Chemical aspects of siderophore mediated iron transport

In this mini-review we describe selected aspects of the coordination chemistry relevant to siderophore mediated iron transport and bioavailability. Specific emphasis is placed on a discussion of in vitro kinetic and thermodynamic data that are relevant to elucidating possible in vivo mechanisms for environmental iron acquisition by microbial cells.     

Stereochemical aspects of iron transport in Mycelia sterilia EP-76.

Chromic complexes of N,N',N'-triacetylfusarinine C have been prepared and examined for biological activity in Mycelia sterilia EP-76. The iron transport system of this organism recognizes only the lambda coordination isomer of Cr(III)-triacetylfusarinine C even though the delta configuration predominates in solution. Chromium is excreted into the medium following triacetylfusarinine C-mediated uptake of the metal. Hydrogenation of the double bonds conjugated to the hydroxamic acid functions of triacetylfusarinine C yields four chromatographically distinct ferric complexes. Two of the complexes have the lambda configuration, while the other two have the opposite (delta) configuration. The complexes differ in effectiveness as siderophores in M. sterilia EP-76, the lambda isomers being most active.     

The membrane potential is the driving force for siderophore iron transport in fungi

Abstract Respiratory inhibitors and uncouplers severely impair [⁵⁵Fe]ferricrocin uptake by Neurospora crassa . parallel measurements of ATP decay and ferricrocin uptake, however, disprove the idea that direct input of metabolic energy in the form of ATP is required for transmembrane movement of siderophores. The role of the membrane potential for siderophore uptake was demonstrated using iron-deficient cells, which were derepressed in the glucose-II uptake system. Addition of high amounts of glucose (1 mM) to glu-II-derepressed cells leads to a membrane depolarization of about 120 mV, followed by a significant inhibition of ferricrocin uptake, which recovered after some minutes. Full transport inhibition occurred after membrane depolarization in the presence of plasma membrane ATP-ase inhibitors (DCCD or DES), indicating that the membrane potential is essential for siderophore transport in fungi.     

Tracking metabolism and imaging transport in arbuscular mycorrhizal fungi. Metabolism and transport in AM fungi

In the last few years the application of modern techniques to the study of arbuscular mycorrhizas has greatly increased our understanding of the mechanisms underlying carbon metabolism in these mutualistic symbioses. Arbuscular mycorrhizal (AM) monoxenic cultures, nuclear magnetic resonance spectroscopy together with isotopic labeling, and analyses of expressed sequence tags (ESTs) have shed light on the metabolic processes taking place in these interactions, particularly in the case of the mycobiont. More recently, in vivo multiphoton microscopy has provided us with some new insights in the allocation and translocation processes which play crucial roles in the distribution of host plant-derived C throughout the fungal colony. In this mini-review we highlight recent advances in these fields, with special attention to the visualization of oleosomes (i.e., lipid bodies) as they move along the long, coenocytic AM fungal hyphae. Volumetric measurements of such oleosomes have allowed us to estimate the flux of triacylglycerides from the intraradical to the extraradical phase of the AM fungal colony. We raise questions and postulate regulatory mechanisms for C metabolism and translocation within the arbuscular mycorrhizal fungal colony.     

Structural determinants of the substrate and stereochemical specificity of phosphotriesterase

Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the S(P)-enantiomer over the corresponding R(P)-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, Leu-271, Leu-303, Phe-306, Ser-308, Tyr-309, and Met-317. These studies have shown that substitution of Gly-60 with an alanine within the small subsite dramatically decreased k(cat) and k(cat)/K(a) for the R(P)-enantiomers, but had little influence on the kinetic constants for the S(P)-enantiomers of the chiral substrates. As a result, the chiral preference for the S(P)-enantiomers was greatly enhanced. For example, the value of k(cat)/K(a) with the mutant G60A for the S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding R(P)-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the S(P)-enantiomers, due to selective increases in the reaction rates for the R(P)-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on k(cat)/K(a) for either the R(P)- or S(P)-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.     

Ribosomal elongation cycle: energetic, kinetic and stereochemical aspects

As a preface to an analysis of the ribosomal elongation cycle, we examine the energetics of macromolecular structural transformations. We show that the kinetic barriers and changes of the energetic levels during these transformations are essentially determined by disruption of hydrogen and cation-ligand bonds, and by uncompensated losses of these bonds (ULBs). The disruption of a hydrogen or cation-ligand bond increases the heights of kinetic barriers by the energy of these bonds. The association and dissociation of macromolecules, and conformational transitions within macromolecules, can change the numbers of ULBs but cannot completely eliminate them. Two important general conclusions are drawn from this analysis. First, occupation of enzyme active centers by substrates should be accompanied by a reduction in the number of ULBs. This reduction decreases the activation barriers in enzyme reactions, and is a major contributor to catalysis. Second, the enzymic reactions of the ribosomal cycle (structural changes caused by transpeptidation and by GTP hydrolyses in EF-Tu and EF-G) disrupt kinetic traps that prevent tRNAs from dissociating into solution during their motion within the ribosome and are necessary for progression of the cycle. These results are general purpose structural-functional blocks for building a molecular model of the ribosomal elongation cycle. Here, we demonstrate the utility of these blocks for analysis of acceptance of cognate tRNAs into the ribosomal elongation cycle.     

Organelle transport and molecular motors in fungi

Polarized growth, secretion of exoenzymes, organelle inheritance, and organelle positioning require vectorial transport along cytoskeletal elements. The discovery of molecular motors and intensive studies on their biological function during the past 3 years confirmed a central role of these mechanoenzymes in morphogenesis and development of yeasts and filamentous fungi. Saccharomyces cerevisiae proved to be an excellent model system, in which the complete set of molecular motors is presumed to be known. Genetic studies combined with cell biological methods revealed unexpected functional relationships between these motors and has greatly improved our understanding of nuclear migration, exocytosis, and endocytosis in yeasts. Tip growth of elongated hyphae, compared to budding, however, does require vectorial transport over long distances. The identification of ubiquitous motors that are not present in yeast indicates that studies on filamentous fungi might be helpful to elucidate the role of motors in long-distance organelle transport within higher eukaryotic cells. Copyright 1998 Academic Press.     

P metabolism and transport in AM fungi

The arbuscular mycorrhizal symbiosis is mutualistic, based on reciprocal transfer of P from the fungus to the plant and carbon from the plant to the fungus. Thus P is a most important `currency' in the symbiosis. After absorbing P from the soil solution, the fungi first incorporate it into the cytosolic pool, and the excess P is transferred to the vacuoles. The vacuolar P pool probably plays a central role in P supply to the plant. The main forms of inorganic P in fungal vacuoles are orthophosphate and polyphosphate, but organic P molecules may also be present. Long distance translocation of P from the site of uptake in the external mycelium to the site of transfer to the plant is probably achieved via transfer of vacuolar components. This transport would be mediated either by protoplasmic streaming or the motile tubular vacuole-like system. The site of release of P into the interfacial apoplast and thence to the plant is most probably the fungal arbuscules. The biochemical and biophysical processes involved in P metabolism and transfer between cellular compartments in the symbiosis are currently not well understood. Some recent investigations of substrate specificities of phosphatase-type enzymes in AM fungi and other eukaryotic microorganisms, however, have shed new light on earlier results and permit the construction of a hypothetical scheme of P-flow, including possible regulatory factors. Steps in this scheme are experimentally testable and should stimulate future research.     

Molecular mechanisms of iron uptake in fungi

Fungi, like all free-living organisms, are in competition for limiting nutrients. In accumulating iron, fungi are faced also with a trace metal whose aqueous and redox chemistry make it both relatively bio-unavailable and strongly cytotoxic. Successful adaptation to this environmental context has provided fungi with an iron uptake strategy that has three features: it relies on redox cycling to enhance iron bio-availability and reduce iron cytotoxicity; it includes both high- and low-affinity pathways that are mechanistically distinct; and it is autoregulating so as to maintain intracellular iron homeostasis. Using Saccharomyces cerevisiae as a paradigm, this review summarizes current knowledge about the four pathways by which this yeast accumulates iron. These four pathways include: siderophore iron accumulation; high affinity iron uptake via an iron permease; and two lower affinity uptake pathways through relatively non-specific divalent metal ion transporters. All of these four pathways are directly or indirectly dependent on the activity of metalloreductase activity expressed extracellularly on the plasma membrane. A variety of experimental and genomics data indicate that this resourcefulness is shared by many, if not most, fungi. On the other hand, while the autoregulation of iron metabolism in Baker's yeast is well-understood, little is known about the apparent homeostatic mechanisms in these other yeasts and fungi. The integration of these multiple uptake mechanisms and their regulation into over-all iron homeostasis in yeast concludes this brief review.     

Biosynthetic intermediates and stereochemical aspects of aldehyde biosynthesis in the marine diatom Thalassiosira rotula

Intermediates of the aldehyde biosynthesis in Thalassiosira rotula are investigated. Use of labeled precursors and cell preparations proves production of 2E,4Z-octadienal from 6Z,9Z,12Z-hexadecatrienoic acid (C16:3 omega-4) through the lipoxygenase-dependent intermediate (9S)-9-hydroperoxyhexadeca-6,10,12-trienoic acid. On the contrary, synthesis of 2E,4Z,7Z-decatrienal involves mainly EPA (C20:5 omega-3) by a 11R-lipoxygenase, as suggested by identification of chiral 11R-HEPE (12% e.e.) in the diatom extracts. Consistently with the necessity to have a rapid transport and metabolization of the intermediate hydroperoxides, we show that lipoxygenase and lyase activities are both found in the same subcellular fraction of the microalga.     

Regulation of sulfate transport in filamentous fungi

Inorganic sulfate enters the mycelia of Aspergillus nidulans, Penicillium chrysogenum, and Penicillium notatum by a temperature-, energy-, pH-, ionic strength-, and concentration-dependent transport system (“permease”). Transport is unidirectional. In the presence of excess external sulfate, ATP sulfurylase-negative mutants will accumulate inorganic sulfate intracellularly to a level of about 0.04 m. The intracellular sulfate can be retained against a concentration gradient. Retention is not energy-dependent, nor is there any exchange between intracellular (accumulated) and extracellular sulfate. The sulfate permease is under metabolic control. Sulfur starvation of high methionine-grown mycelia results in about a 1000-fold increase in the specific sulfate transport activity at low external sulfate concentrations. l-Methionine is a metabolic repressor of the sulfate permease, while intracellular sulfate and possibly l-cysteine (or a derivative of l-cysteine) are feedback inhibitors. Sulfate transport follows hyperbolic saturation kinetics with a Michaelis constant (Km) value of 6 × 10⁻⁵ to 10⁻⁴m and a V_max (for maximally sulfurstarved mycelia) of about 5 micromoles per gram per minute. Refeeding sulfur-starved mycelia with sulfate or cysteine results in about a 10-fold decrease in the V_max value with no marked change in the Km. Azide and dinitrophenol also reduce the V_max.     

Specificity and control of choline-O-sulfate transport in filamentous fungi

Choline-O-sulfate uptake by Penicillium notatum showed the following characteristics. (i) Transport was mediated by a permease which is highly specific for choline-O-sulfate. No significant inhibition of transport was caused by choline, choline-O-phosphate, acetylcholine, ethanolamine-O-phosphate, ethanolamine-O-sulfate, methanesulfonyl choline, 2-aminoethane thiosulfate, or the monomethyl or dimethyl analogues of choline-O-sulfate. Similarly, no significant inhibition was caused by any common sulfur amino acid or inorganic sulfur compound. Mutants lacking the inorganic sulfate permease possessed the choline-O-sulfate permease at wild-type levels. (ii) Choline-O-sulfate transport obeyed saturation kinetics (K(m) = 10(-4) to 3 x 10(-4)m; V(max) = 1 to 6 mumoles per g per min). The kinetics of transport between 10(-9) and 10(-1)m external choline-O-sulfate showed that only one saturable mechanism is present. (iii) Transport was sensitive to 2,4-dinitrophenol, azide, N-ethylmaleimide, p-chloromercuribenzoate, and cyanide. Ouabain, phloridzin, and eserine had no effect. (iv) Transport was pH-dependent with an optimum at pH 6. Variations in the ionic strength of the incubation medium had no effect. (v) Transport was temperature-dependent with a Q(10) of greater than 2 between 3 and 40 C. Transport decreased rapidly above 40 C. (vi) Ethylenediaminetetraacetate (sodium salts, pH 6) had no effect, nor was there any stimulation by metal or nonmetal ions. Cu(++), Ag(+), and Hg(++) were inhibitory. (vii) The initial rate at which the ester is transported was independent of intracellular hydrolysis. After long periods of incubation (> 10 min), a significant proportion of the transported choline-O-sulfate was hydrolyzed intracellulary. In the presence of 5 x 10(-3)m external choline-O-sulfate, the mycelia accumulated choline-O-sulfate to an apparent intracellular concentration of 0.075 m by 3 hr. Transport was unidirectional. No efflux or exchange of (35)S-choline-O-sulfate was observed when preloaded mycelia were suspended in buffer alone or in buffer containing a large excess of unlabeled choline-O-sulfate. (viii) The specific transport activity of the mycelium depended on the sulfur source used for growth. (ix) Sulfur starvation of sulfur-sufficient mycelium resulted in an increase in the specific transport activity of the mycelium. This increase was prevented by cycloheximide, occurred only when a metabolizable carbon source was present, and resulted from an increase in the V(max) of the permease, rather than from a decrease in K(m). The increase could be partially reversed by refeeding the mycelia with unlabeled choline-O-sulfate, sulfide, sulfite, l-homocysteine, l-cysteine, or compounds easily converted to cysteine. The results strongly suggested that the choline-O-sulfate permease is regulated primarily by repression-derepression, but that intracellular choline-O-sulfate and cysteine can act as feedback inhibitors.     

Brain iron transport

Brain iron is a crucial participant and regulator of normal physiological activity. However, excess iron is involved in the formation of free radicals, and has been associated with oxidative damage to neuronal and other brain cells. Abnormally high brain iron levels have been observed in various neurodegenerative diseases, including neurodegeneration with brain iron accumulation, Alzheimer's disease, Parkinson's disease and Huntington's disease. However, the key question of why iron levels increase in the relevant regions of the brain remains to be answered. A full understanding of the homeostatic mechanisms involved in brain iron transport and metabolism is therefore critical not only for elucidating the pathophysiological mechanisms responsible for excess iron accumulation in the brain but also for developing pharmacological interventions to disrupt the chain of pathological events occurring in these neurodegenerative diseases. Numerous studies have been conducted, but to date no effort to synthesize these studies and ideas into a systematic and coherent summary has been made, especially concerning iron transport across the luminal (apical) membrane of the capillary endothelium and the membranes of different brain cell types. Herein, we review key findings on brain iron transport, highlighting the mechanisms involved in iron transport across the luminal (apical) as well as the abluminal (basal) membrane of the blood–brain barrier, the blood–cerebrospinal fluid barrier, and iron uptake and release in neurons, oligodendrocytes, astrocytes and microglia within the brain. We offer suggestions for addressing the many important gaps in our understanding of this important topic, and provide new insights into the potential causes of abnormally increased iron levels in regions of the brain in neurodegenerative disorders.     

Main structural and stereochemical aspects of the antiherpetic activity of nonahydroxyterphenoyl-containing C-glycosidic ellagitannins

Antiherpetic evaluation of five nonahydroxyterphenoyl-containing C-glycosidic ellagitannins, castalagin (1), vescalagin (2), grandinin (3), roburin B (5), and roburin D (7), was performed in cultured cells against four HSV-1 and HSV-2 strains, two of which were resistant to Acyclovir. All five ellagitannins displayed significant anti-HSV activities against the Acyclovir-resistant mutants, but the monomeric structures 1-3 were more active than the dimers 5 and 7. Vescalagin (2) stands out among the five congeners tested as the most potent and selective inhibitor, with an IC50 value in the subfemtomolar range and a selectivity index 5x10(5) times higher than that of Acyclovir. Molecular modeling was used to provide a rationale for the surprisingly lower activity profile of its epimer castalagin (1). These ellagitannins have promising potential as novel inhibitors in the search for non-nucleoside drugs active against Acyclovir-resistant herpes viruses.     

Energetic aspects of spore germination in filamentous fungi

The antifungal activity of substances interfering with the function and biogenesis of mitochondria was studied. Strict anaerobiosis, cyanide, azide, oligomycin, bongkrekic acid and ethidium bromide were found to prevent spore germination ofAspergillus niger andPenicillium italicum in liquid germination medium. The effect of azide, oligomycin and ethidium bromide was fungicidal. Cyanide and azide completely inhibited the incorporation of¹⁴C-leucine and¹⁴C-uracil into germinating conidia ofA. niger. Oligomycin and ethidium bromide reduced the extent of incorporation of both precursors in the first few hours of conidial germination and at later stages stopped it completely. The inhibition of both spore germination and macromolecules synthesis during the germination ofA. niger conidia were in relation to the specific inhibitory effect of the agents on respiratory activity of dormant conidia and mycelial cells. The results indicate that both the function of mitochondrial genetic and protein synthesizing systems and the function of oxidative phosphorylation are essential for normal spore germination and fungal growth.     

Cellular iron transport

Iron has a split personality as an essential nutrient that also has the potential to generate reactive oxygen species. We discuss how different cell types within specific tissues manage this schizophrenia. The emphasis in enterocytes is on regulating the body's supply of iron by regulating transport into the blood stream. In developing red blood cells, adaptations in transport manage the body's highest flux of iron. Hepatocytes buffer the body's stock of iron. Macrophage recycle the iron from effete red cells among other iron management tasks. Pneumocytes provide a barrier to prevent illicit entry that, when at risk of breaching, leads to a need to handle the dangers in a fashion essentially shared with macrophage. We also discuss or introduce cell types including renal cells, neurons, other brain cells, and more where our ignorance, currently still vast, needs to be removed by future research.