ABSTRACT
Evolution of enzymes plays a crucial role in obtaining new biological functions for all life forms. Arsenate reductases (ArsC) are several families of arsenic detoxification enzymes that reduce arsenate to arsenite, which can subsequently be extruded from cells by specific transporters. Among these, the Synechocystis ArsC (SynArsC) is structurally homologous to the well characterized thioredoxin (Trx)-coupled ArsC family but requires the glutaredoxin (Grx) system for its reactivation, therefore classified as a unique Trx/Grx-hybrid family. The detailed catalytic mechanism of SynArsC is unclear and how the "hybrid" mechanism evolved remains enigmatic. Herein, we report the molecular mechanism of SynArsC by biochemical and structural studies. Our work demonstrates that arsenate reduction is carried out via an intramolecular thiol-disulfide cascade similar to the Trx-coupled family, whereas the enzyme reactivation step is diverted to the coupling of the glutathione-Grx pathway due to the local structural difference. The current results support the hypothesis that SynArsC is likely a molecular fossil representing an intermediate stage during the evolution of the Trx-coupled ArsC family from the low molecular weight protein phosphotyrosine phosphatase (LMW-PTPase) family.
Subject(s)
Arsenate Reductases/metabolism , Arsenates/metabolism , Bacterial Proteins/metabolism , Synechocystis/enzymology , Amino Acid Sequence , Arsenate Reductases/chemistry , Arsenate Reductases/genetics , Arsenates/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Disulfides/metabolism , Glutaredoxins/chemistry , Glutaredoxins/genetics , Glutaredoxins/metabolism , Glutathione/metabolism , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Sulfhydryl Compounds/metabolism , Synechocystis/genetics , Thioredoxins/chemistry , Thioredoxins/genetics , Thioredoxins/metabolismABSTRACT
Arsenate reductases (ArsC) are a group of enzymes that play essential roles in biological arsenic detoxification pathways by catalyzing the intracellular reduction of arsenate to arsenite, which is subsequently extruded from the cells by specific transport systems. The ArsC protein from cyanobacterium Synechocystis sp. strain PCC 6803 (SynArsC) is related to the thioredoxin-dependent ArsC family, but uses the glutathione/glutaredoxin system for arsenate reduction. Therefore, it is classified to a novel thioredoxin/glutaredoxin hybrid arsenate reductase family. Herein we report the chemical shift assignments of (1)H, (13)C and (15)N atoms for the reduced form of SynArsC, which provides a starting point for further structural analysis and elucidation of its enzymatic mechanism.
Subject(s)
Arsenate Reductases/chemistry , Nuclear Magnetic Resonance, Biomolecular , Synechocystis/enzymology , Amino Acid Sequence , Carbon Isotopes , Hydrogen , Molecular Sequence Data , Nitrogen IsotopesABSTRACT
Cationic surfactant/anionic surfactant/beta-CD ternary aqueous systems provide a platform for the coexistence of the host-guest (beta-CD/surfactant) equilibrium and the biased aggregation (monomeric/aggregated surfactants) equilibrium. We report here that the interplay between the two equilibria dominates the systems as follows. (1) The biased aggregation equilibrium imposes an apparent selectivity on the host-guest equilibrium, namely, beta-CD has to always selectively bind the major surfactant (molar fraction > 0.5) even if binding constants of beta-CD to the pair of surfactants are quite similar. (2) In return, the host-guest equilibrium amplifies the bias of the aggregation equilibrium, that is, the selective binding partly removes the major surfactant from the aggregates and leaves the aggregate composition approaching the electroneutral mixing stoichiometry. (3) This composition variation enhances electrostatic attractions between oppositely charged surfactant head groups, thus resulting in less-curved aggregates. In particular, the present apparent host-guest selectivity is of remarkably high values, and the selectivity stems from the bias of the aggregation equilibrium rather than the difference in binding constants. Moreover, beta-CD is defined as a "stoichiometry booster" for the whole class of cationic/anionic surfactant systems, which provides an additional degree of freedom to directly adjust aggregate compositions of the systems. The stoichiometry boosting of the compositions can in turn affect or even determine microstructures and macroproperties of the systems.
Subject(s)
Anions/chemistry , Cations/chemistry , Surface-Active Agents/chemistry , beta-Cyclodextrins/chemistry , Calorimetry , ThermodynamicsABSTRACT
The bacterial phosphoenolpyruvate-dependent sugar phosphotransferase system (PEP-PTS) is essential in the coupled transportation and phosphorylation of various types of carbohydrates. The CmtAB proteins of Escherichia coli are sequentially similar to the mannitol-specific phosphotransferase MtlA. The CmtB protein corresponds to the phosphotransferase enzyme IIA component. Here we report the solution structure of CmtB from E. coli at high resolution by NMR spectroscopy. The results show that CmtB adopts a globular fold consisting of a central mixed five-strand beta-sheet flanked by seven helices at both sides. Structural comparison with the IIA domain of MtlA (IIAMtl) reveals high overall similarity, while notable conformational differences at the active site are observed. The active site pocket of CmtB appears to be wider, and the hydrophobic regions around it is larger compared to IIAMtl. Further, the essential arginine residue at the active site of IIAMtl is substituted by a serine in CmtB. Instead, the active pocket of CmtB contains another arginine at a distinct position, suggesting different molecular mechanisms for phosphoryl transfer.
