Biology subjects

Hanzevacki, M.

Publications and source records attributed to Hanzevacki, M..

5 recordsLinked to original sources

bioRxiv2026

Redox Control of S-sulfocysteine Formation in Adenosine Phosphosulfate Reductase

Sulfur assimilation fuels bacterial growth by supplying the reduced sulfur required for the biosynthesis of sulfur-containing biomolecules. Adenosine 5'-phosphosulfate reductase (APSR) catalyzes the first reductive step of this pathway, converting adenosine 5'-phosphosulfate (APS) to adenosine monophosphate. This reaction proceeds through nucleophilic attack by catalytic C256, located in the flexible C-terminal tail, on the sulfur atom of APS, forming a thiosulfonate enzyme intermediate. Here, we investigate this reaction in APSR from Pseudomonas aeruginosa, an opportunistic pathogen associated with severe infections, particularly in patients with cystic fibrosis. APSR contains an iron-sulfur [4Fe-4S] cluster, which participates in redox steps of the reaction. Here, we show that the redox state of the iron-sulfur cluster also controls the catalytic step. Multiscale molecular simulations investigate how oxidized and reduced cluster states affect APS binding, active site organization, and the nucleophilic attack step. Molecular dynamics (MD) simulations show that the oxidized [4Fe-4S]2+; cluster stabilizes substrate interactions and the conformation of the C-terminus, facilitating a catalytically productive orientation of C256. The activation barrier of 17.7 {+/-} 1.7 kcal mol-1 from quantum mechanics/molecular mechanics (QM/MM) umbrella sampling MD simulations at the B3LYP-D3(BJ)/6-31G(d) level of theory is in good agreement with the experimental kinetics. The redox state of the iron-sulfur cluster shows its role in modulating the conformation of conserved K144, which is important for transition state stabilization in the nucleophilic attack. These findings illuminate the mechanism of this P. aeruginosa target and provide broader insight into the roles of iron-sulfur clusters in controlling enzyme reactivity.

biochemistry↗

bioRxiv2025

Time-resolved ligand dynamics revealed in a β-lactamase using room-temperature serial crystallography

{beta}-Lactamases catalyze {beta}-lactam antibiotic hydrolysis and are important contributors to bacterial antimicrobial resistance; {beta}-lactamase inhibitors are widely used to overcome {beta}-lactamase-mediated antibiotic resistance. Nucleophilic serine {beta}-lactamases (SBLs) react with their substrates and clinically available inhibitors via a covalent reaction to give complexes which can undergo further reaction. Using room temperature drop on fixed target serial crystallography, where ligands are rapidly mixed with microcrystals, and classical single-crystal crystallography at cryogenic temperatures, we investigate the reversible covalent reaction of the SBL CTX-M-15 with the diazobicyclooctane inhibitor avibactam. We observe avibactam covalently reacted (ring-opened) with the nucleophilic Ser70, at timepoints from 80 ms to minutes (room temperature) and hours (100 K). These crystallographic data reveal time-dependent movement of the avibactam carbamoyl complex, from 1.3 s onwards, that has implications for the 5-exo-trig recyclization mechanism that determines inhibitor reformation. Combined with molecular dynamics simulations and quantum mechanics calculations at the density functional theory level, the results show that in the first seconds of the reaction the avibactam N-sulfate nitrogen is poorly positioned for recyclization. This subsequently equilibrates after 10 s to a stable endpoint that is in a conformation potentially primed to initiate recyclization through attack of the N-sulfate nitrogen on the carbamoyl carbon. These results further demonstrate the capacity of room-temperature serial crystallography to capture time-resolved changes in ligand conformation at an enzyme active site, complementing discrete classical cryo-crystallography. These data inform on ligand dynamics and the stereoelectronics of diazobicyclooctane inhibition, aiding drug discovery efforts to develop inhibitors of nucleophilic enzymes.

biochemistry↗

bioRxiv2025

Mechanistic Basis for Inhibition of the Extended Spectrum Class A b-Lactamase GES-1 by Tazobactam and Enmetazobactam

Expression of {beta}-lactamases is the primary form of {beta}-lactam antibiotic resistance in Gram-negative bacteria. Enmetazobactam is a penicillanic acid sulfone (PAS) that inhibits extended spectrum {beta}-lactamases (ESBLs) by forming an acyl-enzyme complex that eventually breaks down to an irreversible lysinoalanine crosslink. In contrast, enmetazobactam inhibits the class A carbapenemase KPC-2 via an acyl-enzyme that does not lead to lysinoalanine crosslink formation. This difference correlates with greater inhibitory potency of enmetazobactam against class A ESBLs, compared to carbapenemases. The GES enzymes, unlike other class A {beta}-lactamase families, show progression from carbapenem-inhibited to carbapenem-hydrolysing phenotypes through single point mutations. We present crystal structures of GES-1, a globally disseminated ESBL, as the enmetazobactam- and tazobactam-derived acyl-enzymes. The complexes differ in the identities of their respective covalent adducts, with the catalytic Ser70 acylated by a 214 Da enmetazobactam-derived fragment, whereas tazobactam has fragmented to a 70 Da aldehyde. The tautomeric form of the enmetazobactam-derived ligand is verified by high-level QM/MM calculations, revealing the trans-enamine as the most thermodynamically stable tautomer, that adopts an optimised conformation that best matches the experimentally observed electron density appended to the side chain oxygen of Ser70. In contrast to previous findings for the ESBL CTX-M-15, mass spectrometry provides no evidence for lysinoalanine crosslink formation on reaction of GES-1 with (enme)tazobactam, providing further evidence that PAS inhibitors inhibit different class A {beta}-lactamases by different mechanisms. This work reveals new details of the basis for PAS inhibition of diverse {beta}-lactamases, and will guide development of future {beta}-lactamase inhibitors.

biochemistry↗

bioRxiv2023

Computational investigation of cis-1,4-polyisoprene binding to the latex clearing protein LcpK30

Latex clearing proteins (Lcps) catalyze the oxidative cleavage of the C=C bonds in cis-1,4-polyisoprene (natural rubber), producing oligomeric compounds that can be repurposed to other materials. The active catalytic site of Lcps is buried inside the protein structure, thus raising the question of how the large hydrophobic rubber chains can access the catalytic center. To improve our understanding of hydrophobic polymeric substrate binding to Lcps and subsequent catalysis, we investigated the interaction of a substrate model containing ten carbon-carbon double bonds with the structurally characterized LcpK30, using multiple computational tools. Prediction of the putative tunnels and cavities in the LcpK30 structure, using CAVER-Pymol plugin 3.0.3, fpocket and Molecular Dynamic (MD) simulations provided valuable insights on how substrate enters from the surface to the buried active site. Two dominant tunnels were discovered that provided feasible routes for substrate binding, and the presence of two hydrophobic pockets was predicted near the heme cofactor. The larger of these pockets is likely to accommodate the substrate and to determine the size distribution of the oligomers. Protein-ligand docking was carried out using GOLD software to predict the conformations and interactions of the substrate within the protein active site. Deeper insight into the protein-substrate interactions, including close-contacts, binding energies and potential cleavage sites in the cis-1,4-polyisoprene, were obtained from MD simulations. Our findings provide further justification that the protein-substrate complexation in LcpK30 is mainly driven by the hydrophobic interactions accompanied by mutual conformational changes of both molecules. Two potential binding modes were identified, with the substrate in either extended or folded conformations. Whilst binding in the extended conformation was most favourable, the folded conformation suggested a preference for cleavage of a central double bond, leading to a preference for oligomers with 5 to 6 C=C bonds, as shown by experimental data. The results provide insight into further enzyme engineering studies to improve catalytic activity and diversify the substrate and product scope of Lcps. Author summaryRubber materials are very important in our everyday life, but also lead to a high amount of rubber waste for which there is no sustainable solution. The enzymatic degradation of diene rubbers is an attractive option to revalorise these materials once they reach end of life. Latex clearing proteins (Lcps) have been shown to degrade polyisoprene rubber, however rates of degradation are low and the product is a mixture of oligomers. Enzyme engineering is necessary to develop a useful process leading to valuable materials, but it requires a thorough understanding of how substrate and protein interact. This is difficult to achieve when the substrates are large molecules with conformational flexibility. Here, we employed multiple computational tools to understand the interaction between LcpK30 and its flexible polyisoprene substrate. Our results show that the substrate can access the active site through two hydrophobic tunnels, which can also serve as product exit pathways. The substrate binds in a hydrophobic pocket near the heme, which determines the size of the oligomeric products. We identified two potential binding modes for the substrate and characterized the hydrophobic contacts responsible for protein-substrate complexation in LcpK30. These results shed light on future enzyme engineering investigations to enhance catalytic activity and broaden the substrate and product range of Lcps.

bioinformatics↗

bioRxiv2023

The adaptability of the ion binding site by the Ag(I)/Cu(I) periplasmic chaperone SilF.

The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram negative bacteria. Sil proteins also bind Cu(I), but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry we show that binding of both ions is not only tighter than previously thought, but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment.

biophysics↗