Mis-regulation of Zn and Mn homeostasis is a key phenotype of Cu stress in Streptococcus pyogenes.

All bacteria possess homeostastic mechanisms that control the availability of micronutrient metals within the cell. Cross-talks between different metal homeostasis pathways within the same bacterial organism have been reported widely. In addition, there have been previous suggestions that some metal uptake transporters can promote adventitious uptake of the wrong metal. This work describes the cross-talk between Cu and the Zn and Mn homeostasis pathways in Group A Streptococcus (GAS). Using a ∆copA mutant strain that lacks the primary Cu efflux pump and thus traps excess Cu in the cytoplasm, we show that growth in the presence of supplemental Cu promotes downregulation of genes that contribute to Zn or Mn uptake. This effect is not associated with changes in cellular Zn or Mn levels. Co-supplementation of the culture medium with Zn or, to a lesser extent, Mn alleviates key Cu stress phenotypes, namely bacterial growth and secretion of the fermentation end-product lactate. However, neither co-supplemental Zn nor Mn influences cellular Cu levels or Cu availability in Cu-stressed cells. In addition, we provide evidence that the Zn or Mn uptake transporters in GAS do not promote Cu uptake. Together, the results from this study strengthen and extend our previous proposal that mis-regulation of Zn and Mn homeostasis is a key phenotype of Cu stress in GAS.

Conformational study into N-alkyl-N'-aryl ureas to inform drug discovery.

Ureas are an important functional group in small molecule drugs as well as having wider applications in organic chemistry. Understanding of their conformation is of critical importance for rational design of urea-containing bioactive compounds. Whilst the conformational preferences of biaryl ureas have been extensively studied, very little attention has been paid to alkylated analogues. We carried out a systematic study of N-aryl (phenyl and pyridyl)-N'-cyclopentyl ureas with differing N-methylation patterns using Well Tempered Metadynamics at a semi-empirical level in implicit water (GBSA) using Well-Tempered Metadynamics to generate their conformational free-energy landscapes. Geometries and energetics of the most relevant configurations are further refined using DFT level of theory. Validation for the computation was obtained by synthesis of all 8 analogues followed by conformational studies by X-ray crystallography and NMR. These findings reveal that the methylation pattern significantly affects the conformational preference of the system. Most notably, N-phenyl-N'-cyclopentyl urea is shown to adopt both the trans-trans, and cis-trans conformations with equal energy and that the cis-trans conformation can be significantly stabilised by the presence of an internal hydrogen bond to the N'-hydrogen. This study will be of utility for the design of N-alkyl-N'-aryl ureas as drug candidates.

An evolutionary path to altered cofactor specificity in a metalloenzyme.

Almost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal's redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity.