Antimetabolites are small molecules that inhibit enzymes by mimicking physiological substrates. We report the discovery and structural elucidation of the antimetabolite 7-deoxy-sedoheptulose (7dSh). This unusual sugar inhibits the growth of various prototrophic organisms, including species of cyanobacteria, Saccharomyces, and Arabidopsis. We isolate bioactive 7dSh from culture supernatants of the cyanobacterium Synechococcus elongatus. A chemoenzymatic synthesis of 7dSh using S. elongatus transketolase as catalyst and 5-deoxy-d-ribose as substrate allows antimicrobial and herbicidal bioprofiling. Organisms treated with 7dSh accumulate 3-deoxy-d-arabino-heptulosonate 7-phosphate, which indicates that the molecular target is 3-dehydroquinate synthase, a key enzyme of the shikimate pathway, which is absent in humans and animals. The herbicidal activity of 7dSh is in the low micromolar range. No cytotoxic effects on mammalian cells have been observed. We propose that the in vivo inhibition of the shikimate pathway makes 7dSh a natural antimicrobial and herbicidal agent.
Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86–91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158–163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3–11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.
Prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2), produced essentially by the prostate gland, are 237-amino acid monomeric proteins, with 79% identity in primary structure. Twenty-five anti-PSA monoclonal antibodies (Mabs) were studied for binding to a large array of synthetic linear peptides selected from computer models of PSA and hK2, as well as to biotinylated peptides covering the entire PSA sequence.Sixteen of the Mabs were bound to linear peptides forming four independent binding regions (I-N). Binding region I was localized to amino acid residues 1-13 (identical sequence for PSA and hK2), I1 (a and b) was localized to residues 53-64, 111 (a and b) was localized to residues 80-91 (= kallikrein loop), and N was localized to residues 151-164. Mabs binding to regions I and IIa were reactive with free PSA, PSA-ACT complex, and with hK2; Mabs binding to regions IIb, IIIa, and IV were reactive with free PSA and PSA-ACT complex, but unreactive with hK2; Mabs binding to region IIIb detected free PSA only. All Mabs tested (n = 7) specific for free PSA reacted with kallikrein loop (binding region IIIb). The presence of Mabs interacting with binding region I did not inhibit the catalytic activity of PSA, whereasMabs interacting with other binding regions inhibited the catalysis. Theoretical model structures of PSA, hK2, and the PSA-ACT complex were combined with the presented data to suggest an overall orientation of PSA with regard to ACT.
Iron is a vital mineral for almost all living organisms and has a pivotal role in central metabolism. Despite its great abundance on earth, the accessibility for microorganisms is often limited, because poorly soluble ferric iron (Fe3+) is the predominant oxidation state in an aerobic environment. Hence, the reduction of Fe3+ is of essential importance to meet the cellular demand of ferrous iron (Fe2+) but might become detrimental as excessive amounts of intracellular Fe2+ tend to undergo the cytotoxic Fenton reaction in the presence of hydrogen peroxide. We demonstrate that the complex formation rate of Fe3+ and phenolic compounds like protocatechuic acid was increased by 46% in the presence of HCO3− and thus accelerated the subsequent redox reaction, yielding reduced Fe2+. Consequently, elevated CO2/HCO3− levels increased the intracellular Fe2+ availability, which resulted in at least 50% higher biomass-specific fluorescence of a DtxR-based Corynebacterium glutamicum reporter strain, and stimulated growth. Since the increased Fe2+ availability was attributed to the interaction of HCO3− and chemical iron reduction, the abiotic effect postulated in this study is of general relevance in geochemical and biological environments. IMPORTANCE In an oxygenic environment, poorly soluble Fe3+ must be reduced to meet the cellular Fe2+ demand. This study demonstrates that elevated CO2/HCO3− levels accelerate chemical Fe3+ reduction through phenolic compounds, thus increasing intracellular Fe2+ availability. A number of biological environments are characterized by the presence of phenolic compounds and elevated HCO3− levels and include soil habitats and the human body. Fe2+ availability is of particular interest in the latter, as it controls the infectiousness of pathogens. Since the effect postulated here is abiotic, it generally affects the Fe2+ distribution in nature.
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