QM/MM Description of Newly Selected Catalytic Bioscavengers Against Organophosphorus Compounds Revealed Reactivation Stimulus Mediated by Histidine Residue in the Acyl-Binding Loop

Butyrylcholinesterase (BChE) is considered as an efficient stoichiometric antidote against organophosphorus (OP) poisons. Recently we utilized combination of calculations and ultrahigh-throughput screening (uHTS) to select BChE variants capable of catalytic destruction of OP pesticide paraoxon. The purpose of this study was to elucidate the molecular mechanism underlying enzymatic hydrolysis of paraoxon by BChE variants using hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. Detailed analysis of accomplished QM/MM runs revealed that histidine residues introduced into the acyl-binding loop are always located in close proximity with aspartate residue at position 70. Histidine residue acts as general base thus leading to attacking water molecule activation and subsequent SN2 inline hydrolysis resulting in BChE reactivation. This combination resembles canonical catalytic triad found in active centers of various proteases. Carboxyl group activates histidine residue by altering its pKa, which in turn promotes the activation of water molecule in terms of its nucleophilicity. Observed re-protonation of catalytic serine residue at position 198 from histidine residue at position 438 recovers initial configuration of the enzyme’s active center, facilitating next catalytic cycle. We therefore suggest that utilization of uHTS platform in combination with deciphering of molecular mechanisms by QM/MM calculations may significantly improve our knowledge of enzyme function, propose new strategies for enzyme design and open new horizons in generation of catalytic bioscavengers against OP poisons

Role of κ→λ light-chain constant-domain switch in the structure and functionality of A17 reactibody

The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophosphate-metabolizing reactibody A17 has been re-engineered by replacing its constant κ light chain by the λ chain (A17λ), and the X-ray structure of A17λ has been determined at 1.95 Å resolution. It was found that compared with A17κ the active centre of A17λ is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the VL and VH domains. These VL/VH domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126° compared with 138° in A17κ. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17κ and A17λ variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions

Deep Functional Profiling Facilitates the Evaluation of the Antibacterial Potential of the Antibiotic Amicoumacin

The global spread of antibiotic resistance is forcing the scientific community to find new molecular strategies to counteract it. Deep functional profiling of microbiomes provides an alternative source for the discovery of novel antibiotic producers and probiotics. Recently, we implemented this ultrahigh-throughput screening approach for the isolation of Bacillus pumilus strains efficiently producing the ribosome-targeting antibiotic amicoumacin A (Ami). Proteomics and metabolomics revealed essential insight into the activation of Ami biosynthesis. Here, we applied omics to boost Ami biosynthesis, providing the optimized cultivation conditions for high-scale production of Ami. Ami displayed a pronounced activity against Lactobacillales and Staphylococcaceae, including methicillin-resistant Staphylococcus aureus (MRSA) strains, which was determined using both classical and massive single-cell microfluidic assays. However, the practical application of Ami is limited by its high cytotoxicity and particularly low stability. The former is associated with its self-lactonization, serving as an improvised intermediate state of Ami hydrolysis. This intramolecular reaction decreases Ami half-life at physiological conditions to less than 2 h, which is unprecedented for a terminal amide. While we speculate that the instability of Ami is essential for Bacillus ecology, we believe that its stable analogs represent attractive lead compounds both for antibiotic discovery and for anticancer drug development

Presentation_1_QM/MM Description of Newly Selected Catalytic Bioscavengers Against Organophosphorus Compounds Revealed Reactivation Stimulus Mediated by Histidine Residue in the Acyl-Binding Loop.PDF

<p>Butyrylcholinesterase (BChE) is considered as an efficient stoichiometric antidote against organophosphorus (OP) poisons. Recently we utilized combination of calculations and ultrahigh-throughput screening (uHTS) to select BChE variants capable of catalytic destruction of OP pesticide paraoxon. The purpose of this study was to elucidate the molecular mechanism underlying enzymatic hydrolysis of paraoxon by BChE variants using hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. Detailed analysis of accomplished QM/MM runs revealed that histidine residues introduced into the acyl-binding loop are always located in close proximity with aspartate residue at position 70. Histidine residue acts as general base thus leading to attacking water molecule activation and subsequent SN2 inline hydrolysis resulting in BChE reactivation. This combination resembles canonical catalytic triad found in active centers of various proteases. Carboxyl group activates histidine residue by altering its pK_a, which in turn promotes the activation of water molecule in terms of its nucleophilicity. Observed re-protonation of catalytic serine residue at position 198 from histidine residue at position 438 recovers initial configuration of the enzyme’s active center, facilitating next catalytic cycle. We therefore suggest that utilization of uHTS platform in combination with deciphering of molecular mechanisms by QM/MM calculations may significantly improve our knowledge of enzyme function, propose new strategies for enzyme design and open new horizons in generation of catalytic bioscavengers against OP poisons.</p

A kinase bioscavenger provides antibiotic resistance by extremely tight substrate binding

Microbial communities are self-controlled by repertoires of lethal agents, the antibiotics. In their turn, these antibiotics are regulated by bioscavengers that are selected in the course of evolution. Kinase-mediated phosphorylation represents one of the general strategies for the emergence of antibiotic resistance. A new subfamily of AmiN-like kinases, isolated from the Siberian bear microbiome, inactivates antibiotic amicoumacin by phosphorylation. The nanomolar substrate affinity defines AmiN as a phosphotransferase with a unique catalytic efficiency proximal to the diffusion limit. Crystallographic analysis and multiscale simulations revealed a catalytically perfect mechanism providing phosphorylation exclusively in the case of a closed active site that counteracts substrate promiscuity. AmiN kinase is a member of the previously unknown subfamily representing the first evidence of a specialized phosphotransferase bioscavenger

Deep Functional Profiling of Wild Animal Microbiomes Reveals Probiotic <i>Bacillus pumilus</i> Strains with a Common Biosynthetic Fingerprint

The biodiversity of microorganisms is maintained by intricate nets of interactions between competing species. Impaired functionality of human microbiomes correlates with their reduced biodiversity originating from aseptic environmental conditions and antibiotic use. Microbiomes of wild animals are free of these selective pressures. Microbiota provides a protecting shield from invasion by pathogens in the wild, outcompeting their growth in specific ecological niches. We applied ultrahigh-throughput microfluidic technologies for functional profiling of microbiomes of wild animals, including the skin beetle, Siberian lynx, common raccoon dog, and East Siberian brown bear. Single-cell screening of the most efficient killers of the common human pathogen Staphylococcus aureus resulted in repeated isolation of Bacillus pumilus strains. While isolated strains had different phenotypes, all of them displayed a similar set of biosynthetic gene clusters (BGCs) encoding antibiotic amicoumacin, siderophore bacillibactin, and putative analogs of antimicrobials including bacilysin, surfactin, desferrioxamine, and class IId cyclical bacteriocin. Amicoumacin A (Ami) was identified as a major antibacterial metabolite of these strains mediating their antagonistic activity. Genome mining indicates that Ami BGCs with this architecture subdivide into three distinct families, characteristic of the B. pumilus, B. subtilis, and Paenibacillus species. While Ami itself displays mediocre activity against the majority of Gram-negative bacteria, isolated B. pumilus strains efficiently inhibit the growth of both Gram-positive S. aureus and Gram-negative E. coli in coculture. We believe that the expanded antagonistic activity spectrum of Ami-producing B. pumilus can be attributed to the metabolomic profile predetermined by their biosynthetic fingerprint. Ultrahigh-throughput isolation of natural probiotic strains from wild animal microbiomes, as well as their metabolic reprogramming, opens up a new avenue for pathogen control and microbiome remodeling in the food industry, agriculture, and healthcare

Role of κ→λ light-chain constant-domain switch in the structure and functionality of A17 reactibody

The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophos­phate-metabolizing reactibody A17 has been re-engineered by replacing its constant κ light chain by the λ chain (A17λ), and the X-ray structure of A17λ has been determined at 1.95 Å resolution. It was found that compared with A17κ the active centre of A17λ is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the V(L) and V(H) domains. These V(L)/V(H) domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126° compared with 138° in A17κ. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17κ and A17λ variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions

Epitope-Specific Response of Human Milk Immunoglobulins in COVID-19 Recovered Women

The breastfeeding of infants by mothers who are infected with SARS-CoV-2 has become a dramatic healthcare problem. The WHO recommends that infected women should not abandon breastfeeding; however, there is still the risk of contact transmission. Convalescent donor milk may provide a defense against the aforementioned issue and can eliminate the consequences of artificial feeding. Therefore, it is vital to characterize the epitope-specific immunological landscape of human milk from women who recovered from COVID-19. We carried out a comprehensive ELISA-based analysis of blood serum and human milk from maternity patients who had recovered from COVID-19 at different trimesters of pregnancy. It was found that patients predominantly contained SARS-CoV-2 N-protein-specific immunoglobulins and had manifested the antibodies for all the antigens tested in a protein-specific and time-dependent manner. Women who recovered from COVID-19 at trimester I–II showed a noticeable decrease in the number of milk samples with sIgA specific to the N-protein, linear NTD, and RBD-SD1 epitopes, and showed an increase in samples with RBD conformation-dependent sIgA. S-antigens were found to solely induce a sIgA1 response, whereas N-protein sIgA1 and sIgA2 subclasses were involved in 100% and 33% of cases. Overall, the antibody immunological landscape of convalescent donor milk suggests that it may be a potential defense agent against COVID-19 for infants, conferring them with a passive immunity

Multiscale computation delivers organophosphorus reactivity and stereoselectivity to immunoglobulin scavengers

Quantum mechanics/molecular mechanics (QM/MM) maturation of an immunoglobulin (Ig) powered by supercomputation delivers novel functionality to this catalytic template and facilitates artificial evolution of biocatalysts. We here employ density functional theory-based (DFT-b) tight binding and funnel metadynamics to advance our earlier QM/MM maturation of A17 Ig-paraoxonase (WTIgP) as a reactibody for organophosphorus toxins. It enables regulation of biocatalytic activity for tyrosine nucleophilic attack on phosphorus. The single amino acid substitution l-Leu47Lys results in 340-fold enhanced reactivity for paraoxon. The computed ground-state complex shows substrate-induced ionization of the nucleophilic l-Tyr37, now H-bonded to l-Lys47, resulting from repositioning of l-Lys47. Multiple antibody structural homologs, selected by phenylphosphonate covalent capture, show contrasting enantioselectivities for a P-chiral phenylphosphonate toxin. That is defined by crystallographic analysis of phenylphosphonylated reaction products for antibodies A5 and WTIgP. DFT-b analysis using QM regions based on these structures identifies transition states for the favored and disfavored reactions with surprising results. This stereoselection analysis is extended by funnel metadynamics to a range of WTIgP variants whose predicted stereoselectivity is endorsed by experimental analysis. The algorithms used here offer prospects for tailored design of highly evolved, genetically encoded organophosphorus scavengers and for broader functionalities of members of the Ig superfamily, including cell surface-exposed receptors