stress resistance of the host insect.

     

Kinetic and Functional Analysis of l-Threonine Kinase, the PduX Enzyme of Salmonella enterica

The PduX enzyme of Salmonella enterica is an l-threonine kinase used for the de novo synthesis of coenzyme B₁₂ and the assimilation of cobyric acid. PduX with an N-terminal histidine tag (His₈-PduX) was produced in Esch e richia coli and purified. The recombinant enzyme was soluble and active. Kinetic analysis indicated a steady-state Ordered Bi Bi complex mechanism in which ATP is the first substrate to bind. Based on a multiple sequence alignment of PduX homologues and other GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) family members, 14 PduX variants having changes at 10 conserved serine/threonine and aspartate/glutamate sites were constructed by site-directed mutagenesis. Each variant was produced in E. coli and purified. Comparison of the circular dichroism spectra and kinetic properties of the PduX variants with those of the wild-type enzyme indicated that Glu-24 and Asp-135 are needed for proper folding, Ser-99 and Glu-132 are used for ATP binding, and Ser-253 and Ser-255 are critical to l-threonine binding whereas Ser-100 is essential to catalysis, but its precise role is uncertain. The studies reported here are the first to investigate the kinetic and catalytic mechanisms of l-threonine kinase from any organism.The B₁₂ coenzymes (adenosylcobalamin (AdoCbl)² and methylcobalamin) are the largest cofactors known in biology. They are essential for human health and have important metabolic roles in many microbes (1, 2). The B₁₂ coenzymes are synthesized de novo only by certain prokaryotes and from corrinoid precursors by a broader range of organisms (1, 2). De novo synthesis by prokaryotes is the ultimate source of B₁₂, and this process has been extensively studied because of its importance to diverse biological forms and to the commercial production of B₁₂ as a dietary supplement (1). Salmonella enterica is an important model organism for studies of B₁₂ synthesis (3, 4). This organism carries out de novo synthesis under anaerobic conditions and assimilates corrinoids such as cobinamide and cobyric acid (Cby) under both aerobic and anaerobic conditions (3, 5). We recently showed that the PduX enzyme of S. enterica is an l-threonine (l-Thr) kinase, which is required for the de novo synthesis of AdoCbl and methylcobalamin and the assimilation of Cby (6). PduX catalyzes the conversion of l-Thr and ATP to ADP and l-threonine-O-3-phosphate (l-Thr-P), a required building block for the de novo synthesis of B₁₂ (Fig. 1) (6). By sequence similarity, the PduX enzyme of S. enterica belongs to the galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase (GHMP) family (7), which is a novel family of kinases found in metabolic pathways of small molecules. Although there have been no three-dimensional structures of PduX or other l-Thr kinases reported to date, the crystal structures of several GHMP kinases have been solved (8–22). These structures reveal a unique kinase fold and a novel nucleotide-binding mode that are conserved among members of the GHMP kinase family. Members also contain three common sequence motifs consisting of about 10–15 contiguous amino acids (9, 23). Motif I is at the N-terminal region, and its function is uncertain. Motif II is the most conserved with a typical sequence of Pro-X-X-X-Gly-Leu-X-Ser-Ser-Ala. This region is involved in nucleotide binding and distinguishes the GHMP family from other protein kinase families. Motif III is near the C terminus and contains a small glycine-rich loop that appears well positioned to interact with substrate that is phosphorylated by ATP (24). However, there are highly divergent sequences outside these three motifs, and modes of oligomerization, substrate binding, and catalytic mechanism vary among different GHMP family members (14).Open in a separate windowFIGURE 1.Roles of PduX in B₁₂ synthesis in S. enterica. PduX is required for both the de novo synthesis of AdoCbl and methylcobalamin (MeCbl) and the assimilation of cobyric acid. AdoCbl is required for growth of S. enterica on 1,2-PD and ethanolamine as well for the activity of the MetH methionine synthase that converts homocysteine to methionine. Btu, B₁₂ uptake system; Cbi, cobinamide; AdoCbi, adenosylcobinamide; AdoCby, adenosylcobyric acid; AdoCbi-P, adenosylcobinamide phosphate; AP-P, (R)-1-amino-2-propanol-O-2-phosphate.Based on structural and biochemical data, members of the GHMP family are subdivided into two groups depending on their catalytic mechanisms. In the first group, which includes rat mevalonate kinase, the crystal structure shows an aspartate residue in the active site positioned to act as a catalytic base that abstracts a proton from the substrate hydroxyl group. In addition, there is a lysine residue that is thought to reduce the pK_a of the substrate hydroxyl group and to stabilize the pentacoordinated γ-phosphoryl group of ATP (18). In the second group, such as Escherichia coli homoserine kinase, there appears to be no suitable residue positioned to act as a catalytic base. Consequently it has been hypothesized that homoserine kinase achieves catalysis through transition state stabilization alone (14).In the present study we investigated the catalytic mechanism of PduX by a series of kinetic studies and mutational analyses of 10 invariant serine/threonine and aspartate/glutamate residues. Results indicate a steady-state Ordered Bi Bi mechanism in which ATP binds first. We also identified several amino acids critical to substrate binding and catalysis. To our knowledge, these are the first reported studies on the catalytic mechanism of l-Thr kinase from any organism.     

Characteristic differences in metabolite profile in male and female plants of dioecious <Emphasis Type="Italic">Piper betle</Emphasis> L.

Piper betle is a dioecious pan-Asiatic plant having cultural and medicinal uses. It belongs to the family Piperaceae and is a native of the tropics although it is also cultivated in subtropical areas. Flowering in P. betle occurs only in tropical regions. Due to lack of inductive floral cycles the plant remains in its vegetative state in the subtropics. Therefore, due to lack of flowering, gender distinction cannot be made the in the subtropics. Gender distinction in P. betle in vegetative state can be made using Direct Analysis in Real Time Mass Spectroscopy (DARTMS), a robust high-throughput method. DARTMS analysis of leaf samples of two male and six female plants showed characteristic differences in the spectra between male and female plants. Semi-quantitative differences in some of the identified peaks in male and female landraces showed gender-based differences in metabolites. Cluster analysis using the peaks at m/z 151, 193, 235 and 252 showed two distinct clusters of male and female landraces. It appears that male and female plants besides having flowers of different sexes also have characteristic differences in the metabolites representing two metabolic types.     

Tryptophan Synthase and Production of l-Tryptophan in Regulatory Mutants

A 5-fluorotryptophan-resistant mutant of Brevibacterium flavum, No. 187, accumulated 2.6 g of indole 3-glycerol (InG) in addition to 8.0 g of l-tryptophan per liter in the culture medium. The addition of l-serine to the medium decreased the accumulation of InG and increased that of l-tryptophan up to a concentration of 10.3 g/liter, while the addition of l-tryptophan increased the InG accumulation, suggesting that InG was formed by hydrolysis of indole 3-glycerol phosphate (InGP), the substrate of tryptophan synthase (TS) which catalyzed the final step reaction of tryptophan biosynthesis. Then, in order to examine the mechanism of the InG accumulation, TS was purified from tryptophan auxotroph, TA-60. The reaction mechanism of TS was Ordered Bi Bi with Km’s of 0.63 and 0.038 mm for serine and InGP, respectively. Tryptophan, a product of the TS reaction, inhibited TS competitively with respect to serine and the Ki for tryptophan was estimated to be 2.0 mm. On the other hand, anthranilate synthase (AS), the first enzyme in the tryptophan biosynthetic pathway, was much less sensitive to the feedback inhibition by tryptophan in strain No. 187 than in the wild strain. The tryptophan concentration giving 50% inhibition of AS in strain No. 187 was estimated to be 2.4 mm, almost comparable to that of TS, 7.7 mm. From these results, it was concluded that the accumulation of InG in strain No. 187 would result from the product inhibition of TS by the tryptophan accumulated.     

Pathogen-Triggered Ethylene Signaling Mediates Systemic-Induced Susceptibility to Herbivory in Arabidopsis

Multicellular eukaryotic organisms are attacked by numerous parasites from diverse phyla, often simultaneously or sequentially. An outstanding question in these interactions is how hosts integrate signals induced by the attack of different parasites. We used a model system comprised of the plant host Arabidopsis thaliana, the hemibiotrophic bacterial phytopathogen Pseudomonas syringae, and herbivorous larvae of the moth Trichoplusia ni (cabbage looper) to characterize mechanisms involved in systemic-induced susceptibility (SIS) to T. ni herbivory caused by prior infection by virulent P. syringae. We uncovered a complex multilayered induction mechanism for SIS to herbivory. In this mechanism, antiherbivore defenses that depend on signaling via (1) the jasmonic acid–isoleucine conjugate (JA-Ile) and (2) other octadecanoids are suppressed by microbe-associated molecular pattern–triggered salicylic acid (SA) signaling and infection-triggered ethylene signaling, respectively. SIS to herbivory is, in turn, counteracted by a combination of the bacterial JA-Ile mimic coronatine and type III virulence-associated effectors. Our results show that SIS to herbivory involves more than antagonistic signaling between SA and JA-Ile and provide insight into the unexpectedly complex mechanisms behind a seemingly simple trade-off in plant defense against multiple enemies.     

Genome Analysis of Legionella pneumophila Strains Using a Mixed-Genome Microarray

Background

Legionella, the causative agent for Legionnaires’ disease, is ubiquitous in both natural and man-made aquatic environments. The distribution of Legionella genotypes within clinical strains is significantly different from that found in environmental strains. Developing novel genotypic methods that offer the ability to distinguish clinical from environmental strains could help to focus on more relevant (virulent) Legionella species in control efforts. Mixed-genome microarray data can be used to perform a comparative-genome analysis of strain collections, and advanced statistical approaches, such as the Random Forest algorithm are available to process these data.

Methods

Microarray analysis was performed on a collection of 222 Legionella pneumophila strains, which included patient-derived strains from notified cases in the Netherlands in the period 2002–2006 and the environmental strains that were collected during the source investigation for those patients within the Dutch National Legionella Outbreak Detection Programme. The Random Forest algorithm combined with a logistic regression model was used to select predictive markers and to construct a predictive model that could discriminate between strains from different origin: clinical or environmental.

Results

Four genetic markers were selected that correctly predicted 96% of the clinical strains and 66% of the environmental strains collected within the Dutch National Legionella Outbreak Detection Programme.

Conclusions

The Random Forest algorithm is well suited for the development of prediction models that use mixed-genome microarray data to discriminate between Legionella strains from different origin. The identification of these predictive genetic markers could offer the possibility to identify virulence factors within the Legionella genome, which in the future may be implemented in the daily practice of controlling Legionella in the public health environment.     

The Wall Teichoic Acid Polymerase TagF Is Non-processive in Vitro and Amenable to Study Using Steady State Kinetic Analysis

Wall teichoic acids are a chemically diverse group of anionic polymers that constitute up to 50% of the Gram-positive cell wall. These polymers play a pivotal role in virulence and have been implicated in a diverse range of physiological functions. The TagF-like family of enzymes has been shown to be responsible for wall teichoic acid priming and polymerization events. Although many such enzymes are well validated therapeutic targets, a mechanistic understanding of this enzyme family has remained elusive. TagF is the prototypical teichoic acid polymerase and uses CDP-glycerol to catalyze synthesis of the linear (1,3)-linked poly(glycerol phosphate) teichoic acid in Bacillus subtilis 168. Here we used a synthetic soluble analog of the natural substrate of the enzyme, Lipid ϕ, to conduct the first detailed mechanistic investigation of teichoic acid polymerization. Through the use of a new high pressure liquid chromatography-based assay to monitor single glycerol phosphate incorporations into the Lipid ϕ analog, we conducted a detailed analysis of reaction product formation patterns and unequivocally showed TagF to be non-processive in vitro. Furthermore by monitoring the kinetics of polymerization, we showed that Lipid ϕ analog species varying in size have the same K_m value of 2.6 μm and validated use of Bi Bi velocity expressions to model the TagF enzyme system. Initial rate analysis showed that TagF catalyzes a sequential Bi Bi mechanism where both substrates are added to the enzyme prior to product release consistent with a single displacement chemical mechanism.Wall teichoic acids are a group of phosphate-containing anionic carbohydrate polymers that constitute up to 50% of the dry weight of the Gram-positive cell wall (1). Teichoic acids play a pivotal role in virulence and have been implicated in a diverse range of physiological functions including cation homeostasis, nutrient trafficking, binding of envelope proteins, and regulation of autolysins (2–4). Our knowledge of wall teichoic acid synthesis largely stems from studies conducted in the model bacterium Bacillus subtilis 168, which expresses a linear (1,3)-linked poly(glycerol phosphate) teichoic acid (5–7). Through these studies, our group and others have identified the genetic requirements for poly(glycerol phosphate) synthesis; however, until recently, a biochemical understanding of priming and polymerization events have been confounded by the interfacial localization of these enzymatic steps (8, 9). Indeed interfacial localization has hindered the understanding of the synthesis of many other important cell wall components such as O-antigen, polysialic acid, lipoarabinomannan, oligomers for N-linked glycosylation, and others (10–13). A breakthrough in our ability to study the enzymes involved in the lipid-linked steps of wall teichoic acid synthesis was made by Ginsberg et al. (14) with the development of synthetic substrate analogs of lipid-linked wall teichoic acid intermediates. These substrate analogs have since facilitated the detailed mechanistic study of uncharacterized teichoic acid enzymes and were used to reconstitute all intracellular steps in Staphylococcus aureus wall teichoic acid synthesis in vitro (15, 16).Genetic and biochemical studies have given rise to a model for the synthesis of poly(glycerol phosphate) wall teichoic acid in B. subtilis 168 in which polymer synthesis is carried out on the intracellular surface of the cytoplasmic membrane by stepwise additions of sugars to an undecaprenol phosphate lipid carrier via the tag (teichoic acid glycerol) gene products (7, 17). Polymer synthesis is initiated by TagO, which catalyzes the transfer of N-acetylglucosamine-1-phosphate from UDP-GlcNAc to undecaprenol phosphate to create Lipid α (18) (the new nomenclature for lipid-linked teichoic acid intermediates proposed by Pereira and Brown (17) is summarized in 2 is transferred to Lipid α from UDP-ManNAc by TagA, producing Lipid β that is “primed” with sn-glycerol-3-phosphate by TagB to create the polymerization substrate Lipid ϕ.1 (9, 14, 16). Some 30–50 glycerol phosphate residues are subsequently added to Lipid ϕ.1 by TagF, and the intracellular steps of teichoic acid synthesis are completed via polymer glucosylation by TagE (6, 8). Intracellular teichoic acid is then exported to the outer leaflet of the cytoplasmic membrane by the TagG/H ATP-binding cassette transport system and transferred to peptidoglycan by a currently unknown enzyme (19).

TABLE 1

Recently proposed nomenclature for wall teichoic acid intermediatesShown is the nomenclature proposed for wall teichoic acid biosynthetic intermediates (17). Intermediates were named according to the enzyme utilizing the molecule as a substrate. Lipid α is the substrate for TagA, Lipid β is the substrate for TagB. Lipid ϕ.n species are substrates for TagF where n indicates the number of glycerol phosphate residues in the molecule. For example, Lipid ϕ.1 is the product of the TagB-catalyzed priming reaction where a single glycerol phosphate residue is added. und, undecaprenol; P, phosphate; GroP, sn-glycerol-3-phosphate.

Oxido-reduction is not the only mechanism allowing ions to traverse the ferritin protein shell

Background

Most models for ferritin iron release are based on reduction and chelation of iron. However, newer models showing direct Fe(III) chelation from ferritin have been proposed. Fe(III) chelation reactions are facilitated by gated pores that regulate the opening and closing of the channels.

Scope of review

Results suggest that iron core reduction releases hydroxide and phosphate ions that exit the ferritin interior to compensate for the negative charge of the incoming electrons. Additionally, chloride ions are pumped into ferritin during the reduction process as part of a charge balance reaction. The mechanism of anion import or export is not known but is a natural process because phosphate is a native component of the iron mineral core and non-native anions have been incorporated into ferritin in vitro. Anion transfer across the ferritin protein shell conflicts with spin probe studies showing that anions are not easily incorporated into ferritin. To accommodate both of these observations, ferritin must possess a mechanism that selects specific anions for transport into or out of ferritin. Recently, a gated pore mechanism to open the 3-fold channels was proposed and might explain how anions and chelators can penetrate the protein shell for binding or for direct chelation of iron.

Conclusions and general significance

These proposed mechanisms are used to evaluate three in vivo iron release models based on (1) equilibrium between ferritin iron and cytosolic iron, (2) iron release by degradation of ferritin in the lysosome, and (3) metallo-chaperone mediated iron release from ferritin.