Hydroxamic acids in nature.

The hydroxamic acid bond occurs in products from fungi, yeast, bacteria, and plants. The -CON(OH)-bond arises by oxidation of a free or bound amino group in a unit structure which is often closely related to conventional amino acids. Products are known with one, two, or three hydroxamic acid groups per molecule. The chemistry of the ferrichrome type compounds, which are ferric trihydroxamate-containing peptides, has been worked out in detail and includes a complete crystallographic analysis of the ferrichrome A molecule. The trihydroxamates form potent complexes with ferric ion, called siderochromes, and these are believed to play a role in the metabolism of the metal ion in microorganisms. The actual physiological activity observed ranges from that of growth factor, antibiotic, antibiotic antagonist, tumor inhibitor or cell-division factor. The precise molecular mechanism whereby these substances exert their potent beological activity remains to be elucidated.

Zinc and Cobalt: Effect on the Iron Metabolism of Ustilago sphaerogena.

The ferrichrome content of cells of the smut fungus Ustilago sphaerogena during growth in a medium deficient in zinc increases with increase in the cobalt of the medium from 0 to 3 X 10(-5)M. The addition of zinc to such cultures prevents the accumulation of ferrichrome. The results suggest that zinc is involved in the utilization of iron via a process which can be blocked by cobalt.

Leaf infections: siderochromes (natural polyhydroamates) mimic the "green island" effect.

Topical application of as little as 5x10(-12) moles of pure siderochromes (polyhydroxamate iron-transport compounds from bacteria and fungi) onto detached bean leaves causes spots of chlorophyll retention that are surrounded by chlorotic halos. These spots appear similar to "green islands" that are caused by certain fungal infections on leaves and are somewhat similar to zones where senescence is delayed by cytokinin.

Metal ion regulation of gene expression. Fur repressor-operator interaction at the promoter region of the aerobactin system of pColV-K30.

Transcription of the iron-controlled aerobactin operon of the enterobacterial plasmid pColV-K30 is negatively regulated through the interaction of a Fe2+-binding repressor (the Fur protein) with operator sequences within the promoter region of the operon. The DNA sequences essential for interaction with the repressor were located by site-directed mutagenesis of specific regions within the 31 base-pair protected by the repressor from DNase I nicking. Occupation of two contiguous repressor-binding sites appears to be required for the complete repression of the system. Contacts of the Fur repressor with the corresponding operator sequences were analyzed with hydroxyl radical footprint and methylation protection experiments. These indicate that DNA-protein contacts approach a symmetrical mode and take place at all sides of the DNA helix.

Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12.

We have cloned genes responsible for enterobactin synthesis (entD) and transport (fepA,fes) from Escherichia coli K-12. Relevant recombinant plasmids enabled EntD- and transport-defective mutants to grow on iron-limiting medium. Subcloning and deletion analysis demonstrated that the gene order is entD-fepA-fes. Protein synthesis studies in minicells suggest that FepA is first translated as an Mr 84 000 precursor, which is subsequently cleaved to the active Mr 81 000 receptor; the fes gene product is an Mr 44 000 protein; no polypeptide has been identified as the entD gene product.

Artificial siderophores. 1. Synthesis and microbial iron transport capabilities.

Several di- and trihydroxamate analogues of natural microbial iron chelators have been prepared. The syntheses involved linkage of core structural units, including pyridinedicarboxylic acid, benzenetricarboxylic acid, nitrilotriacetic acid, and tricarballylic acid, by amide bonds to 1-amino-omega-(hydroxyamino)alkanes to provide the polyhydroxamates 1-5. The required protected (hydroxyamino)alkanes 8, 16, and 21 were prepared by different routes. 1-Amino-3-[(benzyloxy)amino]propane di-p-toluenesulfonate (8) was prepared from the N-protected aminopropanol 6 by oxidation to the aldehyde, formation of the substituted oxime, and reduction with NaBH3CN followed by deprotection of the Boc group. The pentyl derivatives 16 and 21 were made by direct alkylation with either benzyl acetohydroxamate or N-carbobenzoxy-O-benzylhydroxylamine. In Escherichia coli RW193 most of the analogues behaved nutritionally as ferrichrome. However, in E. coli AN193, a mutant lacking the ferrichrome receptor, capacity to use other natural siderophores was retained while response to all analogues was lost.

Artificial siderophores. 2. Syntheses of trihydroxamate analogues of rhodotorulic acid and their biological iron transport capabilities in Escherichia coli.

Tris[(acetylhydroxyamino)alkyl] isocyanurates 2a-c were synthesized from alpha, omega-dibromoalkanes 5 in four steps. The alkylation of the bromides 5a-c with O-benzyl-N-[(trichloroethoxy)carbonyl]hydroxylamine in the presence of DBU gave N-alkylation products 7a-c. The (trichloroethoxy)cabronyl protecting group of 7a-c was easily removed by Zn dust in acetic acid. When the reaction was performed with acetic anhydride, the desired N-acetylated materials 10a-c were obtained. The alkylation of cyanuric acid with 12 in the presence of base provided the N-alkylated materials 13, which were hydrogenated to provide 2a-c. In order to determine the affect of structural modifications on biological activity, various chain lengths of the side arms were utilized and the retroanalogue 3 was prepared. Most of the compounds examined acted as ferrichrome in supporting the iron nutrition of Escherichia coli. However, tris[(acetylhydroxyamino)butyl] isocyanurate 2b, and to some extent its pentyl analogue, 2c, displayed the unique and remarkable property of supporting growth of fhuB mutants, the latter unresponsive to the other analogues and to all natural siderophores tested.

An electron spin resonance study of the Mn(II) and Cu(II) complexes of the Fur repressor protein.

EPR spectra of Mn(II) Fur complex suggested the presence of Mn(II) in one site per Fur monomer in which Mn(II) is present in a low symmetry environment. The binding of the Mn(II) Fur complex to a DNA fragment "iron box" has a slight broadening effect on the Mn(II) signal and hence it altered the symmetry of the Mn(II) environment. We also report EPR spectra of Cu(II) Fur and Cu(II) C92S C95S mutant Fur complexes as models for Fe(II) complexes; the anisotropic g values and A values observed indicate the presence of Cu(II) in two different environments in the protein; a major axially distorted Cu(II) site bound to nitrogen and a minor distorted tetrahedral sulfur bound to the Cu(II) site. The effect of metal ion on Fur DNA binding is also discussed.

A new generation of antibodies.

Antibodies, proteins produced in animals that bind with high specificity and affinity to a seemingly limitless variety of biomolecules, have an essential role in clinical medicine and basic biological research. Methods to produce antibody fragments on the surface of bacterial viruses were surveyed for their ability to replace animal-dependent antibodies. The use of filamentous phage to display antibody fragments derived from semisynthetic antibody genes was found to produce proteins that bind to antigens with a variety, specificity, and affinity similar to those produced in animal systems.