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ELife Jun 2024Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range...
Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA's association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-β4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-C preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-β4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-β4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.
Topics: Molecular Dynamics Simulation; Allosteric Regulation; Adenosine Triphosphate; Catalytic Domain; Cyclic AMP-Dependent Protein Kinases; Protein Conformation; Protein Binding; Nucleotides; Substrate Specificity; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits
PubMed: 38913408
DOI: 10.7554/eLife.91506 -
Faraday Discussions Jun 2024The effective management of plastic waste has become a global imperative, given our reliance on a linear model in which plastics are manufactured, used once, and then...
The effective management of plastic waste has become a global imperative, given our reliance on a linear model in which plastics are manufactured, used once, and then discarded. This has led to the pervasive accumulation of plastic debris in landfills and environmental contamination. Recognizing this issue, numerous initiatives are underway to address the environmental repercussions associated with plastic disposal. In this study, we investigate the possible molecular mechanism of polyurethane esterase A (PueA), which has been previously identified as responsible for the degradation of a polyester polyurethane (PU) sample in , as an effort to develop enzymatic biodegradation solutions. After generating the unsolved 3D structure of the protein by AlphaFold2 from its known genome, the enzymatic hydrolysis of the same model PU compound previously used in experiments has been explored employing QM/MM molecular dynamics simulations. This required a preliminary analysis of the 3D structure of the apo-enzyme, identifying the putative active site, and the search for the optimal protein-substrate binding site. Finally, the resulting free energy landscape indicates that wild-type PueA can degrade PU chains, although with low-level activity. The reaction takes place by a characteristic four-step path of the serine hydrolases, involving an acylation followed by a diacylation step. Energetics and structural analysis of the evolution of the active site along the reaction suggests that PueA can be considered a promising protein scaffold for further development to achieve efficient biodegradation of PU.
PubMed: 38836643
DOI: 10.1039/d4fd00022f -
BioRxiv : the Preprint Server For... May 2024In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal...
In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from , with and without an inactive analog of the FPP substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP complex was solved to 2.65 Å resolution and showed the ligand in a linear, elongated orientation, incapable of undergoing the initial cyclization event to form a bond between carbons C1 and C11. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case density that was well fit with a curled-up tetraethylene glycol molecule presumably recruited from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotamer shifts among active-site residues. Most notably, W56 was observed to undergo a 90° rotation between the 2FFPP complex and apo-enzyme structures, suggesting that it contributes to steric interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L variant lost the ability to cyclize the FPP substrate and produced only the linear terpene products farnesol and α- and β-farnesene.
PubMed: 38746203
DOI: 10.1101/2024.05.04.592530 -
Journal of Inherited Metabolic Disease Apr 2024Sulfite intoxication is the hallmark of four ultrarare disorders that are caused by impaired sulfite oxidase activity due to genetic defects in the synthesis of the... (Review)
Review
Sulfite intoxication is the hallmark of four ultrarare disorders that are caused by impaired sulfite oxidase activity due to genetic defects in the synthesis of the molybdenum cofactor or of the apoenzyme sulfite oxidase. Delays on the diagnosis of these disorders are common and have been caused by their unspecific presentation of acute neonatal encephalopathy with high early mortality, followed by the evolution of dystonic cerebral palsy and also by the lack of easily available and reliable diagnostic tests. There is significant variation in survival and in the quality of symptomatic management of affected children. One of the four disorders, molybdenum cofactor deficiency type A (MoCD-A) has recently become amenable to causal treatment with synthetic cPMP (fosdenopterin). The evidence base for the rational use of cPMP is very limited. This prompted the formulation of these clinical guidelines to facilitate diagnosis and support the management of patients. The guidelines were developed by experts in diagnosis and treatment of sulfite intoxication disorders. It reflects expert consensus opinion and evidence from a systematic literature search.
PubMed: 38627985
DOI: 10.1002/jimd.12730 -
International Journal of Molecular... Mar 2024Enzymes reliant on pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B, hold significant importance in both biology and medicine. They facilitate... (Review)
Review
Enzymes reliant on pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B, hold significant importance in both biology and medicine. They facilitate various biochemical reactions, particularly in amino acid and neurotransmitter metabolisms. Vitamin B is absorbed by organisms in its non-phosphorylated form and phosphorylated within cells via pyridoxal kinase (PLK) and pyridox-(am)-ine 5'-phosphate oxidase (PNPOx). The flavin mononucleotide-dependent PNPOx enzyme converts pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate into PLP. PNPOx is vital for both biosynthesis and salvage pathways in organisms producing B vitamers. However, for those depending on vitamin B as a nutrient, PNPOx participates only in the salvage pathway. Transferring the PLP produced via PNPOx to client apo-enzymes is indispensable for their catalytic function, proper folding and targeting of specific organelles. PNPOx activity deficiencies due to inborn errors lead to severe neurological pathologies, particularly neonatal epileptic encephalopathy. PNPOx maintains PLP homeostasis through highly regulated mechanisms, including structural alterations throughout the catalytic cycle and allosteric PLP binding, influencing substrate transformation at the active site. Elucidation at the molecular level of the mechanisms underlying PNPOx activity deficiencies is a requirement to develop personalized approaches to treat related disorders. Finally, despite shared features, the few PNPOx enzymes molecularly and functionally studied show species-specific regulatory properties that open the possibility of targeting it in pathogenic organisms.
Topics: Humans; Infant, Newborn; Oxidoreductases; Phosphates; Pyridoxaminephosphate Oxidase; Pyridoxal Phosphate; Vitamin B 6; Pyridoxine; Metabolic Diseases; Vitamins
PubMed: 38542149
DOI: 10.3390/ijms25063174 -
Journal of Bacteriology Apr 2024A hallmark of infection of the urinary tract is the formation of stones. The ability to induce urinary stone formation requires urease, a nickel metalloenzyme that...
UNLABELLED
A hallmark of infection of the urinary tract is the formation of stones. The ability to induce urinary stone formation requires urease, a nickel metalloenzyme that hydrolyzes urea. This reaction produces ammonia as a byproduct, which can serve as a nitrogen source and weak base that raises the local pH. The resulting alkalinity induces the precipitation of ions to form stones. Transcriptional regulator UreR activates expression of urease genes in a urea-dependent manner. Thus, urease genes are highly expressed in the urinary tract where urea is abundant. Production of mature urease also requires the import of nickel into the cytoplasm and its incorporation into the urease apoenzyme. Urease accessory proteins primarily acquire nickel from one of two nickel transporters and facilitate incorporation of nickel to form mature urease. In this study, we performed a comprehensive RNA-seq to define the urea-induced transcriptome as well as the UreR regulon. We identified UreR as the first defined regulator of nickel transport in . We also offer evidence for the direct regulation of the Ynt nickel transporter by UreR. Using bioinformatics, we identified UreR-regulated urease loci in 15 family species across three genera. Additionally, we located two mobilized UreR-regulated urease loci that also encode the transporter, implying that UreR regulation of nickel transport is a conserved regulatory relationship. Our study demonstrates that UreR specifically regulates genes required to produce mature urease, an essential virulence factor for uropathogenesis.
IMPORTANCE
Catheter-associated urinary tract infections (CAUTIs) account for over 40% of acute nosocomial infections in the USA and generate $340 million in healthcare costs annually. A major causative agent of CAUTIs is , an understudied Gram-negative pathogen noted for its ability to form urinary stones via the activity of urease. Urease mutants cannot induce stones and are attenuated in a murine UTI model, indicating this enzyme is essential to pathogenesis. Transcriptional regulation of urease genes by UreR is well established; here, we expand the UreR regulon to include regulation of nickel import, a function required to produce mature urease. Furthermore, we reflect on the role of urea catalysis in metabolism and provide evidence for its importance.
Topics: Animals; Mice; Proteus mirabilis; Urease; Nickel; Bacterial Proteins; Escherichia coli; Urinary Tract Infections; Urea; Proteus Infections
PubMed: 38534115
DOI: 10.1128/jb.00031-24 -
Cell Feb 2024Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an...
Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions. Our data show the PEP core adopts a typical fold as bacterial RNAP. Fifteen PAPs bind at the periphery of the PEP core, facilitate assembling the PEP-PAP supercomplex, protect the complex from oxidation damage, and likely couple gene transcription with RNA processing. Our results report the high-resolution architecture of the chloroplast transcription apparatus and provide the structural basis for the mechanistic and functional study of transcription regulation in chloroplasts.
Topics: Chloroplasts; Cryoelectron Microscopy; DNA-Directed RNA Polymerases; Nicotiana; Photosynthesis; Plastids
PubMed: 38428393
DOI: 10.1016/j.cell.2024.01.026 -
Frontiers in Cellular and Infection... 2024Monkeypox or mpox virus (mpox) is a double-stranded DNA virus that poses a significant threat to global public health security. The F3 protein, encoded by mpox, is an...
BACKGROUND
Monkeypox or mpox virus (mpox) is a double-stranded DNA virus that poses a significant threat to global public health security. The F3 protein, encoded by mpox, is an apoenzyme believed to possess a double-stranded RNA-binding domain (dsRBD). However, limited research has been conducted on its function. In this study, we present data on the transcriptomics and proteomics of F3L-transfected HEK293T cells, aiming to enhance our comprehension of F3L.
METHODS
The gene expression profiles of pCAGGS-HA-F3L transfected HEK293T cells were analyzed using RNA-seq. Proteomics was used to identify and study proteins that interact with F3L. Real-time PCR was used to detect mRNA levels of several differentially expressed genes (DEGs) in HEK293T cells (or Vero cells) after the expression of F3 protein.
RESULTS
A total of 14,822 genes were obtained in cells by RNA-Seq and 1,672 DEGs were identified, including 1,156 up-regulated genes and 516 down-regulated genes. A total of 27 cellular proteins interacting with F3 proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and 19 cellular proteins with large differences in abundance ratios were considered to be candidate cellular proteins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the DEGs were significantly enriched in immune-related pathways, including type I interferon signaling pathway, response to virus, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, etc. Moreover, some selected DEGs were further confirmed by real-time PCR and the results were consistent with the transcriptome data. Proteomics data show that cellular proteins interacting with F3 proteins are mainly related to RNA splicing and protein translation.
CONCLUSIONS
Our analysis of transcriptomic and proteomic data showed that (1) F3L up-regulates the transcript levels of key genes in the innate immune signaling pathway, such as , and elicits a broad spectrum of antiviral immune responses in the host. F3L also increases the expression of the FOS and JNK genes while decreasing the expression of TNFR2, these factors may ultimately induce apoptosis. (2) F3 protein interacts with host proteins involved in RNA splicing and protein translation, such as SNRNP70, POLR2H, HNRNPA1, DDX17, etc. The findings of this study shed light on the function of the F3 protein.
Topics: Animals; Chlorocebus aethiops; Humans; Transcriptome; Monkeypox virus; Vero Cells; Chromatography, Liquid; HEK293 Cells; Mpox (monkeypox); Proteomics; Tandem Mass Spectrometry; Gene Expression Profiling; Ribonucleoprotein, U1 Small Nuclear
PubMed: 38415010
DOI: 10.3389/fcimb.2024.1354410 -
Chemical Communications (Cambridge,... Feb 2024Hydrogenases are enzymes that catalyze the reversible conversion of protons to hydrogen gas, using earth-abundant metals such as nickel and/or iron. This characteristic...
Hydrogenases are enzymes that catalyze the reversible conversion of protons to hydrogen gas, using earth-abundant metals such as nickel and/or iron. This characteristic makes them promising for sustainable energy applications, particularly in clean hydrogen production. However, their widespread use faces challenges, including a limited pH range and susceptibility to oxygen. In response to these issues, SacCoMyo is introduced as an artificial enzyme. SacCoMyo is designed by replacing the native metal in the myoglobin (Myo) scaffold with a hydroxocobalamin (Co) porphyrin core and complemented by a protective heteropolysaccharide-linked (Sac) shell. This engineered protein proves to be resilient, maintaining robust functionality even in acidic environments and preventing denaturation in a pH 1 electrolyte. The cobalt porphyrin core of SacCoMyo reduces the activation overpotential for hydrogen generation. A high turnover frequency of about 2400 H s is demonstrated in the presence of molecular oxygen, showcasing its potential in biohydrogen production and its ability to overcome the limitations associated with natural hydrogenases.
Topics: Hydrogen; Cobalt; Oxygen; Apoenzymes; Hydrogenase; Hydrogen-Ion Concentration; Porphyrins
PubMed: 38333929
DOI: 10.1039/d3cc06185j -
Journal of Medical Virology Feb 2024Cap RNA methylations play important roles in the replication, evasion of host RNA sensor recognition, and pathogenesis. Coronaviruses possess both guanine N7- and...
Cap RNA methylations play important roles in the replication, evasion of host RNA sensor recognition, and pathogenesis. Coronaviruses possess both guanine N7- and 2'-O-ribose methyltransferases (N7-MTase and 2'-O-MTase) encoded by nonstructural protein (nsp) 14 and nsp16/10 complex, respectively. In this study, we reconstituted the two-step RNA methylations of N7-MTase and 2'-O-MTase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and demonstrated its common and different features in comparison with that of SARS-CoV. We revealed that the nsp16/10 2'-O-MTase of SARS-CoV-2 has a broader substrate selectivity than the counterpart of SARS-CoV and can accommodate both unmethylated and uncapped RNA substrates in a sequence-independent manner. Most intriguingly, the substrate selectivity of nsp16/10 complex is not determined by the apoenzyme of nsp16 MTase but by its cofactor nsp10. These results provide insight into the unique features of SARS-CoV-2 MTases and may help develop strategies to precisely intervene in the methylation pathway and pathogenesis of SARS-CoV-2.
Topics: Humans; Methyltransferases; SARS-CoV-2; COVID-19; RNA Methylation; RNA Caps
PubMed: 38285434
DOI: 10.1002/jmv.29411