ABSTRACT
The earth is thought to have gone through complex physicochemical changes during the accretion and magma ocean stages. To better understand this evolution process at the fundamental level, we investigate the behavior of a bulk earth melt system by simulating the composition Fe35.7Mg19.0Si15.2O30.2 (in wt%) at high pressure. A deep neural network potential trained by first-principles data can enable accurate molecular dynamics simulation of large supercells that greatly enhances sampling for reliable evaluation of elemental partitioning. Our simulated system undergoes a phase separation in which the four elements clump together to different extents into two major domains. Based on the coordination and space-decomposition analyses, the inferred composition at 3000 K and 29.1 GPa contains 96.2, 0.1, 1.9 and 1.7 wt% of Fe, Mg, Si, and O, respectively, for the one domain and the corresponding elemental proportions are 3.0, 29.7, 22.0, and 45.3 wt% for the other domain. The predicted segregation thus leads to the formation of an iron-rich phase which corresponds to the metallic core and a magma ocean phase which corresponds to the silicate mantle. The metallic domain incorporates more silicon and more oxygen whereas the magma ocean domain gains more iron oxides at higher temperatures. Our predicted compositions compare favorably with those derived from experimental work for the equilibrium state metal and silicate reacting under high-pressure conditions.
ABSTRACT
Most organophosphates (OPs) are hydrophobic, and after exposure, can sequester into lipophilic regions within the body, such as adipose tissue, resulting in long term chronic effects. Consequently, there is an urgent need for therapeutic agents that can decontaminate OPs in these hydrophobic regions. Accordingly, an enzyme-polymer surfactant nanocomplex is designed and tested comprising chemically supercharged phosphotriesterase (Agrobacterium radiobacter; arPTE) electrostatically conjugated to amphiphilic polymer surfactant chains ([cat.arPTE][S-]). Experimentally-derived structural data are combined with molecular dynamics (MD) simulations to provide atomic level detail on conformational ensembles of the nanocomplex using dielectric constants relevant to aqueous and lipidic microenvironments. These show the formation of a compact admicelle pseudophase surfactant corona under aqueous conditions, which reconfigures to yield an extended conformation at a low dielectric constant, providing insight into the mechanism underpinning cell membrane binding. Significantly, it demonstrated that [cat.arPTE][S-] spontaneously binds to human mesenchymal stem cell membranes (hMSCs), resulting in on-cell OP hydrolysis. Moreover, the nanoconstruct can endocytose and partition into the intracellular fatty vacuoles of adipocytes and hydrolyze sequestered OP.
ABSTRACT
The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.
Subject(s)
Burkholderiales , Burkholderiales/enzymology , Burkholderiales/genetics , Burkholderiales/metabolism , Phthalic Acids/metabolism , Phthalic Acids/chemistry , Hydrolases/metabolism , Hydrolases/genetics , Hydrolases/chemistry , Solubility , Polyethylene Terephthalates/metabolism , Polyethylene Terephthalates/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/chemistry , Protein Engineering/methods , Protein Folding , Escherichia coli/genetics , Escherichia coli/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Models, MolecularABSTRACT
The development of novel antivirals is crucial not only for managing current COVID-19 infections but for addressing potential future zoonotic outbreaks. SARS-CoV-2 main protease (Mpro) is vital for viral replication and viability and therefore serves as an attractive target for antiviral intervention. Herein, we report the optimization of a cyclic peptide inhibitor that emerged from an mRNA display selection against the SARS-CoV-2 Mpro to enhance its cell permeability and inâ vitro antiviral activity. By identifying mutation-tolerant amino acid residues within the peptide sequence, we describe the development of a second-generation Mpro inhibitor bearing five cyclohexylalanine residues. This cyclic peptide analogue exhibited significantly improved cell permeability and antiviral activity compared to the parent peptide. This approach highlights the importance of optimizing cyclic peptide hits for activity against intracellular targets such as the SARS-CoV-2 Mpro.
Subject(s)
Antiviral Agents , Coronavirus 3C Proteases , Hydrophobic and Hydrophilic Interactions , Peptides, Cyclic , SARS-CoV-2 , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , Coronavirus 3C Proteases/chemistry , Humans , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Amino Acids/chemistry , COVID-19 Drug TreatmentABSTRACT
Oxygen plays a crucial role in human embryogenesis, homeostasis, and tissue regeneration. Emerging engineered regenerative solutions call for novel oxygen delivery systems. To become a reality, these systems must consider physiological processes, oxygen release mechanisms and the target application. In this review, we explore the biological relevance of oxygen at both a cellular and tissue level, and the importance of its controlled delivery via engineered biomaterials and devices. Recent advances and upcoming trends in the field are also discussed with a focus on tissue-engineered constructs that could meet metabolic demands to facilitate regeneration.
Subject(s)
Oxygen , Regeneration , Tissue Engineering , Humans , Oxygen/metabolism , Tissue Engineering/methods , Regeneration/physiology , Animals , Biocompatible Materials/chemistryABSTRACT
Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.
Subject(s)
Amides , Periplasmic Binding Proteins , Amides/metabolism , Amides/chemistry , Crystallography, X-Ray , Periplasmic Binding Proteins/metabolism , Periplasmic Binding Proteins/chemistry , Periplasmic Binding Proteins/genetics , ATP-Binding Cassette Transporters/metabolism , ATP-Binding Cassette Transporters/chemistry , Amino Acids/metabolism , Mesorhizobium/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Models, Molecular , Amidohydrolases/metabolism , Amidohydrolases/chemistry , Calcium/metabolism , Protein BindingABSTRACT
Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.
Subject(s)
Leucine , Leucine/chemistry , Escherichia coli/metabolism , Protein Conformation , Models, Molecular , Crystallography, X-Ray , Peptidylprolyl Isomerase/chemistry , Peptidylprolyl Isomerase/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolismABSTRACT
Plastic degrading enzymes have immense potential for use in industrial applications. Protein engineering efforts over the last decade have resulted in considerable enhancement of many properties of these enzymes. Directed evolution, a protein engineering approach that mimics the natural process of evolution in a laboratory, has been particularly useful in overcoming some of the challenges of structure-based protein engineering. For example, directed evolution has been used to improve the catalytic activity and thermostability of polyethylene terephthalate (PET)-degrading enzymes, although its use for the improvement of other desirable properties, such as solvent tolerance, has been less studied. In this review, we aim to identify some of the knowledge gaps and current challenges, and highlight recent studies related to the directed evolution of plastic-degrading enzymes.
Subject(s)
Directed Molecular Evolution , Protein Engineering , Directed Molecular Evolution/methods , Plastics/chemistry , Plastics/metabolism , Polyethylene Terephthalates/chemistry , Polyethylene Terephthalates/metabolism , Enzymes/genetics , Enzymes/chemistry , Enzymes/metabolismABSTRACT
Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.
Subject(s)
Carbonic Anhydrases , Cyanobacteria , Ribulose-Bisphosphate Carboxylase , Ribulose-Bisphosphate Carboxylase/metabolism , Ribulose-Bisphosphate Carboxylase/chemistry , Ribulose-Bisphosphate Carboxylase/genetics , Carbonic Anhydrases/metabolism , Carbonic Anhydrases/genetics , Carbonic Anhydrases/chemistry , Cyanobacteria/metabolism , Cyanobacteria/genetics , Cyanobacteria/enzymology , Allosteric Regulation , Phylogeny , Ribulosephosphates/metabolism , Models, Molecular , Protein Multimerization , Carbon Dioxide/metabolism , Substrate Specificity , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistryABSTRACT
Plastic waste has become a substantial environmental issue. A potential strategy to mitigate this problem is to use enzymatic hydrolysis of plastics to depolymerize post-consumer waste and allow it to be reused. Over the last few decades, the use of enzymatic PET-degrading enzymes has shown promise as a great solution for creating a circular plastic waste economy. PsPETase from Piscinibacter sakaiensis has been identified as an enzyme with tremendous potential for such applications. But to improve its efficiency, enzyme engineering has been applied aiming at enhancing its thermal stability, enzymatic activity, and ease of production. Here, we combine different strategies such as structure-based rational design, ancestral sequence reconstruction and machine learning to engineer a more highly active Combi-PETase variant with a melting temperature of 70 °C and optimal performance at 60 °C. Furthermore, this study demonstrates that these approaches, commonly used in other works of enzyme engineering, are most effective when utilized in combination, enabling the improvement of enzymes for industrial applications.
Subject(s)
Protein Engineering , Polyethylene Terephthalates/chemistry , Polyethylene Terephthalates/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Enzyme Stability , BurkholderialesABSTRACT
Lanthanide ions have ideal chemical properties for catalysis, such as hard Lewis acidity, fast ligand-exchange kinetics, high coordination-number preferences and low geometric requirements for coordination. As a result, many small-molecule lanthanide catalysts have been described in the literature. Yet, despite the ability of enzymes to catalyse highly stereoselective reactions under gentle conditions, very few lanthanoenzymes have been investigated. In this work, the mononuclear binding of europium(III) and gadolinium(III) to the active site of a mutant of the model enzyme phosphotriesterase are described using X-ray crystallography at 1.78 and 1.61â Å resolution, respectively. It is also shown that despite coordinating a single non-natural metal cation, the PTE-R18 mutant is still able to maintain esterase activity.
Subject(s)
Lanthanoid Series Elements , Metalloproteins , Phosphoric Triester Hydrolases , Phosphoric Triester Hydrolases/chemistry , Phosphoric Triester Hydrolases/metabolism , Catalytic Domain , Gadolinium , Europium , CationsABSTRACT
How a protein's function influences the shape of its fitness landscape, smooth or rugged, is a fundamental question in evolutionary biochemistry. Smooth landscapes arise when incremental mutational steps lead to a progressive change in function, as commonly seen in enzymes and binding proteins. On the other hand, rugged landscapes are poorly understood because of the inherent unpredictability of how sequence changes affect function. Here, we experimentally characterize the entire sequence phylogeny, comprising 1,158 extant and ancestral sequences, of the DNA-binding domain (DBD) of the LacI/GalR transcriptional repressor family. Our analysis revealed an extremely rugged landscape with rapid switching of specificity, even between adjacent nodes. Further, the ruggedness arises due to the necessity of the repressor to simultaneously evolve specificity for asymmetric operators and disfavors potentially adverse regulatory crosstalk. Our study provides fundamental insight into evolutionary, molecular, and biophysical rules of genetic regulation through the lens of fitness landscapes.
Subject(s)
PhylogenyABSTRACT
Cytochrome-P450-mediated cross-linking of ribosomally encoded peptides (RiPPs) is rapidly expanding and displays great potential for biocatalysis. Here, we demonstrate that active site engineering of the biarylitide cross-linking enzyme P450Blt enables the formation of His-X-Tyr and Tyr-X-Tyr cross-linked peptides, thus showing how such P450s can be further exploited to produce alternate cyclic tripeptides with controlled cross-linking states.
Subject(s)
Peptides, Cyclic , Peptides , Peptides, Cyclic/metabolism , Peptides/chemistry , Cytochrome P-450 Enzyme System , Biocatalysis , Catalytic DomainABSTRACT
Freshwater mussels are important indicators of the overall health of their environment but have suffered declines that have been attributed to factors such as habitat degradation, a loss of fish hosts, climate change, and excessive nutrient inputs. The loss of mussel biodiversity can negatively impact freshwater ecosystems such that understanding the mussel's gut microbiome has been identified as a priority topic for developing conservation strategies. In this study, we determine whether ethanol-stored specimens of freshwater mussels can yield representative information about their gut microbiomes such that changes in the microbiome through time could potentially be determined from museum mussel collections. A short-term preservation experiment using the invasive clam Corbicula fluminea was used to validate the use of ethanol as a method for storing the bivalve microbiome, and the gut microbiomes of nine native mussel species that had been preserved in ethanol for between 2 and 9 years were assessed. We show that ethanol preservation is a valid storage method for bivalve specimens in terms of maintaining an effective sequencing depth and the richness of their gut bacterial assemblages and provide further insight into the gut microbiomes of the invasive clam C. fluminea and nine species of native mussels. From this, we identify a "core" genus of bacteria (Romboutsia) that is potentially common to all freshwater bivalve species studied. These findings support the potential use of ethanol-preserved museum specimens to examine patterns in the gut microbiomes of freshwater mussels over long periods.
ABSTRACT
WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine (NMet-Dht) residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase (NRPS). The cytochrome P450 encoded by sas16 (P450Sas) has been shown to be essential for the formation of the alkene moiety in NMet-Dht, but the timing and mechanism of the P450Sas-mediated α,ß-dehydrogenation of Dht remained unclear. Here, we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein (PCP)-bound dipeptide intermediate (Z)-2-pent-1'-enyl-cinnamoyl-Thr-N-Me-Tyr. We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS, and further that P450Sas appears to be specific for substrates containing the (Z)-2-pent-1'-enyl-cinnamoyl group. A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates, including the substitution of the canonical active site alcohol residue with a phenylalanine (F250), which in turn is critical to P450Sas activity and WS9326A biosynthesis. Together, our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate, thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.
ABSTRACT
Geochronology indicates a rapid transition (tens of Myrs) from primary to secondary crust building on the Moon. The processes responsible for initiating secondary magmatism, however, remain in debate. Here we test the hypothesis that the earliest secondary crust (Mg-suite) formed as a direct consequence of density-driven mantle overturn, and advance 3D mantle convection models to quantify the resulting extent of lower mantle melting. Our modeling demonstrates that overturn of thin ilmenite-bearing cumulates ≤ 100 km triggers a rapid and short-lived episode of lower mantle melting which explains the key volume, geochronological, and spatial characteristics of early secondary crust building without contributions from other energy sources, namely KREEP (potassium, rare earth elements, phosphorus, radiogenic U, Th). Observations of globally distributed Mg-suite eliminate degree-1 overturn scenarios. We propose that gravitational instabilities in magma ocean cumulate piles are major driving forces for the onset of mantle convection and secondary crust building on differentiated bodies.
ABSTRACT
The biological significance of a small supernumerary marker chromosome that results in dosage alterations to chromosome 9p24.1, including triplication of the GLDC gene encoding glycine decarboxylase, in two patients with psychosis is unclear. In an allelic series of copy number variant mouse models, we identify that triplication of Gldc reduces extracellular glycine levels as determined by optical fluorescence resonance energy transfer (FRET) in dentate gyrus (DG) but not in CA1, suppresses long-term potentiation (LTP) in mPP-DG synapses but not in CA3-CA1 synapses, reduces the activity of biochemical pathways implicated in schizophrenia and mitochondrial bioenergetics, and displays deficits in prepulse inhibition, startle habituation, latent inhibition, working memory, sociability and social preference. Our results thus provide a link between a genomic copy number variation, biochemical, cellular and behavioral phenotypes, and further demonstrate that GLDC negatively regulates long-term synaptic plasticity at specific hippocampal synapses, possibly contributing to the development of neuropsychiatric disorders.
ABSTRACT
Heparanase (HPSE) is the only mammalian endo-ß-glucuronidase known to catalyze the degradation of heparan sulfate. Dysfunction of HPSE activity has been linked to several disease states, resulting in HPSE becoming the target of numerous therapeutic programs, yet no drug has passed clinical trials to date. Pentosan polysulfate sodium (PPS) is a heterogeneous, FDA-approved drug for the treatment of interstitial cystitis and a known HPSE inhibitor. However, due to its heterogeneity, characterization of its mechanism of HPSE inhibition is challenging. Here, we show that inhibition of HPSE by PPS is complex, involving multiple overlapping binding events, each influenced by factors such as oligosaccharide length and inhibitor-induced changes in the protein secondary structure. The present work advances our molecular understanding of the inhibition of HPSE and will aid in the development of therapeutics for the treatment of a broad range of pathologies associated with enzyme dysfunction, including cancer, inflammatory disease, and viral infections.
Subject(s)
Glucuronidase , Heparitin Sulfate , Animals , Heparitin Sulfate/chemistry , Glucuronidase/chemistry , Mammals/metabolismABSTRACT
The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Pandemics/prevention & control , Peptides/pharmacologyABSTRACT
Nonribosomal peptide synthetases produce many important peptide natural products and are centred around carrier proteins (CPs) that deliver intermediates to various catalytic domains. We show that the replacement of CP substrate thioesters by stabilised ester analogues leads to active condensation domain complexes, whereas amide stabilisation generates non-functional complexes.