We have synthesized, separated, and purified =10 mg of a deoxyundecanucleotide duplex containing a single centrally positioned covalent adduct between (+)-antibenzo[a]pyrene (BP) diol epoxide and the exocyclic amino group of guanosine. Excellent proton NMR spectra are observed for the (+)-trans-anti-BP diol epoxide-N2-dG adduct positioned opposite dC and flanked by G-C pairs in theWe have determined the solution structure centered about the BP covalent adduct site in the (BP)G-C ll-mer duplex by incorporating intramolecular and intermolecular proton-proton distance bounds deduced from the NMR data sets as constraints in energy minimization computations. The BP ring is positioned in the minor groove and directed toward the 5' end of the modified strand. One face of the BP ring of (BP)G6 is stacked over the G18 and A19 sugarphosphate backbone on the partner strand and the other face is exposed to solvent. A minimally perturbed B-DNA helix is observed for the d[T4-C5-(BP)G6-C7-T8]Jd[A15-G16-C17-G18-A19] segment centered about the adduct site with WatsonCrick alignment for both the (BP)G6-C17 pair and flanking GC pairs. A widening of the minor groove at the adduct site is detected that accommodates the BP ring whose long axis makes an angle of =45°with the average direction of the DNA helix axis. Our study holds future promise for the characterization of other stereoisomerically pure adducts of BP diol epoxides with DNA to elucidate the molecular basis of structure-activity relationships associated with the stereoisomerdependent spectrum of mutational and carcinogenic activities.Benzo[a]pyrene (BP), a ubiquitous environmental pollutant, is metabolized in mammalian cells to highly reactive, mutagenic, and tumorigenic diol epoxide derivatives (the field of carcinogen-DNA adducts is reviewed in refs.

The structural characteristics of a mucin glycopeptide motif derived from the N-terminal fragment STTAV of the cell surface glycoprotein CD43 have been investigated by NMR. In this study, a series of molecules prepared by total synthesis were examined, consisting of the peptide itself, three glycopeptides having clustered sites of alpha-O-glycosylation on the serine and threonine side chains with the Tn, TF, and STF carbohydrate antigens, respectively, and one with the beta-O-linked TF antigen. Additionally, a glycopeptide having the sequence SSSAVAV, triglycosylated with the Le(y) epitope, was investigated. NMR data for the tri-STF-STTAV glycopeptide were used to solve the structure of this construct through restrained molecular dynamics calculations. The calculations revealed a defined conformation for the glycopeptide core rooted in the interaction of the peptide and the first N-acetylgalactosamine residue. The similarity of the NMR data for each of the alpha-O-linked glycopeptides demonstrates that this structure persists for each construct and that the mode of attachment of the first sugar and the peptide is paramount in establishing the organization of the core. The core provides a common framework on which a variety of glycans may be displayed. Remarkably, while there is a profound organizational effect on the peptide backbone with the alpha-linked glycans, attachment via a beta-linkage has little apparent consequence.

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This paper reports on a combined two-dimensional NMR and energy minimization computational characterization of the conformation of the N-(deoxyguanosyl-8-yl)aminofluorene adduct [(AF)G] positioned across adenosine in a DNA oligomer duplex as a function of pH in aqueous solution. This study was undertaken on the d[C1-C2-A3-T4-C5-(AF)G6-C7-T8-A9-C10-C11].[G12-G13-T14 -A15-G16-A17-G18- A19-T20-G21-G22] complementary undecamer [(AF)G 11-mer duplex]. The modification of the single G6 on the pyrimidine-rich strand was accomplished by reaction of the oligonucleotide with N-acetoxy-2-(acetylamino)fluorene and subsequent deacetylation under alkaline conditions. The HPLC-purified modified strand was annealed with the unmodified purine-rich strand to generate the (AF)G 11-mer duplex. The exchangeable and nonexchangeable protons are well resolved and narrow in the NMR spectra of the (AF)G 11-mer duplex so that the base and the majority of sugar nucleic acid protons, as well as several aminofluorene ring protons, have been assigned following analysis of two-dimensional NOESY and COSY data sets at pH 6.9, 30 degrees C in H2O and D2O solution. The NOE distance constraints establish that the glycosidic torsion angle is syn at (AF)G6 and anti at A17, which results in the aminofluorene ring being positioned in the minor groove. A very large downfield shift is detected at the H2' sugar proton of (AF)G6 associated with the (AF)G6[syn].A17[anti] alignment in the (AF)G 11-mer duplex. The NMR parameters demonstrate formation of Watson-Crick C5.G18 and C7.G16 base pairs on either side of the (AF)G6[syn].A17[anti] modification site with the imino proton of G18 more stable to exchange than the imino proton of G16. Several nonexchangeable aminofluorene protons undergo large downfield shifts as do the imino and H8 protons of G16 on lowering of the pH from neutrality to acidic values for the (AF)G 11-mer duplex. Both the neutral and acidic pH conformations have been defined by assigning the NOE constraints in the [C5-(AF)G6-C7].[G16-A17-G18] segment centered about the modification site and incorporating them in distance constrained minimized potential energy calculations in torsion angle space with the DUPLEX program. A series of NOEs between the aminofluorene protons and the DNA sugar protons in the neutral pH conformation establish that the aminofluorene ring spans the minor groove and is directed toward the G16-A17-G18 sugar-phosphate backbone on the partner strand.(ABSTRACT TRUNCATED AT 400 WORDS)

Virtually all microorganisms require iron for growth. The paucity of iron in surface ocean water (approximately 0.02-1.0 nM (refs 1, 2)) has spurred a lively debate concerning iron limitation of primary productivity, yet little is known about the molecular mechanisms used by marine microorganisms to sequester iron. Terrestrial bacteria use a siderophore-mediated ferric uptake system. A siderophore is a low-molecular-mass compound with a high affinity for ferric ion which is secreted by microorganisms is response to low-iron environments; siderophore biosynthesis is regulated by iron levels, with repression by high iron. Although open-ocean marine microorganisms (such as phytoplankton and bacteria) produce siderophores, the nature of these siderophores has not been investigated. We report here the first structure determination, to our knowledge, of the siderophores from an open-ocean bacterium, alterobactin A and B from Alteromonas luteoviolacea. A. luteoviolacea is found in oligotrophic and coastal waters. Alterobactin A has an exceptionally high affinity constant for ferric ion. We suggest that at least some marine microorganisms may have developed higher-affinity iron chelators as part of an efficient iron-uptake mechanism which is more effective than that of their terrestrial counterparts.

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Petrobactin is a bis-catecholate, alpha-hydroxy acid siderophore produced by the oil-degrading marine bacterium Marinobacter hydrocarbonoclasticus. The Fe(III)-complexed form of petrobactin is photoreactive in natural sunlight, mediated by the Fe(III)-citrate moiety. The reaction results in decarboxylation of the petrobactin ligand and reduction of Fe(III) to Fe(II). This report is one of the first to show the photoreactivity of Fe(III)-siderophores mediated by the ferric ion-alpha-hydroxy acid group. The demonstration of light-mediated decarboxylation of an Fe(III)-siderophore complex raises questions about a possible functional role for photoreactivity in siderophore-mediated iron uptake.

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