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In this paper we have theoretically predicted a novel pathway for the mutagenic tautomerization of the classical A∙T DNA base pairs in the free state, the Watson-Crick A·Т(WC), reverse Watson-Crick A·Т(rWC), Hoogsteen A·Т(H) and reverse Hoogsteen A·Т(rH) pairs, via sequential proton transfer accompanied by a significant change in the mutual orientation of the bases. Quantum-mechanical (QM) calculations were performed at the MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level in vacuum phase, along with Bader’s quantum theory of Atoms in Molecules (QTAIM). These processes involve transition states (TSs) with quasi-orthogonal structures (symmetry C1), which are highly polar, tight ion pairs (A-, N6H2-deprotonated)∙(T+, O4/O2-protonated). Gibbs free energies of activation for the A∙T(WC) / A∙T(rWC) ↔ A*∙Т(rwWC) / A*∙Т(wWC) tautomeric transitions (~43.5 kcal∙mol-1) are lower than for the A∙T(H) / A∙T(rH) ↔ A*N7∙Т(rwH) / A*N7∙Т(wH) tautomerisations (~53.0 kcal∙mol-1) (rare tautomers are marked by an asterisk; w—wobble configured tautomerisation products). The (T)N3+H⋯N1-(A), (T)O4+H⋯N1-(A) / (T)N3+H⋯N1-(A) and (T)O2+H⋯N1-(A) H-bonds are found in the transition states TSA-·T+A·T(WC)↔A*·T(rwWC) / TSA-·T+A·T(rWC)↔A*·T(wWC). However, in the transition state TSA-·T+A·Т(H)↔A*N7·T(rwH) / TSA-·T+A·Т(rH)↔A*N7·T(wH), the (T)N3+H⋯N7-(A), (T)O4+H⋯N7-(A) / (T)N3+H⋯N7-(A) and (T)O2+H⋯N7-(A) H-bonds are supplemented by the attractive (T)O4+/O2+⋯N6-(A) van der Waals contacts. It was demonstrated that the products of the tautomerization of the classical A∙T DNA base pairs—A*∙Т(rwWC), A*N7∙Т(rwH) and A*N7∙Т(wH) (symmetry Cs)–further transform via double proton transfer into the energetically favorable wobble A∙T*(rwWC), A∙T*(rwH) and A∙T*O2(wH) base mispairs (symmetry Cs). [ABSTRACT FROM AUTHOR]

Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel. [ABSTRACT FROM AUTHOR]