A high-throughput screening assay for pyruvate carboxylase

Abstract

Pyruvate carboxylase (PC) catalyzes the conversion of pyruvate to oxaloacetate (OAA), an important metabolic reaction in a wide range of organisms. Small molecules directed against PC would enable detailed studies on the metabolic role of this enzyme and would have the potential to be developed into pharmacological agents. Currently, specific and potent small molecule regulators of PC are unavailable. To assist in efforts to find, develop, and characterize small molecule effectors of PC, a novel fixed-time assay has been developed based on the reaction of OAA with the diazonium salt, Fast Violet B (FVB), which produces a colored adduct with an absorbance maximum at 530 nm. This fixed time assay is reproducible, sensitive and responsive to known effectors of Rhizobium etli PC, Staphylococcus aureus PC, and Listeria monocytogenes PC, and is highly amenable to high-throughput screening. The assay was validated using a plate uniformity assessment test and a pilot screen of a library of 1280 compounds. The results indicate that the assay is suitable for screening small molecule libraries to find novel small molecule effectors of PC.

Solving the Conundrum: Widespread Proteins Annotated for Urea Metabolism in Bacteria Are Carboxyguanidine Deiminases Mediating Nitrogen Assimilation from Guanidine

“…After 6 h of the reaction incubation, EDTA was added as a quenching agent to 10 mM, denoted by an asterisk. The presence of EDTA terminates the enzymatic reaction, as previously demonstrated for pyruvate carboxylase . The signal corresponding to the carboxy carbon atom of allophanate is indicated at 160.9 ppm.…”

Identification of Pyruvate Carboxylase as the Cellular Target of Natural Bibenzyls with Potent Anticancer Activity against Hepatocellular Carcinoma via Metabolic Reprogramming

“…As shown in Figure S18, the IC 50 values of oxalate were 100 (cell-based PC activity assay) and 88.8 μM (cell lysate-based PC activity assay), which were similar to the reported IC 50 value of oxalate (97 μM) for Rhizobium etli PC. 23 Erianin at noncytotoxic concentrations had no effect on PC expression (Figures S19 and S20).…”

“…Fluorescence probe 22 was synthesized according to the same procedure as described for the preparation of probe 7 to give the title compound as a croci powder. Inactive Fluorescence Probe (23). The fluorescence probe 23 was synthesized according to the same procedure as described for the preparation of probe 7 to give the title compound as a croci powder.…”

Sustained Control of Pyruvate Carboxylase by the Essential Second Messenger Cyclic di-AMP in Bacillus subtilis

“…When the cultures reached an OD 600 of 1.2, MnCl 2 was added (final concentration, 10 mM) and the cultures were transferred to 28°C. After 20 min, expression of the recombinant proteins was induced by the addition of isopropyl 1-thio-β- d -galactopyranoside (final concentration, 0.3 mM), and the cultures were grown for 3 h. PC-6×His and PC CT+BCCP -6×His were purified as described previously ( 48 ) in buffer A (20 mM Tris, pH 7.8, 200 mM NaCl, 0.5 mM EGTA, 6 mM β-mercaptoethanol). The cells were lysed and purified as described above.…”