Theoretical Study of the Phosphoryl Transfer Reaction from ATP to Dha Catalyzed by DhaK from Escherichia coli

Protein kinases, representing one of the largest protein families involved in almost all aspects of cell life, have become one of the most important targets for the development of new drugs to be used in, for instance, cancer treatments. In this article an exhaustive theoretical study of the phosphoryl transfer reaction from adenosine triphosphate (ATP) to dihydroxyacetone (Dha) catalyzed by DhaK from Escherichia coli (E. coli) is reported. Two different mechanisms, previously proposed for the phosphoryl transfer from ATP to the hydroxyl side chain of specific serine, threonine, or tyrosine residues, have been explored based on the generation of free energy surfaces (FES) computed with hybrid QM/MM potentials. The results suggest that the substrate-assisted phosphoryl and proton-transfer mechanism is kinetically more favorable than the mechanism where an aspartate would be activating the Dha. Although the details of the mechanisms appear to be dramatically dependent on the level of theory employed in the calculations (PM3/MM, B3LYP:PM3/MM, or B3LYP/MM), the transition states (TSs) for the phosphoryl transfer step appear to be described as a concerted step with different degrees of synchronicity in the breaking and forming bonds process in both explored mechanisms. Residues of the active site belonging to different subunits of the protein, such as Gly78B, Thr79A, Ser80A, Arg178B, and one Mg2+ cation, would be stabilizing the transferred phosphate in the TS. Asp109A would have a structural role by posing the Dha and other residues of the active site in the proper orientation. The information derived from our calculations not only reveals the role of the enzyme and the particular residues of its active site, but it can assist in the rational design of new more specific inhibitors

Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis

Biomass deconstruction remains integral for enabling second‐generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long‐term stability at 75 °C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism

The Escherichia coli MarA protein regulates the ycgZ-ymgABC operon to inhibit biofilm formation

The Escherichia coli marRAB operon is a paradigm for chromosomally encoded antibiotic resistance. The operon exerts its effect via an encoded transcription factor called MarA that modulates efflux pump and porin expression. In this work, we show that MarA is also a regulator of biofilm formation. Control is mediated by binding of MarA to the intergenic region upstream of the ycgZ-ymgABC operon. The operon, known to influence the formation of curli fibres and colanic acid, is usually expressed during periods of starvation. Hence, the ycgZ-ymgABC promoter is recognised by σ38 (RpoS)-associated RNA polymerase (RNAP). Surprisingly, MarA does not influence σ38 -dependent transcription. Instead, MarA drives transcription by the housekeeping σ70 -associated RNAP. The effects of MarA on ycgZ-ymgABC expression are coupled with biofilm formation by the rcsCDB phosphorelay system, with YcgZ, YmgA and YmgB forming a complex that directly interacts with the histidine kinase domain of RcsC

Human immunodeficiency virus and other sexually transmitted diseases in Cuban women

ABSTRACTA cross-sectional study was performed in 60 Cuban women of child-bearing age who were sero-positive for human immunodeficiency virus (HIV) and 60 controls. Human papillomavirus (HPV) was identified most frequently, with oncogenic HPV serotypes 16, 33 and 58 detected in HIV-positive patients, and serotypes 11, 33 and 51 in the controls (relative risk 4.41; 95% CI 2.21– 8.29). Syphilis and hepatitis B and C viruses were detected exclusively in HIV-sero-positive women (p < 0.05). Sexually transmitted diseases (STDs) appeared to pose a substantial health problem, especially for HIV-positive women. Clinics should consider screening and treatment for STDs as part of their HIV prevention programmes

Modelling and Analysis of Central Metabolism Operating Regulatory Interactions in Salt Stress Conditions in a L-Carnitine Overproducing E. coli Strain

Based on experimental data from E. coli cultures, we have devised a mathematical model in the GMA-power law formalism that describes the central and L-carnitine metabolism in and between two steady states, non-osmotic and hyperosmotic (0.3 M NaCl). A key feature of this model is the introduction of type of kinetic order, the osmotic stress kinetic orders (gOSn), derived from the power law general formalism, which represent the effect of osmotic stress in each metabolic process of the model

Sample cartridge with built in miniature molecule evaporator for in situ measurement with a photoemission electron microscope

We present a new sample holder that is compatible with Photoemission Electron Microscopes PEEMs and contains a molecule evaporator. With the integrated low cost evaporator, a local and controlled material deposition in clean ultra high vacuum conditions can be achieved minimizing the contamination of the analysis chamber. Different molecule systems can easily be studied by exchanging the sample holder. This opens up new possibilities for in situ investigation of thin film growth by means of spectromicroscopy and element selective imaging at the nanometer scale. As an example of the performances of the setup, we present a study of the hybrid inorganic organic system HIOS consisting of the organic acceptor molecule 2,2 amp; 8242; perfluoronaphthalene 2,6 diylidene dimalononitrile F6TCNNQ and ZnO, which is of great interest for novel HIOS based optoelectronic devices. Here, the ZnO surface work function modification by F6TCNNQ adsorption is investigated in situ in a spatially resolved manner. In addition, we employ PEEM to selectively probe the chemical state of F6TCNNQ molecules in contact with ZnO in the first monolayer and those molecules located in multilayers in 3D island

Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis

Biomass deconstruction remains integral for enabling second‐generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long‐term stability at 75 °C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism