Biology subjects

Savchenko, A.

Publications and source records attributed to Savchenko, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Genomic, Clinical, and Spatial Predictors of Durable Response to BRAF/MEK Inhibition in BRAF-Mutant Melanoma

BRAF-targeted therapy (BRAFi/MEKi) and immune checkpoint blockade (anti-PD-1/anti-CTLA-4) have transformed the treatment of BRAF-mutant metastatic melanoma. While most patients who respond to targeted therapy eventually progress, a subset derives durable benefit, and biomarkers to identify this subset would inform optimal treatment selection. In this study, we analyzed pre-treatment tumor samples from a clinically annotated cohort of 155 patients with BRAF-mutant metastatic melanoma treated with first-line BRAFi/MEKi and followed for up to five years. We stratified patients into durable responders (PFS [≥] 24 months) and rapid progressors (PFS < 6 months with progression) and found that a global metric of tumor genomic heterogeneity, rather than individual gene alterations, distinguished these groups. Combining genomic heterogeneity with baseline tumor burden (e.g., lactate dehydrogenase (LDH) or radiographic lesion dimensions), we developed a parsimonious model that predicted durable responders with high precision and specificity. Notably, the analogous population of patients treated instead with immunotherapy were not durable responders, suggesting that the selected predictors of durable responders are targeted therapy specific. Spatial profiling of a subset of pre-treatment biopsies (n = 47) demonstrated that high intratumoral, but not peritumoral, CD8+ T-cell infiltration correlated with prolonged survival on BRAF-targeted therapy and served as an independent predictive factor when considered with genomic heterogeneity and features of clinical tumor burden. Together, these findings highlight the distinct baseline intrinsic and extrinsic features underlying durable response to BRAF-targeted therapy and support their potential implication in guiding treatment selection for patients with BRAF-mutant metastatic melanoma. One-Sentence SummaryIntegrated clinical, tumor genomic, and immune microenvironmental features predict durable responses to BRAF-targeted therapy.

cancer biology↗

bioRxiv2025

Dormant viral pathways underlie space-induced neural senescence: a neuroprotective strategy for spaceflight and neurological diseases

Long-duration spaceflight is associated with neurological symptoms in astronauts, yet the underlying molecular mechanisms remain unclear. Using human brain organoids cultured aboard the International Space Station, we analyzed three independent spaceflights to demonstrate that exposure to the space environment triggers Space-Induced Neural Senescence (SINS), characterized by chromatin remodeling, mitochondrial dysfunction, and activation of viral-like transcriptional programs in the absence of infection. Multi-omics analyses identified upregulation of endogenous LINE-1 (L1) retroelements, whose activity was markedly enhanced in organoids lacking MECP2, a known L1 repressor implicated in Rett syndrome. The resulting accumulation of cytoplasmic L1 DNA elicited an IL-6-mediated inflammatory and neurotoxic response, which was reversed by reverse transcriptase inhibitors (RTi) such as lamivudine or stavudine. Parallel preclinical experiments in Mecp2-deficient mice confirmed that RTi treatment restored neuronal morphology, synaptogenesis, function, cognition, and survival. These findings reveal that the space environment reactivates dormant genomic retroelements, providing an unexpected mechanistic insight into astronaut neurobiology and identifying a potential therapeutic strategy for both space-induced and terrestrial neurological conditions. Our pioneering study demonstrates the value of space-enabling research in accelerating drug discovery and disease treatment on Earth.

neuroscience↗

bioRxiv2025

Structural analysis of 23S rRNA methylating enzyme Cfr reveals RNA binding determinants for methylation regiospecificity and antibiotic resistance

The 23S rRNA methylating enzyme Cfr, found in pathogens including Staphylococcus aureus, Clostridium difficile, Escherichia coli and Klebsiella pneumoniae, confers resistance to phenicols, lincosamides, oxazolidinones (including linezolid), pleuromutilins, and streptogramins A (the PhLOPSA phenotype). Cfr catalyses methylation of the C8 position of the A2503 base in 23S rRNA; the recognition site of the above antibiotic classes. Along with RlmN housekeeping enzyme, Cfr can also promote methylation of the C2 position of the same base. The molecular and structural basis of Cfrs dual substrate specificity is not known, which hinders our ability to design Cfr-targeting inhibitors necessary to curb the PhLOPSA resistance. Here, we present the first crystal structure of Cfr and a detailed analysis of its possible interactions with rRNA. Using structure-guided mutagenesis, mass spectrometry analysis of in-cellulo 23S rRNA methylated species, and in-cellulo resistance studies, we identify the key amino acids essential for Cfr methylation and multi-drug resistance activity. In particular, we found that Cfrs Q329 residue is important for C8-specific methylation. These data provide a framework for further studies of the biochemistry, structure and inhibition of this important resistance determinant.

microbiology↗

bioRxiv2024

Torsional Twist of the SARS-CoV and SARS-CoV-2 SUD-N and SUD-M domains

Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vacuole that houses the replication-transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated, but rather, have significant rotational flexibility. Phylogenetic analysis supports that the disulfide bond cysteines are also absent in pangolin-SARS and closely related viruses, consistent with pangolins being the presumed intermediate host in the emergence of SARS-CoV-2. The absence of these cysteines does not impact viral replication or protein translation.

microbiology↗

bioRxiv2024

Cryo-EM Structure of Salmonella typhimurium ArnC; the Key Enzyme in Lipid-A Modification Conferring Polymyxin Resistance

1Polymyxins are last-resort antimicrobial peptides administered clinically against multi-drug resistant bacteria, including Gram-negative ESKAPE pathogens. However, an increasing number of pathogens employ a defense strategy involving a relay of enzymes encoded by the pmrE(ugd) loci and the arnBCDTEF operon. As a result, an Ara-4N headgroup is added to the lipid-A component of outer membrane (OM) lipopolysaccharides (LPS) rendering polymyxins ineffective. Here, we report the cryo-EM structures of glycosyltransferase ArnC from Salmonella typhimurium resolved in both apo and UDP-bound forms at resolutions 2.75 [A] and 3.8 [A], respectively. The structure of the ArnC protomer comprises of three distinct regions: an N-terminal glycosyltransferase domain, transmembrane region, and the interface helices (IHs). ArnC forms a stable tetramer with C2 symmetry through interactions in the C-terminal region, which is expected to protrude into the cytosol, where the {beta}8 strand inserts into the adjacent protomer. ArnC protomers have two distinct types of interfaces involving multiple hydrogen bonds and salt bridges. The binding of UDP induces conformational changes that stabilizes structurally labile A-loop, spanning residues 201 to 213, and part of the putative catalytic pocket formed by IH1 and IH2. The comparative analysis of ArnC structures with homologs GtrB and DPMS suggests the key residues involved in ArnC catalytic activity.

biochemistry↗

bioRxiv2024

Global survey of secondary metabolism in Aspergillus niger via activation of specific transcription factors

Genomics analysis confirmed the status of the filamentous fungi as a rich source of novel secondary metabolites; however, the discovery of these compounds is hampered by the cryptic nature of their biosynthetic pathways under laboratory conditions. Consequently, despite substantial research effort over the past decades, much of the secondary metabolome remains uncharacterized in fungal organisms. Our manual curation of biosynthetic gene clusters (BGCs) in the Aspergillus niger NRRL3 genome revealed that only 13 of 86 BGCs have had their cognate secondary metabolite products confirmed or reliably inferred. We also identified 60 transcription factors associated with cryptic BGCs. To further characterize A. niger secondary metabolism, we created a collection of strains each overexpressing a single BGC-associated transcription factor. We analyzed the strain collection using a standardized pipeline where we monitored phenotypic changes and compound production using mass spectrometry. Strains showing evidence of secondary metabolism activation were selected for gene expression analysis. Our approach resulted in the production of multiple potentially novel secondary metabolites and linked a specific BGC to tensidol production in A. niger. More broadly, this study found evidence counter to the existing paradigm of BGC expression controlled by co-localized transcription factors, lending credence to the emerging picture of a complex regulatory network governing fungal secondary metabolism. Significance StatementFungi produce an array of chemically diverse compounds that are routinely found to harbour valuable bioactivity. The products of secondary metabolism, these compounds have been a source of antimicrobials, anti-cancer agents, and other biopharmaceutical compounds termed natural products. Despite their demonstrated economic value, much is still unknown about the biosynthesis, regulation, and identities of these compounds. This study adopted a genome-wide approach to improve our understanding of the regulatory mechanisms that control fungal secondary metabolism, improving our ability to investigate the pathways responsible for natural product production.

microbiology↗

bioRxiv2024

Structural architecture of TolQ-TolR inner membrane protein complex from opportunistic pathogen Acinetobacter baumannii

Gram-negative bacteria harness the proton motive force (PMF) within their inner membrane (IM) to uphold the integrity of their cell envelope, an indispensable aspect for both division and survival. The IM TolQ-TolR complex is the essential part of the Tol-Pal system, serving as a conduit for PMF energy transfer to the outer membrane. Here we present cryo-EM reconstructions of Acinetobacter baumannii TolQ in apo and TolR- bound forms at atomic resolution. The apo TolQ configuration manifests as a symmetric pentameric pore, featuring a trans-membrane funnel leading towards a cytoplasmic chamber. In contrast, the TolQ-TolR complex assumes a proton non-permeable stance, characterized by the TolQ pentamers flexure to accommodate the TolR dimer, where two protomers undergo a translation-based relationship. Our structure-guided analysis and simulations support the rotor-stator mechanism of action, wherein the rotation of the TolQ pentamer harmonizes with the TolR protomers interplay. These findings broaden our mechanistic comprehension of molecular stator units empowering critical functions within the Gram-negative bacterial cell envelope. TeaserApo TolQ and TolQ-TolR structures depict structural rearrangements required for cell envelope organization in bacterial cell division.

molecular biology↗

bioRxiv2024

Structural and functional analyses of SARS-CoV-2 Nsp3 and its specific interactions with the 5' UTR of the viral genome

Non-structural protein 3 (Nsp3) is the largest open reading frame encoded in the SARS-CoV-2 genome, essential for formation of double-membrane vesicles (DMV) wherein viral RNA replication occurs. We conducted an extensive structure-function analysis of Nsp3 and determined the crystal structures of the Ubiquitin-like 1 (Ubl1), Nucleic Acid Binding (NAB), {beta}-coronavirus-Specific Marker ({beta}SM) domains and a sub-region of the Y domain of this protein. We show that the Ubl1, ADP-ribose phosphatase (ADRP), human SARS Unique (HSUD), NAB, and Y domains of Nsp3 bind the 5 UTR of the viral genome and that the Ubl1 and Y domains possess affinity for recognition of this region, suggesting high specificity. The Ubl1-Nucleocapsid (N) protein complex binds the 5 UTR with greater affinity than the individual proteins alone. Our results suggest that multiple domains of Nsp3, particularly Ubl1 and Y, shepherd the 5 UTR of viral genome during translocation through the DMV membrane, priming the Ubl1 domain to load the genome onto N protein.

microbiology↗

bioRxiv2024

Global atlas of predicted functional domains in Legionella pneumophila Dot/Icm translocated effectors

Legionella pneumophila utilizes the Dot/Icm type IVB secretion system to deliver hundreds of effector proteins inside eukaryotic cells to ensure intracellular replication. Our understanding of the molecular functions of this largest pathogenic arsenal known to the bacterial world remains incomplete. By leveraging advancements in 3D protein structure prediction, we provide a comprehensive structural analysis of 368 L. pneumophila effectors, representing a global atlas of predicted functional domains summarized in a database (https://pathogens3d.org/legionella-pneumophila). Our analysis identified 157 types of diverse functional domains in 287 effectors, including 159 effectors with no prior functional annotations. Furthermore, we identified 35 unique domains in 30 effector models that have no similarity with experimentally structurally characterized proteins, thus, hinting at novel functionalities. Using this analysis, we demonstrate the activity of thirteen domains, including three unique folds, predicted in L. pneumophila effectors to cause growth defects in the Saccharomyces cerevisiae model system. This illustrates an emerging strategy of exploring synergies between predictions and targeted experimental approaches in elucidating novel effector activities involved in infection.

microbiology↗

bioRxiv2024

A random mutagenesis screen enriched for missense mutations in bacterial effector proteins.

To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode for them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae. For many pathogens, the heterologous expression of effectors in yeast is growth inhibitory at a frequency much higher than housekeeping genes, an observation ascribed to targeting conserved eukaryotic proteins. Abrogation of yeast growth inhibition has been used to identify bacterial suppressors of effector activity, host targets, and functional residues and domains within effector proteins. We present here a yeast-based method for enriching for informative, in-frame, missense mutations in a pool of random effector mutants. We benchmark this approach against three effectors from Legionella pneumophila, an intracellular bacterial pathogen that injects a staggering >330 effectors into the host cell. For each protein, we show how in silico protein modeling (AlphaFold2) and missense- directed mutagenesis can be combined to reveal important structural features within effectors. We identify known active site residues within the metalloprotease RavK, highly conserved residues in SdbB, and previously unidentified functional motifs within the C-terminal domain of SdbA. We show that this domain has structural similarity with glycosyltransferases and exhibits in vitro activity consistent with this predicted function.

microbiology↗

bioRxiv2023

Functional and Structural Characterization of an IclR Family Transcription Factor for the Development of Dicarboxylic Acid Biosensors

Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work we characterize the PcaR TF belonging to the IclR family. We present in vitro functional analysis of PcaRs ligand profile and construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this TF. This work provides key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/550818v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@16bcdd2org.highwire.dtl.DTLVardef@95ba9forg.highwire.dtl.DTLVardef@f598borg.highwire.dtl.DTLVardef@1bfe5f2_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPcaR is an IclR family transcription regulator responsive to dicarboxylic acids C_LIO_LIPcaR was established as an in vivo biosensor in yeast C_LIO_LICrystal structure of PcaR in the apo form was solved C_LIO_LICrystal structure with PcaR in complex with succinate was solved C_LIO_LISequence alignments unveil ligand-binding positions in the IclR family C_LI

synthetic biology↗

bioRxiv2023

Structural characterization of the Sel1-like repeat protein LceB from Legionella pneumophila

Legionella are freshwater Gram-negative bacteria that in their normal environment infect protozoa. However, this adaptation also allows Legionella to infect human alveolar macrophages and cause pneumonia. Central to Legionella pathogenesis are more than 330 secreted effectors, of which there are 9 core effectors that are conserved in all pathogenic species. Despite their importance, the biochemical function of several core effectors remains unclear. To address this, we have taken a structural approach to characterize the core effector of unknown function LceB, or Lpg1356, from Legionella pneumophila. Here we solve an X-ray crystal structure of LceB using an AlphaFold model for molecular replacement. The experimental structure shows that LceB adopts a Sel1-like repeat fold as predicted. However, the crystal structure captured multiple conformations of LceB all of which differed from the AlphaFold model. Comparison of the predicted model and the experimental models suggests that LceB is highly flexible in solution. Additionally, molecular analysis of LceB using its close structural homologues reveals sequence and structural motifs of known biochemical function. Specifically, LceB harbors a repeated KAAEQG motif that both stabilizes the Sel1-like repeat fold and is known to participate in protein-protein interactions with eukaryotic host proteins. We also observe that LceB forms several higher-order oligomers in solution. Overall, our results have revealed that LceB has conformational flexibility, self-associates, and contains a molecular surface for binding a target host-cell protein. Additionally, our data provides structural insights into the Sel1-like repeat family of proteins that remain poorly studied.

biochemistry↗

bioRxiv2023

Environmental life cycle assessment of recombinant growth factor production for cultivated meat applications

Growth factors are critical components of current serum-supplemented and serum-free media formulations for cultivated meat production. However, growth factors have been excluded, estimated using proxies, or modeled using proprietary data in existing environmental assessments of cultivated meat products. Cell culture media has been identified as a hotspot in such studies, therefore it is important to accurately quantify the environmental impacts of growth factor supplementation. To address this gap, this study applied life cycle assessment (LCA) methodology to comparatively assess the environmental impacts of recombinant growth factor production for cultivated meat applications. Life cycle inventories were developed for four recombinant growth factors (IGF-1, FGF, TGF-{beta}, and PDGF) produced using a novel bench- scale process. The functional unit of the product output was selected as 1 mg of produced growth factor. The results indicate that recombinant growth factors can have significant environmental impacts within cultivated meat systems, despite being used in very small quantities. For example, the global warming potential of production of 1 mg of IGF-1, FGF, TGF-{beta}, and PDGF was estimated to be 0.1, 0.04, 0.2 and 0.2 kg CO2 eq, respectively. Future research should explore the sustainability of producing these growth factors at scale to meet the needs of the expanding cultivated meat industry or identifying alternatives to these growth factors that have a lower impact on the environment. Nomenclature O_TBL View this table: org.highwire.dtl.DTLVardef@1f0c19org.highwire.dtl.DTLVardef@534179org.highwire.dtl.DTLVardef@1ef86b1org.highwire.dtl.DTLVardef@73fd9eorg.highwire.dtl.DTLVardef@10fb642_HPS_FORMAT_FIGEXP M_TBL C_TBL

ecology↗

bioRxiv2022

Monomer and dimer structures of cytochrome bo3 ubiquinol oxidase from Escherichia coli

The E. coli cytochrome bo3 ubiquinol oxidase is a four-subunit heme-copper oxidase that serves as a proton pump in the E. coli aerobic respiratory chain. Despite many mechanistic studies on this protein, it is unclear whether this ubiquinol oxidase functions as a monomer, or as a dimer in a manner similar to its eukaryotic counterparts - the mitochondrial electron transport complexes. In this study, we determined the monomeric and dimeric structures of the E. coli cytochrome bo3 ubiquinol oxidase reconstituted in amphipol by cryogenic electron microscopy single particle reconstruction (cryo-EM SPR) to a resolution of 3.15 [A] and 3.46 [A], respectively. We have discovered that the protein can form a dimer in C2 symmetry, with the dimerization interface maintained by interactions between the subunit II of one monomer and the subunit IV of the other monomer. Moreover, the dimerization does not induce significant structural changes in each monomer, except the movement of a loop in subunit IV (residues 67-74).

biophysics↗

bioRxiv2022

Functional diversification despite structural congruence in the HipBST toxin-antitoxin system of Legionella pneumophila

Toxin-antitoxin (TA) systems are abundant genetic modules in bacterial chromosomes and on mobile elements. They are often patchily distributed and their physiological functions remain poorly understood. Here, we characterize a TA system in Legionella pneumophila that is highly conserved across Legionella species. This system is distantly related to Escherichia coli HipBST and we demonstrate that it is a functional tripartite TA system (denoted HipBSTLp). We identify HipBSTLp homologs in diverse taxa, yet in the Gammaproteobacteria these are almost exclusively found in Legionella species. Notably, the toxin HipTLp was previously reported to be a pathogenic effector protein that is translocated by L. pneumophila into its eukaryotic hosts. Contrary to this, we find no signal of HipTLp translocation beyond untranslocated control levels and make several observations consistent with a canonical role as a bacterial toxin. We present structural and biochemical insights into the regulation and neutralization of HipBSTLp, and identify key variations between this system and HipBSTEc. Finally, we show that the target of HipTLp is likely not conserved with any characterized HipA or HipT toxin. This work serves as a unique comparison of a TA system across bacterial species and illustrates the molecular diversity that exists within a single TA family.

microbiology↗

bioRxiv2022

Ni(II) binding affinity and specificity of solute binding proteins: the importance of the double His motif and variable loop revealed by structural and mutational studies

Extracytoplasmic solute binding proteins (SBPs) are molecular shuttles involved in the cellular uptake of various small molecules and metal ions including Ni(II). Our previous study with the Ni(II) binding proteins (NiBPs) CjNikZ from Campylobacter jejuni and CcNikZ-II from Clostridium carboxidivorans demonstrated they were able to bind Ni(II) at low micromolar affinity without the need for additional chelators. Here, we determined the crystal structure of apo CcNikZ-II, which revealed a Ni(II) binding site comprised of the highly conserved double His (HH-)prong (His511, His512) and a short variable (v-)loop nearby (Thr59-Thr64, TEDKYT). Alanine scanning mutagenesis of the CcNikZ-II Ni(II) binding site identified Glu60 and His511 as essential for high affinity binding to Ni(II). Phylogenetic analysis of >4000 SBP sequences demonstrated the presence of two clusters of proteins containing the HH-prong with CcNikZ-II and CjNikZ. To provide insights into the role of the double His-prong and v-loop sequence in Ni(II) binding of NiBPs, nine purified CcNikZ-II homologues containing the HH-prong and v-loop were screened using an automated screening workflow. Metal binding assays with purified homologous NiBPs revealed high Ni(II) binding affinity without requirement for chelators indicating that the double His prong represents a signature motif for the presence of Ni(II) binding activity in SBPs. The engineered CcNikZ-II variants with the wild type v-loop (TEDKYT) replaced with v-loops from NiBPs with higher affinity showed up to an order of magnitude higher affinity for Ni(II). In addition, the v-loop appears to play a role in metal ion specificity as purified wild type and engineered NiBPs with different v-loop sequences showed distinct metal profiles. This work paves way for metalloprotein engineering of NiBPs towards biocatalytic and metal recovery applications.

biochemistry↗

bioRxiv2022

Thermophilic carboxylesterases from hydrothermal vents of the volcanic island of Ischia active on synthetic and biobased polymers and mycotoxins

Hydrothermal vents have a widespread geographical distribution and are of high interest for investigating microbial communities and robust enzymes for various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes (Geobacillus and Brevibacillus spp.), Proteobacteria and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate (PHB) and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus spp., and to some extent, Fontimonas and Schleiferia spp. The screening at 37-70{o}C of metagenomic fosmid library from above enrichment cultures resulted in identification and successful production in Escherichia coli of three hydrolases (IS10, IS11 and IS12), all derived from yet uncultured Chloroflexota and showing low sequence identity (33-56%) to characterized enzymes. Enzymes exhibited maximal esterase activity at temperatures 70-90{o}C, with IS11 showing the highest thermostability (90% activity after 20 min incubation at 80{o}C). IS10 and IS12 were highly substrate-promiscuous and hydrolysed all 51 monoester substrates tested. Enzymes were active with polyesters (PLA and polyethylene terephthalate model substrate, 3PET) and mycotoxin T-2 (IS12). IS10 and IS12 had a classical /{beta} hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in the hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 [A] revealed the presence of the N-terminal {beta}-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 includes catalytic residues Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain encloses the catalytic cleft like a lid contributing to substrate binding. Thus, this study has identified novel thermotolerant carboxylesterases with a broad substrate range including polyesters and mycotoxins for potential applications in biotechnology. IMPORTANCEHigh-temperature-active microbial enzymes are important biocatalysts for many industrial applications including recycling of synthetic and biobased polyesters increasingly used in textiles, fibres, coatings and adhesives. Here, we have discovered three novel thermotolerant carboxylesterases (IS10, IS11 and IS12) from high-temperature enrichment cultures from the Ischia hydrothermal vents incubated with biobased polymers. The identified metagenomic enzymes originated from uncultured Chloroflexota and showed low sequence similarity to known carboxylesterases. Active sites of IS10 and IS12 had the largest "effective volumes" among the characterized prokaryotic carboxylesterases and exhibited high substrate promiscuity, including hydrolysis of polyesters and mycotoxin T-2 (IS12). Though less promiscuous compared to IS10 and IS12, IS11 had a higher thermostability with high temperature optimum (80-90 {o}C) for activity, hydrolysed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity in these enzymes makes them attractive candidates for further optimisation and potential application in plastics recycling.

microbiology↗

bioRxiv2022

Molecular mechanism of plasmid-borne resistance to sulfonamides

The sulfonamides (sulfas) are the oldest class of synthetic antibacterial that target the essential, conserved dihydropteroate synthase (DHPS) enzyme, encoded by folP, through chemical mimicry of its substrate p-aminobenzoic acid (pABA). Resistance has complicated their clinical utility and is widespread in pathogenic species. Resistance is mediated by acquisition of sul genes on mobile genetic elements, which code for the so-called Sul enzymes that are divergent DHPS enzymes with intrinsic sulfa-insensitivity. Even decades after the discovery of this resistance mechanism, its molecular details have not been understood. In this study, we elucidate the molecular basis for intrinsic resistance of Sul enzymes using x-ray crystallography, enzymology, mutagenesis, intrinsic tryptophan fluorescence, antibiotic susceptibility of a contemporary {Delta}folP strain, and adaptive laboratory evolution of folP. We show that the active sites of Sul enzymes possess a modified pABA-interaction region based on insertion of a Phe-Gly sequence. This insertion is necessary for discrimination between pABA and sulfonamides, more than 1000-fold loss in binding affinity of sulfas to Sul enzymes, and robust pan-sulfonamide resistance. We detect no fitness cost due to this active site modification, as it does not compromise the rate of dihydropteroate biosynthesis and complements the thymidine-auxotrophy of an E. coli folP deletion strain. Lab-evolved sulfa-resistance folP recapitulated this mechanism through the same active site insertion. Finally, we show that this insertion and a nearby loop confer increased active site flexibility of Sul enzymes relative to DHPS. These results provide a molecular foundation for revisiting DHPS-targeted antibacterials to evade resistance.

microbiology↗