An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Aminoglycoside 3'-phosphotransferases [APH(3')s] are important bacterial resistance enzymes for aminoglycoside antibiotics. These enzymes phosphorylate the 3'-hydroxyl of these antibiotics, a reaction that inactivates the drug. A series of experiments were carried out to shed light on the details of the turnover chemistry by these enzymes. Quench-flow pre-steady-state kinetic analyses of the reactions of Gram-negative APH(3') types Ia and IIa with kanamycin A, neamine, and their respective difluorinated analogues 4'-deoxy-4',4'-difluorokanamycin A and 4'-deoxy-4',4'-difluoroneamine were carried out, in conjunction with measurements of thio effect and viscosity studies. The fluorinated analogues were shown to be severely impaired as substrates for these enzymes. The magnitude of the effect of the impairment of the fluorinated substrates was in the same range as when the D198A mutant APH(3')-Ia was studied with nonfluorinated substrates. Residue 198 is the proposed active site base that promotes the aminoglycoside hydroxyl for phosphorylation. These findings collectively argue that the Gram-negative APH(3')s show significant nucleophilic participation in the transition state for the phosphate transfer reaction.
Shiga toxin (STX) is a ribosome-inactivating cytotoxin produced by Shigella dysenteriae serotype 1. The enzymatic domain of the STX A polypeptide has been defined by introducing amino- and carboxy-terminal deletions in the polypeptide and assessing activity in a cell-free translation system. Three recombinant forms of StxA which possess enzymatic activity were genetically fused to a 165-amino-acid polypeptide derived from CD4, the cellular receptor for human immunodeficiency virus type 1 (HIV-1). This strategy eliminated the STX receptor-binding subunit and directed the hybrid toxins to cells expressing the HIV-1 surface glycoprotein gp120. A bacterial lysate containing these toxin chimeras killed the HIV-1-infected T-cell line 8E5 but was not cytotoxic toward the uninfected parental cell line A3.01. This cytotoxic activity was specifically inhibited by monoclonal antibodies which block the interaction between CD4 and gp120. These StxA-CD4 hybrids add to the repertoire of recombinant fusion proteins which possess the capacity to selectively kill HIV-1-infected T cells.
Recent X-ray crystallographic analyses have demonstrated that the receptor-binding (B) subunits of Shiga toxin (STX) are arranged as a doughnut-shaped pentamer. The C terminus of the enzymatic (A) subunit presumably penetrates the nonpolar pore of the STX B pentamer, and the holotoxin is stabilized by noncovalent interactions between the polypeptides. We identified a stretch of nine nonpolar amino acids near the C terminus of StxA which were required for subunit association by using site-directed mutagenesis to introduce progressive C-terminal deletions in the polypeptide and assessing holotoxin formation by a receptor analog enzyme-linked immunosorbent assay, immunoprecipitation, and a cytotoxicity assay. Tryptophan and aspartic acid residues which form the N-terminal boundary, as well as two arginine residues which form the C-terminal boundary of the nine-amino-acid sequence, were implicated as the stabilizers of subunit association. Our model proposes that residues 279 to 287 of the 293-amino-acid STX A subunit penetrate the pore while the tryptophan, aspartic acid, and 2 arginine residues interact with other charged or aromatic amino acids outside the pore on the planar surfaces of the STX B pentamer.
The novel 7-(N-formyl-, 7-(N-acetyl-, and 7-(N-isobutyrylamino)-2-methylquinoline-5,8-diones were synthesized in excellent overall yields in three steps via the nitration of the commercially available 8-hydroxy-2-methylquinoline followed by a reduction-acylation step and then oxidation. Acid hydrolysis of 7-(N-acetylamino)-2-methylquinoline-5,8-dione (14a) afforded the novel 7-aminoquinoline-5,8-dione 7 in excellent yields. Due to our efficient preparation of dione 14a, we now report a short and practical method for the total synthesis of the potent antitumor agent lavendamycin methyl ester (1b) with an excellent overall yield.
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