ABSTRACT
Vicinal disulfide rings (VDRs) occur when a disulfide bond forms between adjacent cysteine residues in a protein and results in a rare eight-membered ring structure. This eight-membered ring has been found to exist in four major conformations in solution, divided between cis and trans conformers. Some selenoenzymes use a special type of VDR in which selenium replaces sulfur, generating a vicinal selenosulfide ring (VSeSR). Here, we provide evidence that this substitution reduces ring strain, resulting in a strong preference for the trans conformation relative to cis in a VSeSR (cis:trans - 9:91). This was determined by using the 'γ-gauche effect', which makes use of both (1) H-NMR and two-dimensional (2D) NMR techniques for determining the amide bond conformeric ratio. The presence of selenium in a VSeSR also lowers the dihedral strain energy (DSE) of the selenosulfide bond relative to the disulfide bond of VDRs. While cis amide geometry decreases strain on the amide bond, it increases strain on the scissile disulfide bond of the VDR found in thioredoxin reductase from Drosophila melanogaster (DmTR). We hypothesize that the cis conformation of the VDR is the catalytically competent conformer for thiol/disulfide exchange. This hypothesis was investigated by computing the DSE of VDR and VSeSR conformers, the structure of which was determined by 2D NMR spectroscopy and energy minimization. The computed values of the VDR from DmTR are 16.5 kJ/mol DSE and 14.3 kJ/mol for the C+ and T- conformers, respectively, supporting the hypothesis that the enzyme uses the C+ conformer for thiol/disulfide exchange.
Subject(s)
Peptide Fragments/chemistry , Thioredoxin-Disulfide Reductase/chemistry , Animals , Drosophila melanogaster/enzymology , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Organoselenium Compounds/chemistry , Organoselenium Compounds/metabolism , Oxidation-Reduction , Peptide Fragments/metabolism , Protein Conformation , Stereoisomerism , Thioredoxin-Disulfide Reductase/metabolismABSTRACT
Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolism in vitro and in situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation in Pseudomonas aeruginosa. We used genetic analyses and 13C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor in P. aeruginosa but did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, including Pseudomonas mendocina, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Burkholderia ambifaria, and Sinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolism in situ.
Subject(s)
Bacteria, Aerobic/drug effects , Burkholderia/drug effects , Choline/metabolism , Enzyme Inhibitors/metabolism , Metabolic Networks and Pathways/drug effects , Pseudomonas/drug effects , Sinorhizobium meliloti/drug effects , Bacteria, Aerobic/growth & development , Bacteria, Aerobic/metabolism , Burkholderia/growth & development , Burkholderia/metabolism , Carbon/metabolism , Choline/analogs & derivatives , Energy Metabolism/drug effects , Nitrogen/metabolism , Pseudomonas/growth & development , Pseudomonas/metabolism , Sarcosine/analogs & derivatives , Sarcosine/antagonists & inhibitors , Sinorhizobium meliloti/growth & development , Sinorhizobium meliloti/metabolismABSTRACT
A vicinal disulfide ring (VDR) results from disulfide bond formation between two adjacent cysteine residues. This 8-membered ring is a rare motif in protein structures and is functionally important to those few proteins that posses it. This article focuses on the construction of strained and unstrained VDR mimics, discernment of the preferred conformation of these mimics, and the determination of their respective disulfide redox potentials.
