Synthesis and Evaluation of 1,3-Disubstituted Imidazolidine-2,4,5-triones as Inhibitors of Pyruvate Carboxylase.

Substituted imidazolidinetriones (IZTs) have been identified as potent inhibitors of pyruvate carboxylase (PC) through an in silico screening approach. Alkyl 2-(2,4,5-trioxo-3-substituted imidazolidin-1-yl)acetates (6i-6r) are the most potent of the series, with IC50 values between 3 and 12 µM, and several IZTs demonstrate high passive permeability across an artificial membrane. IZTs are mixed-type inhibitors with respect to pyruvate and noncompetitive with respect to ATP. This class of inhibitors appears to be selective for PC. Inhibitors in the IZT series do not inhibit the metalloenzymes human carbonic anhydrase II and matrix metalloprotease-12, and they do not inhibit the related biotin-dependent enzyme, guanidine carboxylase. Altogether, IZTs offer promise as PC inhibitors with potential downstream applications in cellular and in vivo systems.

Synthesis and Evaluation of 5-(Heteroarylmethylene)hydantoins as Glycogen Synthase Kinase-3ß Inhibitors.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase which plays a center role in the phosphorylation of a wide variety of proteins, generally leading to their inactivation. As such, GSK-3 is viewed as a therapeutic target. An ever-increasing number of small organic molecule inhibitors of GSK-3 have been reported. Phenylmethylene hydantoins are known to exhibit a wide range of inhibitory activities including for GSK-3ß. A family of fourteen 2-heterocycle substituted methylene hydantoins (14, 17-29) were prepared and evaluated for the inhibition of GSK-3ß at 25 µM. The IC50 values of five of these compounds was determined; the two best inhibitors are 5-[(4'-chloro-2-pyridinyl)methylene]hydantoin (IC50 = 2.14 ± 0.18 µM) and 5-[(6'-bromo-2-pyridinyl)methylene]hydantoin (IC50 = 3.39 ± 0.16 µM). The computational docking of the compounds with GSK-3ß (pdb 1q41) revealed poses with hydrogen bonding to the backbone at Val135. The 5-[(heteroaryl)methylene]hydantoins did not strongly inhibit other metalloenzymes, demonstrating poor inhibitory activity against matrix metalloproteinase-12 at 25 µM and against human carbonic anhydrase at 200 µM, and were not inhibitors for Staphylococcus aureus pyruvate carboxylase at concentrations >1000 µM.

Allosteric Site at the Biotin Carboxylase Dimer Interface Mediates Activation and Inhibition in Staphylococcus aureus Pyruvate Carboxylase.

Allosteric regulation of the essential anaplerotic enzyme, pyruvate carboxylase (PC), is vital for metabolic homeostasis. PC catalyzes the bicarbonate- and ATP-dependent carboxylation of pyruvate to form oxaloacetate. Dysregulation of PC activity can impact glucose and redox metabolism, which contributes to the pathogenicity of many diseases. To maintain homeostasis, PC is allosterically activated by acetyl-CoA and allosterically inhibited by l-aspartate. In this study, we further characterize the molecular basis of allosteric regulation in Staphylococcus aureus PC (SaPC) using slowly/nonhydrolyzable dethia analogues of acetyl-CoA and site-directed mutagenesis of residues at the biotin carboxylase homodimer interface. The dethia analogues fully activate SaPC but demonstrate significantly reduced binding affinities relative to acetyl-CoA. Residues Arg21, Lys46, and Glu418 of SaPC are located at the biotin carboxylase dimer interface and play a critical role in both allosteric activation and inhibition. A structure of R21A SaPC in complex with acetyl-CoA reveals an intact molecule of acetyl-CoA bound at the allosteric site, offering new molecular insights into the acetyl-CoA binding site. This study demonstrates that the biotin carboxylase domain dimer interface is a critical allosteric site in PC, serving as a convergence point for allosteric activation by acetyl-CoA and inhibition by l-aspartate.

Dethiosulfovibrio faecalis sp. nov., a novel proteolytic, non-sulphur-reducing bacterium isolated from a marine aquaculture solid waste bioreactor.

A new Dethiosulfovibrio strain, designated F2BT, was isolated from an anaerobic digester for treating solid waste from a marine recirculating aquaculture system. The motile, Gram-negative, non-spore-forming curved rods were 2-7 µm long and 1 µm in diameter. Growth occurred at temperatures ranging from 20 to 40 °C with a maximum rate of growth at 30 °C. The pH range for growth was pH 6.0-8.0, with a maximum rate of growth at pH 7.5. This isolate was halotolerant growing in NaCl concentrations ranging from 0 to 1.6 M with a maximum rate of growth at 0.4 M. Similarly to the five described Dethiosulfovibrio species, this obligate anaerobe isolate was fermentative, capable of utilizing peptides, amino acids and some organic acids for growth, but unlike described strains in the genus did not reduce thiosulphate or elemental sulphur to hydrogen sulphide during fermentation of organic substrates. The G+C content of 55 mol% is similar to the described Dethiosulfovibrio species. The average nucleotide identity analysis between whole genome sequences showed less than 93.15% sequence similarity between strain F2BT and the five other described Dethiosulfovibrio species. Differences in the physiological and phylogenetic characteristics between the new strain and other Dethiosulfovibrio specied indicate that F2BT represents a novel species of this genus and the epithet Dethiosulfovibrio faecalis sp. nov. is proposed. The type strain is F2BT (=DSM 112078T=KCTC25378T).

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

Free guanidine is increasingly recognized as a relevant molecule in biological systems. Recently, it was reported that urea carboxylase acts preferentially on guanidine, and consequently, it was considered to participate directly in guanidine biodegradation. Urea carboxylase combines with allophanate hydrolase to comprise the activity of urea amidolyase, an enzyme predominantly found in bacteria and fungi that catalyzes the carboxylation and subsequent hydrolysis of urea to ammonia and carbon dioxide. Here, we demonstrate that urea carboxylase and allophanate hydrolase from Pseudomonas syringae are insufficient to catalyze the decomposition of guanidine. Rather, guanidine is decomposed to ammonia through the combined activities of urea carboxylase, allophanate hydrolase, and two additional proteins of the DUF1989 protein family, expansively annotated as urea carboxylase-associated family proteins. These proteins comprise the subunits of a heterodimeric carboxyguanidine deiminase (CgdAB), which hydrolyzes carboxyguanidine to N-carboxyurea (allophanate). The genes encoding CgdAB colocalize with genes encoding urea carboxylase and allophanate hydrolase. However, 25% of urea carboxylase genes, including all fungal urea amidolyases, do not colocalize with cgdAB. This subset of urea carboxylases correlates with a notable Asp to Asn mutation in the carboxyltransferase active site. Consistent with this observation, we demonstrate that fungal urea amidolyase retains a strong substrate preference for urea. The combined activities of urea carboxylase, carboxyguanidine deiminase and allophanate hydrolase represent a newly recognized pathway for the biodegradation of guanidine. These findings reinforce the relevance of guanidine as a biological metabolite and reveal a broadly distributed group of enzymes that act on guanidine in bacteria.