RESUMO
Cps2L, a thymidylytransferase, is the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis and an antibacterial target. We herein report the evaluation of six sugar phosphate analogues selected to further probe Cps2L substrate tolerance. A modified continuous spectrophotometric assay was employed for facile detection of pyrophosphate (PPi) released from nucleotidylyltransfase-catalysed condensation of sugar 1-phosphates and nucleoside triphosphates to produce sugar nucleotides. Additionally, experiments using waterLOGSY NMR spectroscopy were investigated as a complimentary method to evaluate binding affinity to Cps2L.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Glucofosfatos/química , Nucleotidiltransferases/química , Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Difosfatos/análise , Ensaios Enzimáticos , Inibidores Enzimáticos/síntese química , Cinética , Nucleotidiltransferases/antagonistas & inibidores , Proteínas Recombinantes/química , Espectrofotometria , Streptococcus pneumoniae/química , Streptococcus pneumoniae/enzimologiaRESUMO
NtdA is a putative sugar aminotransferase that is required for the synthesis of 3,3'-neotrehalosadiamine. The enzyme was purified to homogeneity by means of Ni(2+)-affinity chromatography and was crystallized using the microbatch method. X-ray diffraction data were collected from a single crystal to 2.3 A resolution at the Canadian Light Source (CLS). The crystals belonged to space group P2(1), with unit-cell parameters a = 50.3, b = 106.7, c = 96.7 A, beta = 96.2 degrees, and contained two molecules per asymmetric unit.
Assuntos
Bacillus subtilis/enzimologia , Fosfato de Piridoxal/metabolismo , Transaminases/química , Transaminases/isolamento & purificação , Cristalização , Cristalografia por Raios X , Eletricidade Estática , Trealose/análogos & derivados , Trealose/químicaRESUMO
Inositol dehydrogenase (IDH) is an enzyme that catalyses the NAD(+)-dependent oxidation of myo-inositol to scyllo-inosose. The enzyme has been purified to homogeneity by means of Ni(2+)-affinity chromatography and was crystallized in both native and selenomethionine (SeMet) labelled forms using the microbatch method. SAD X-ray diffraction data were collected to 2.0 A resolution from a SeMet-labelled crystal at the Advanced Photon Source (APS) and a MAD data set was collected to 1.75 A resolution at the Canadian Light Source (CLS); this is the first reported anomalous diffraction experiment from the CLS. The crystals belong to space group I222 and contain one molecule per asymmetric unit.
Assuntos
Bacillus subtilis/enzimologia , Oxirredutases/química , Catálise , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Cinética , Oxirredutases/metabolismo , Microbiologia do Solo , Espectrofotometria UltravioletaRESUMO
Fungi synthesize lysine via the alpha-aminoadipate pathway, which is not found in plants or animals. This pathway has been proposed as a target for antifungal agents, but until now no reports have appeared to test this proposal. Hampering studies on the susceptibility of filamentous fungi such as those of the clinically important genus Aspergillus is the fact that growth quantitation is notoriously difficult. We have used the recently-reported XTT-based method of biomass quantitation to measure the susceptibility of Aspergillus nidulans strain A28 to growth suppression by novel compounds designed to target early steps in the alpha-aminoadipate lysine biosynthesis pathway, specifically those steps involving (R)-homocitrate and (2R,3S)-homoisocitrate. Three compounds show moderate inhibition of fungal growth, which can be partially restored by the presence of lysine in the growth medium.