Time-resolved crystallography of boric acid binding to the active site serine of the ß-lactamase CTX-M-14 and subsequent 1,2-diol esterification.

The emergence and spread of antibiotic resistance represent a growing threat to public health. Of particular concern is the appearance of ß-lactamases, which are capable to hydrolyze and inactivate the most important class of antibiotics, the ß-lactams. Effective ß-lactamase inhibitors and mechanistic insights into their action are central in overcoming this type of resistance, and in this context boronate-based ß-lactamase inhibitors were just recently approved to treat multidrug-resistant bacteria. Using boric acid as a simplified inhibitor model, time-resolved serial crystallography was employed to obtain mechanistic insights into binding to the active site serine of ß-lactamase CTX-M-14, identifying a reaction time frame of 80-100 ms. In a next step, the subsequent 1,2-diol boric ester formation with glycerol in the active site was monitored proceeding in a time frame of 100-150 ms. Furthermore, the displacement of the crucial anion in the active site of the ß-lactamase was verified as an essential part of the binding mechanism of substrates and inhibitors. In total, 22 datasets of ß-lactamase intermediate complexes with high spatial resolution of 1.40-2.04 Å and high temporal resolution range of 50-10,000 ms were obtained, allowing a detailed analysis of the studied processes. Mechanistic details captured here contribute to the understanding of molecular processes and their time frames in enzymatic reactions. Moreover, we could demonstrate that time-resolved crystallography can serve as an additional tool for identifying and investigating enzymatic reactions.

Comparative Study of Physicochemical Properties and Antibacterial Potential of Cyanobacteria Spirulina platensis-Derived and Chemically Synthesized Silver Nanoparticles.

The "green synthesis" of nanoparticles (NPs) offers cost-effective and environmentally friendly advantages over chemical synthesis by utilizing biological sources such as bacteria, algae, fungi, or plants. In this context, cyanobacteria and their components are valuable sources to produce various NPs. The present study describes the comparative analysis of physicochemical and antibacterial properties of chemically synthesized (Chem-AgNPs) and cyanobacteria Spirulina platensis-derived silver NPs (Splat-AgNPs). The physicochemical characterization applying complementary dynamic light scattering and transmission electron microscopy revealed that Splat-AgNPs have an average hydrodynamic radius of ∼ 28.70 nm and spherical morphology, whereas Chem-AgNPs are irregular-shaped with an average radius size of ∼ 53.88 nm. The X-ray diffraction pattern of Splat-AgNPs confirms the formation of face-centered cubic crystalline AgNPs by "green synthesis". Energy-dispersive spectroscopy analysis demonstrated the purity of the Splat-AgNPs. Fourier transform infrared spectroscopy analysis of Splat-AgNPs demonstrated the involvement of some functional groups in the formation of NPs. Additionally, Splat-AgNPs demonstrated high colloidal stability with a zeta-potential value of (-50.0 ± 8.30) mV and a pronounced bactericidal activity against selected Gram-positive (Enterococcus hirae and Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa and Salmonella typhimurium) bacteria compared with Chem-AgNPs. Furthermore, our studies toward understanding the action mechanism of NPs showed that Splat-AgNPs alter the permeability of bacterial membranes and the energy-dependent H+-fluxes via FoF1-ATPase, thus playing a crucial role in bacterial energetics. The insights gained from this study show that Spirulina-derived synthesis is a low-cost, simple approach to producing stable AgNPs for their energy-metabolism-targeted antibacterial applications in biotechnology and biomedicine.

Structure and dynamics of the staphylococcal pyridoxal 5-phosphate synthase complex reveal transient interactions at the enzyme interface.

Infectious diseases are a significant cause of death, and recent studies estimate that common bacterial infectious diseases were responsible for 13.6% of all global deaths in 2019. Among the most significant bacterial pathogens is Staphylococcus aureus, accounting for more than 1.1 million deaths worldwide in 2019. Vitamin biosynthesis has been proposed as a promising target for antibacterial therapy. Here, we investigated the biochemical, structural, and dynamic properties of the enzyme complex responsible for vitamin B6 (pyridoxal 5-phosphate, PLP) biosynthesis in S. aureus, which comprises enzymes SaPdx1 and SaPdx2. The crystal structure of the 24-mer complex of SaPdx1-SaPdx2 enzymes indicated that the S. aureus PLP synthase complex forms a highly dynamic assembly with transient interaction between the enzymes. Solution scattering data indicated that SaPdx2 typically binds to SaPdx1 at a substoichiometric ratio. We propose a structure-based view of the PLP synthesis mechanism initiated with the assembly of SaPLP synthase complex that proceeds in a highly dynamic interaction between Pdx1 and Pdx2. This interface interaction can be further explored as a potentially druggable site for the design of new antibiotics.

SARS-CoV-2 Mpro responds to oxidation by forming disulfide and NOS/SONOS bonds.

The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein's crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.

Molecular insights into the dynamic modulation of bacterial ClpP function and oligomerization by peptidomimetic boronate compounds.

Bacterial caseinolytic protease P subunit (ClpP) is important and vital for cell survival and infectivity. Recent publications describe and discuss the complex structure-function relationship of ClpP and its processive activity mediated by 14 catalytic sites. Even so, there are several aspects yet to be further elucidated, such as the paradoxical allosteric modulation of ClpP by peptidomimetic boronates. These compounds bind to all catalytic sites, and in specific conditions, they stimulate a dysregulated degradation of peptides and globular proteins, instead of inhibiting the enzymatic activity, as expected for serine proteases in general. Aiming to explore and explain this paradoxical effect, we solved and refined the crystal structure of native ClpP from Staphylococcus epidermidis (Se), an opportunistic pathogen involved in nosocomial infections, as well as ClpP in complex with ixazomib at 1.90 Å and 2.33 Å resolution, respectively. The interpretation of the crystal structures, in combination with complementary biochemical and biophysical data, shed light on how ixazomib affects the ClpP conformational state and activity. Moreover, SEC-SAXS and DLS measurements show, for the first time, that a peptidomimetic boronate compound also induces the assembly of the tetradecameric structure from isolated homomeric heptameric rings of a gram-positive organism.