Some factors affecting metal ion-monohydroxamate interactions in aqueous solution

The chelating properties exhibited by a series of monohydroxamic acids (propanohydroxamic acid (Pha), hexanohydroxamic acid (Hha), benzohydroxamic acid (Bha), N-methyl-acetohydroxamic acid (MAha), N-phenyl-acetohydroxamic acid (PhAha) and 2-hydroxypyridine-N-oxide (PYRha)) towards copper(II), nickel(II), zinc(II), calcium(II), magnesium(II) and aluminium(III) ions were studied by pH-metric, spectrophotometric and, in one case, by 27Al NMR methods. The results were compared with the corresponding data for metal ion-acetohydroxamate (Aha) and metal ion-desferrioxamine B (DFB) complexes. Changes of the substituents either on the carbon or on the nitrogen of the hydroxamate moiety caused a measurable effect on the chelate stability only in the case of aluminium(III) complexes. The aromatic derivative, PYRha, formed significantly more stable complexes than expected on the basis of the ligand basicity. The higher complex-forming ability of DFB compared to monohydroxamic acids diminishes in the case of the largest calcium(II) ion.     

Iron uptake in Salmonella typhimurium: utilization of exogenous siderochromes as iron carriers

Aerobic microorganisms have evolved a variety of siderochromes, special ligands which can dissolve insoluble ferric iron and facilitate its transport into the cell. We have found that enb mutants of Salmonella typhimurium blocked in the biosynthesis of enterobactin (its natural iron carrier) are able to utilize siderochromes of different types made by other microorganisms as iron carriers. The antibiotic albomycin delta(2) was used to select mutants defective in ferrichrome-mediated iron uptake. Twelve classes of albomycin-resistant mutants, named sid, were defined on the basis of their growth responses to other siderochromes. Most of these classes have genetic lesions in loci that are cotransduced with panC (represented at 9 min on the genetic map). The locus designated sidJ is cotransduced with enb, whereas sidK and sidL are linked with neither panC nor enb. Genetic and physiological data indicate that S. typhimurium has several transport systems of high specificity for a variety of siderochromes produced by other microorganisms.     

Siderochromes from Pseudomonas fluorescens. I. Isolation and characterization

Several iron-binding pigments (siderochromes) produced by Pseudomonas fluorescens have been isolated and partially characterized. They include ferribactin and various forms of pyoverdine, as well as some previously unreported compounds. In particular, the existence of ferribactin has been independently confirmed for the first time. Column and thin layer chromatographic procedures have been developed to fractionate, purify, and identify the siderochromes. We find ferribactin to contain nine amino acids, one residue each of glutamine, tyrosine, and glycine, and two each of serine, lysine, and N-hydroxyornithine, rather than 10 as earlier reported. Pyoverdine is a peptide with the same composition as ferribactin except for the absence of glutamine and the substitution of a fluorescent chromophore for tyrosine. Paper electrophoresis reveals an extra ionizable group in ferric pyoverdine relative to pyoverdine or ferribactin which provides that complex a definite cathodic mobility at pH 3. Optical spectra of the pyoverdine fluorescent component indicate that, in conjunction with the two hydroxamate groups, it is involved in the metal ion coordination, conferring on pyoverdine a dramatically increased affinity for Fe(III) relative to ferribactin.     

A spectrophotometric assay for the degradation of the siderophore deferrioxamine B

A spectrophotometric assay using ferric perchlorate in a perchloric acid solution has been developed to monitor the degradation of the trihydroxamate siderophore deferrioxamine B to monohydroxamates. Using the ferric perchlorate solution and employing various concentrations of acetohydroxamic acid (as the model monohydroxamic acid) while maintaining a constant amount of deferrioxamine B resulted in the shifting of the absorption maximum from that of ferrioxamine B to longer wavelengths and toward that of a pure ferri-acetomonohydroxamic acid solution. A similar result was noted when a cell-free extract, from a bacterium capable of using deferrioxamine B as its sole carbon source, was given the siderophore in a phosphate buffer and aliquots of the enzyme-deferrioxamine B solution were removed for analysis. The assay may thus be used to monitor the formation of the monohydroxamic acid degradation products of the siderophore by the enzyme(s) in the cell-free extract.     

Succinylhydroxamic derivatives of alpha-amino acids as MMP inhibitors. Study of complex-formation equilibria with Cu2+, Ni2+ and Zn2+

A series of Pro- and Phe-succinyl hydroxamate derivatives, whose nanomolar inhibitory activity towards a series of matrix metalloproteinases (MMPs) was previously reported, have been studied and described herein in their interaction with Cu(2+), Zn(2+), Ni(2+) in aqueous solution, by using potentiometric, spectroscopic and ESI-MS (electrospray ionization mass) spectrometric techniques. A systematic study at various ligand-to-metal molar ratios allowed the determination of the stability constants of the complexes as well as the estimation of the coordination modes. The similarity in the biological activity of these compounds seems to be paralleled by the identical metal-complexation behaviour at neutral pH, namely in terms of chelating effectiveness and coordination modes, irrespective of the presence of one carboxylic or hydroxamate as extra groups, or also of the type of amino-acid residue at the other flank of the succinyl chain, which seems to be enough away from the succinyl hydroxamate metal-binding group. The stability order of the metal complexes with these ligands follows the Irving-Williams trend for this type of complex systems. Noteworthy is the identification of an interesting pentanuclear copper(II) species with the monohydroxamic ligands which structure was ascribed to a 12-metallacrown-4.     

Isolation and identification of the conidial germination factor of Neurospora crassa.

The germination-essential substance (germination factor [GF]) that is lost from conidia of Neurospora crassa on exposure to solutions of low water activity has been isolated and identified as a group of iron-transport compounds, or siderochromes. The principal siderochrome of conidia is ferricrocin, a cyclic hexapeptide. A closely related substance, ferrichrome C, is tentatively identified as a minor constituent. The same substances are also present in extracts of mycelium along with small amounts of a third siderochrome, which has not been identified. The GF activity of culture filtrates is due to coprogen, the only siderochrome previously identified with N. crassa.     

Relationship between the tonB locus and iron transport in Escherichia coli.

When a strain (arcB-) of Escherichia coli, unable to synthesize the iron transport compound enterochelin, was transduced to tonB-, it became resistant to phage phi80 and simultaneously lost the growth response to enterochelin and the ability to transport its iron complex. However, enterochelin precursors (shikimate and 2,3-dihydroxybenzoate) still supported growth, via the synthesis of enterochelin. Dihydroxybenzoate was a better growth factor at a low concentration than it was at higher levels. The evidence suggests that tonB- strains lack an outer membrane component necessary both for the uptake of ferric-enterochelin and for the adsorption of phage phi80. Thus, although ferric-enterochelin cannot penetrate the cell surface from outside, the complex that is formed within the envelope is transported normally into the cell. The aroB-, tonB- mutant also lacked growth reponses to citrate and various hydroxamate siderochromes, which supported growth in the tonB+ parent strain via inducible transport systems for their ferric complexes. The aroB-, tonB- mutant was unable to transport iron in the presence of citrate, but the low-affinity uptake of uncomplexed iron and the transport of amino acids and phosphate were unimpaired. The tonB locus, thus, affects all the known active transport systems for iron, possibly indicating that they share some common outer membrane component.     

Inhibition of Bacillus anthracis metallo-β-lactamase by compounds with hydroxamic acid functionality

Abstract

Metallo-β-lactamases (MBLs) that catalyze hydrolysis of β-lactam antibiotics are an emerging threat due to their rapid spread. A strain of the bacterium Bacillus anthracis has its ability to produce and secrete a MBL, referred to Bla2. To address this challenge, novel hydroxamic acid-containing compounds such as 3-(heptyloxy)-N-hydroxybenzamide (compound 4) and N-hydroxy-3-((6-(hydroxyamino)-6-oxohexyl)oxy)benzamide (compound 7) were synthesized. Kinetic analysis of microbial inhibition indicated that the both sides of hydroxamic acids containing compound 7 revealed a reversible, competitive inhibition with a K_i value of 0.18?±?0.06?μM. The result has reflected that the both sides of dihydroxamic acids in a molecule play a crucial role in the binding affinity rather than monohydroxamic containing compound 4 which was unable to inhibit Bla2. In addition, in silico analysis suggested that compound 7 was coordinated with a zinc ion in the active site of enzyme. These observations suggest that the dihydroxamic acid-containing compound may be a promising drug candidate, and a further implication for designing new inhibitors of Bla2.     

Partial purification and some properties of pyruvate carboxylase from the flight muscle of the locust (Schistocerca gregaria)

A procedure is described for the partial purification of pyruvate carboxylase (pyruvate:CO2 ligase (ADP-forming), EC 6.4.1.1) from the flight muscle of the locust (Schistocerca gregaria). Characterisation of the kinetic properties of this enzyme indicates that it is activated by acetyl-CoA, is insensitive to inhibition by di- and tricarboxylic acids and exhibits an apparent Km for HCO3-(16 mM) which differs by an order of magnitude from that observed for other pyruvate carboxylases. It is suggested that activation of this locust flight muscle pyruvate carboxylase during the rest leads to flight transition may result from increases in the concentrations of pyruvate and HCO3- under these conditions.     

Further characterization and proposed pathway of deferrioxamine B catabolism

Most siderophores are catabolism resistant because of their non-peptidic or cyclic peptide chemical structures. Siderophore degradation is thus a rare event, and one which has received little attention. Based on literature precedence and the generation of monohydroxamic acid(s) by cell-free extracts of a deferrioxamine B degrading bacterium, a catabolic scheme of the siderophore is proposed. Data are also presented concerning parameters such as the pH profile, inducible nature of the siderophore-degrading metabolism and the ability of the deferrioxamine B degrading bacterium to metabolize some of the hypothesized products of deferrioxamine B dissimilation.     

An ethidium-acrylamide affinity medium for recovery of nucleic acids from free solution and from polyacrylamide and agarose gels

The simple preparation of an ethidium-bromide-based nucleic acid affinity medium is described. The medium is composed of an acrylamide matrix to which ethidium bromide is attached. Its use in preparative purification and fractionation of nucleic acids in solution and in electrophoretic elution of nucleic acids from gels is reported. Nucleic acids can be eluted from this medium with a buffered salt solution and concentrated by ethanol precipitation without persistent contamination with undesirable impurities.     

Extraction to 2,4,6-trinitrophenyl derivatives of amino acids and peptides from aqueous acid. A simple method for determining aminotripeptidase activity

2,4,6-Trinitrophenyl (TNP) amino acids and peptides vary greatly in the extent to which they are extracted from acid solution into organic solvents. A method based on this observation is described, in which leu is determined specifically in the presence of gly-gly and an excess of leu-gly-gly. This method has been applied to the assay of aminotripeptidase activity in some tissue extracts, using leu-gly-gly as substrate. Extraction characteristics of many TNP-amino acids and -peptides are recorded. No solvent tested extracted all TNP-amino acids quantitatively from acid solution. The application of this information to the solution of other analytical problems is discussed.     

Tools for investigating peptide-protein interactions: peptide incorporation of environment-sensitive fluorophores via on-resin derivatization

This protocol presents the peptide incorporation of environment-sensitive fluorophores derived from the dimethylaminophthalimide family. The procedure utilizes anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol from these authors. In this protocol, the fluorophores are directly incorporated after solid-phase peptide synthesis (SPPS) via on-resin derivatization of peptides prepared using commercially available diamino acids, which are Alloc-protected on the side-chain amino group. The time required to complete the procedure depends on the size and number of peptides targeted. As an alternative to this approach, the corresponding fluorescent amino acids can be obtained in an Fmoc-protected form for convenient use as building blocks in SPPS. This option is described in a related protocol by these authors.     

Estimation of hormone receptor affinity by competitive displacement of labeled ligand: effect of concentration of receptor and of labeled ligand.

Ferrichrome is shown to compete in vitro for the partially purified outer membrane phage T5 receptor complex of Escherichia coli K-12. Among siderochromes tested the competition is confined to ferrichrome and its analogs. Thus reversal of receptor inhibition of plaque formation is achieved by ferrichrome, ferricrocin, and the aluminum and chromium(III) derivatives of deferriferrichrome but not by rhodotorulic acid, Desferal, or ferrichrome A.     

Malonichrome,a new iron chelate from Fusarium roseum

The predominant iron chelates, or siderochromes, produced by the fungus, Fusarium roseum during culture periods up to seven days are the ester type fusarinine compounds. During longer periods of incubation, the fusarinine compounds completely disappear from the culture medium and are replaced by a new siderochrome. The new compound has been isolated, purified, and its structure determined. It is a cyclic hexapeptide containing one residue of l-alanine, two residues of glycine and three residues of δ-N-hydroxyornithine. The hydroxylamino groups of the ornithine residues are acylated with 3 mol of malonic acid to form a negatively charged ferrichrome type chelate. The circular dichroism spectrum indicates that the stereochemistry about the iron is Λ-cis. This compound, which we name malonichrome, is not an efficient iron donor to F. roseum nor does it show growth factor activity towards Arthrobacter flavescens.     

原子力显微镜在生物学研究中的应用进展

原子力显微镜（atomic force microscope,AFM)具有原子级分辨率,能够在生理条件下对生物样品进行观察,本综述了AFM的原理及技术要点,举例说明了它在核酸,蛋白质,微生物及细胞等领域的应用进展,相信AFM必将在生物学研究中起到越来越重要的作用。     

New microbial growth factor.

A screening procedure was used to isolate from soil a Penicillium sp., two bacterial isolates, and a Streptomyces sp. that produced a new microbial growth factor. This factor was an absolute growth requirement for three soil bacteria. The Penicillium sp. and one of the bacteria requiring the factor, an Arthrobacter sp., were selected for more extensive study concerning the production and characteristics of the growth factor. It did not seem to be related to the siderochromes. It was not present in soil extract, rumen fluid, or any other medium component tested. It appears to be a glycoprotein of high molecular weight, and it has high specific activity. When added to the diets for a meadow vole mammalian test system, it caused an increased consumption of diet without a concurrent increase in rate of weight gain.     

Which Amino Acids Should Be Used in Prebiotic Chemistry Studies?

The adsorption of amino acids on minerals and their condensation under conditions that resemble those of prebiotic earth is a well studied subject. However, which amino acids should be used in these experiments is still an open question. The main goal of this review is to attempt to answer this question. There were two sources of amino acids for the prebiotic earth: (1) exogenous -- meaning that the amino acids were synthesized outside the earth and delivered to our planet by interplanetary dust particles (IDPs), meteorites, comets, etc. and (2) endogenous -- meaning that they were synthesized on earth in atmospheric mixtures, hydrothermal vents, etc. For prebiotic chemistry studies, the use of a mixture of amino acids from both endogenous and exogenous sources is suggested. The exogenous contribution of amino acids to this mixture is very different from the average composition of proteins, and contains several non-protein amino acids. On the other hand, the mixture of amino acids from endogenous sources is seems to more closely resemble the amino acid composition of terrestrial proteins.     

Identifying your enemies – could envelope stress trigger microbial immunity?

Microbes utilize defence systems with fundamental similarities to our innate and adaptive immune responses to protect themselves from harmful invaders. One system, made up of CRISPR loci & Cas proteins, incorporates recognizable features from the genomes of viruses (bacteriophages) and plasmids into bacterial genomes, where they are later used to direct a ribonucleoprotein complex to destroy invading nucleic acids upon re-exposure. CRISPR-mediated defence against invasive nucleic acids is found in most archaea and many eubacteria. Many aspects of this newly described defence system have not been worked out, including the molecular mechanisms by which foreign nucleic acids are incorporated into microbial genomes during adaption and destroyed during interference. In this issue of Molecular Microbiology, DeLisa and colleagues provide insight into how this form of microbial immunity might be regulated in eubacteria. They demonstrate that Escherichia coli CRISPR-mediated immunity requires the presence of the BaeSR two-component system under certain conditions. Since BaeSR regulate an envelope stress response, their data imply that immunity against invading, foreign nucleic acids may be somehow linked to stresses to the bacterial membrane. These observations will help pave the way to understanding how and when CRISPR-based immunity may be important in driving evolution and adaptation in eubacteria.     

Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev

Non-specifically bound nucleic acid contaminants are an unwanted feature of recombinant RNA-binding proteins purified from Escherichia coli (E. coli). Removal of these contaminants represents an important step for the proteins’ application in several biological assays and structural studies. The method described in this paper is a one-step protocol which is effective at removing tightly bound nucleic acids from overexpressed tagged HIV-1 Rev in E. coli. We combined affinity chromatography under denaturing conditions with subsequent on-column refolding, to prevent self-association of Rev while removing the nucleic acid contaminants from the end product. We compare this purification method with an established, multi-step protocol involving precipitation with polyethyleneimine (PEI). As our tailored protocol requires only one-step to simultaneously purify tagged proteins and eliminate bound cellular RNA and DNA, it represents a substantial advantage in time, effort, and expense.