Search

Bacteria contain sigma (σ) factors that control gene expression in response to various environmental stimuli. The alternative sigma factors σ FpvI and σ PvdS bind specifically to the antisigma factor FpvR. These proteins are an essential component of the pyoverdine-based system for iron uptake in Pseudomonas aeruginosa. Due to the uniqueness of this system, where the activities of both the σ FpvI and σ PvdS sigma factors are regulated by the same antisigma factor, the interactions between the antisigma protein FpvR 20 and the σ FpvI and σ PvdS proteins have been widely studied in vivo. However, difficulties in obtaining soluble, recombinant preparations of the σ FpvI and σ PvdS proteins have limited their biochemical and structural characterizations. In this study, we describe a purification protocol that resulted in the production of soluble, recombinant His 6 -σ FpvI /FpvR 1-67 , His 6 -σ FpvI /FpvR 1-89 , His 6 -σ PvdS /FpvR 1-67 and His 6 -σ PvdS /FpvR 1-89 protein complexes (where FpvR 1-67 and FpvR 1-89 are truncated versions of FpvR 20 ) at high purities and concentrations, appropriate for biophysical analyses by circular dichroism spectroscopy and analytical ultracentrifugation. These results showed the proteins to be folded in solution and led to the determination of the affinities of the protein-protein interactions within the His 6 -σ FpvI /FpvR 1-67 and His 6 -σ PvdS /FpvR 1-67 complexes. A comparison of these values with those previously reported for the His 6 -σ FpvI /FpvR 1-89 and His 6 -σ PvdS /FpvR 1-89 complexes is made., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Given the emergence of multi drug resistant Vibrio cholerae strains, there is an urgent need to characterize new anti-cholera targets. One such target is the enzyme dihydrodipicolinate synthase (DHDPS; EC 4.3.3.7), which catalyzes the first committed step in the diaminopimelate pathway. This pathway is responsible for the production of two key metabolites in bacteria and plants, namely meso-2,6-diaminopimelate and L-lysine. Here, we report the cloning, expression and purification of untagged and His-tagged recombinant DHDPS from V. cholerae (Vc-DHDPS) and provide comparative structural and kinetic analyses. Structural studies employing circular dichroism spectroscopy and analytical ultracentrifugation demonstrate that the recombinant enzymes are folded and exist as dimers in solution. Kinetic analyses of untagged and His-tagged Vc-DHDPS show that the enzymes are functional with specific activities of 75.6 U/mg and 112 U/mg, K M (pyruvate) of 0.14 mM and 0.15 mM, K M (L-aspartate-4-semialdehyde) of 0.08 mM and 0.09 mM, and k cat of 34 and 46 s -1 , respectively. These results demonstrate there are no significant changes in the structure and function of Vc-DHDPS upon the addition of an N-terminal His tag and, hence, the tagged recombinant product is suitable for future studies, including screening for new inhibitors as potential anti-cholera agents. Additionally, a polyclonal antibody raised against untagged Vc-DHDPS is validated for specifically detecting recombinant and native forms of the enzyme., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Here, we review recent studies aimed at defining the importance of quaternary structure to a model oligomeric enzyme, dihydrodipicolinate synthase. This will illustrate the complementary and synergistic outcomes of coupling the techniques of analytical ultracentrifugation with enzyme kinetics, in vitro mutagenesis, macromolecular crystallography, small angle X-ray scattering, and molecular dynamics simulations, to demonstrate the role of subunit self-association in facilitating protein dynamics and enzyme function. This multitechnique approach has yielded new insights into the molecular evolution of protein quaternary structure., (© 2015 Elsevier Inc. All rights reserved.)

Given the rise of multi drug resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA), there is an urgent need to discover new antimicrobial agents. A validated but as yet unexplored target for new antibiotics is dihydrodipicolinate reductase (DHDPR), an enzyme that catalyzes the second step of the lysine biosynthesis pathway in bacteria. We report here the cloning, expression and purification of N-terminally his-tagged recombinant DHDPR from MRSA (6H-MRSA-DHDPR) and compare its secondary and quaternary structure with the wild type (MRSA-DHDPR) enzyme. Comparative analyses demonstrate that recombinant 6H-MRSA-DHDPR is folded and adopts the native tetrameric quaternary structure in solution. Furthermore, kinetic studies show 6H-MRSA-DHDPR is functional, displaying parameters for K(m)(NADH) of 6.0 μM, K(m)(DHDP) of 22 μM, and k(cat) of 21s(-1), which are similar to those reported for the native enzyme. The solution properties and stability of the 6H-MRSA-DHDPR enzyme are also reported in varying physicochemical conditions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Csk-homologous kinase (CHK) is an important endogenous inhibitor constraining the oncogenic actions of Src-family kinases (SFKs) in cells. It suppresses SFK activity by specifically phosphorylating the conserved regulatory tyrosine near the C-terminus of SFKs. In addition to phosphorylation, CHK employs a novel non-catalytic inhibitory mechanism to suppress SFK activity. This mechanism involves direct binding of CHK to the active forms of SFKs to form stable protein complexes. Since aberrant activation of SFKs contributes to cancer formation and progression, small-molecule inhibitors mimicking the non-catalytic inhibitory mechanism of CHK are potential anti-cancer therapeutics. Elucidation of the catalytic and regulatory properties and the structural basis of the CHK non-catalytic inhibitory mechanism would facilitate the development of these small-molecule inhibitors. To this end, we developed procedures for higher level expression in insect cells of active recombinant CHK with a hexa-histidine tag attached to its C-terminus (referred to as CHK-His(6)) and its rapid purification by a two-step method. Analyses by size-exclusion column chromatography and analytical ultracentrifugation revealed that the purified CHK-His(6) exists as a monomeric species in solution. Biochemical analyses demonstrated that CHK-His(6) exhibits efficiencies comparable to those of CSK in phosphorylating artificial protein and peptide substrates as well as an intact SFK protein. Our results indicate that the recombinant CHK-His(6) can be used for future studies to decipher the three-dimensional structure, and regulatory and catalytic properties of CHK., (Copyright © 2010 Elsevier Inc. All rights reserved.)