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Structure (London, England : 1993) May 2017The macrolides are a class of antibiotic, characterized by a large macrocyclic lactone ring that can be inactivated by macrolide phosphotransferase enzymes. We present...
The macrolides are a class of antibiotic, characterized by a large macrocyclic lactone ring that can be inactivated by macrolide phosphotransferase enzymes. We present structures for MPH(2')-I and MPH(2')-II in the apo state, and in complex with GTP analogs and six different macrolides. These represent the first structures from the two main classes of macrolide phosphotransferases. The structures show that the enzymes are related to the aminoglycoside phosphotransferases, but are distinguished from them by the presence of a large interdomain linker that contributes to an expanded antibiotic binding pocket. This pocket is largely hydrophobic, with a negatively charged patch located at a conserved aspartate residue, rationalizing the broad-spectrum resistance conferred by the enzymes. Complementary mutation studies provide insights into factors governing substrate specificity. A comparison with macrolides bound to their natural target, the 50S ribosome, suggests avenues for next-generation antibiotic development.
Topics: Bacteria; Bacterial Proteins; Binding Sites; Drug Resistance, Bacterial; Guanosine Triphosphate; Macrolides; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Substrate Specificity
PubMed: 28416110
DOI: 10.1016/j.str.2017.03.007 -
Methods in Molecular Biology (Clifton,... 2015The prediction of protein-protein interactions and kinase-specific phosphorylation sites on individual proteins is critical for correctly placing proteins within...
The prediction of protein-protein interactions and kinase-specific phosphorylation sites on individual proteins is critical for correctly placing proteins within signaling pathways and networks. The importance of this type of annotation continues to increase with the continued explosion of genomic and proteomic data, particularly with emerging data categorizing posttranslational modifications on a large scale. A variety of computational tools are available for this purpose. In this chapter, we review the general methodologies for these types of computational predictions and present a detailed user-focused tutorial of one such method and computational tool, Scansite, which is freely available to the entire scientific community over the Internet.
Topics: Algorithms; Amino Acid Motifs; Binding Sites; Computational Biology; Databases, Protein; Molecular Biology; Phosphorylation; Phosphotransferases; Protein Binding; Protein Interaction Maps; Proteins; Proteomics; Software
PubMed: 25859943
DOI: 10.1007/978-1-4939-2425-7_4 -
Current Opinion in Chemical Biology Dec 2021Advancements in chemical proteomics and mass spectrometry lipidomics are providing new opportunities to understand lipid kinase activity, specificity, and regulation on... (Review)
Review
Advancements in chemical proteomics and mass spectrometry lipidomics are providing new opportunities to understand lipid kinase activity, specificity, and regulation on a global cellular scale. Here, we describe recent developments in chemical biology of lipid kinases with a focus on those members that phosphorylate diacylglycerols. We further discuss future implications of how these mass spectrometry-based approaches can be adapted for studies of additional lipid kinase members with the aim of bridging the gap between protein and lipid kinase-focused investigations.
Topics: Diacylglycerol Kinase; Lipids; Mass Spectrometry; Phosphotransferases; Proteomics
PubMed: 34311404
DOI: 10.1016/j.cbpa.2021.06.007 -
The Journal of Biological Chemistry Oct 2023The bacterial cell envelope is the structure with which the bacterium engages with, and is protected from, its environment. Within this envelop is a conserved...
The bacterial cell envelope is the structure with which the bacterium engages with, and is protected from, its environment. Within this envelop is a conserved peptidoglycan polymer which confers shape and strength to the cell envelop. The enzymatic processes that build, remodel, and recycle the chemical components of this cross-linked polymer are preeminent targets of antibiotics and exploratory targets for emerging antibiotic structures. We report a comprehensive kinetic and structural analysis for one such enzyme, the Pseudomonas aeruginosa anhydro-N-acetylmuramic acid (anhNAM) kinase (AnmK). AnmK is an enzyme in the peptidoglycan-recycling pathway of this pathogen. It catalyzes the pairing of hydrolytic ring opening of anhNAM with concomitant ATP-dependent phosphoryl transfer. AnmK follows a random-sequential kinetic mechanism with respect to its anhNAM and ATP substrates. Crystallographic analyses of four distinct structures (apo AnmK, AnmK:AMPPNP, AnmK:AMPPNP:anhNAM, and AnmK:ATP:anhNAM) demonstrate that both substrates enter the active site independently in an ungated conformation of the substrate subsites, with protein loops acting as gates for anhNAM binding. Catalysis occurs within a closed conformational state for the enzyme. We observe this state crystallographically using ATP-mimetic molecules. A remarkable X-ray structure for dimeric AnmK sheds light on the precatalytic and postcatalytic ternary complexes. Computational simulations in conjunction with the high-resolution X-ray structures reveal the full catalytic cycle. We further report that a P. aeruginosa strain with disrupted anmK gene is more susceptible to the β-lactam imipenem compared to the WT strain. These observations position AnmK for understanding the nexus among peptidoglycan recycling, susceptibility to antibiotics, and bacterial virulence.
Topics: Anti-Bacterial Agents; Catalysis; Crystallography, X-Ray; Pseudomonas aeruginosa; Bacterial Proteins; Phosphotransferases; Recombinant Proteins; Models, Molecular; Protein Structure, Tertiary; Enzyme Activation; Drug Resistance, Bacterial
PubMed: 37660917
DOI: 10.1016/j.jbc.2023.105198 -
Functional characterization of the phosphotransferase system in Parageobacillus thermoglucosidasius.Scientific Reports May 2023Parageobacillus thermoglucosidasius is a thermophilic bacterium characterized by rapid growth, low nutrient requirements, and amenability to genetic manipulation. These...
Parageobacillus thermoglucosidasius is a thermophilic bacterium characterized by rapid growth, low nutrient requirements, and amenability to genetic manipulation. These characteristics along with its ability to ferment a broad range of carbohydrates make P. thermoglucosidasius a potential workhorse in whole-cell biocatalysis. The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) catalyzes the transport and phosphorylation of carbohydrates and sugar derivatives in bacteria, making it important for their physiological characterization. In this study, the role of PTS elements on the catabolism of PTS and non-PTS substrates was investigated for P. thermoglucosidasius DSM 2542. Knockout of the common enzyme I, part of all PTSs, showed that arbutin, cellobiose, fructose, glucose, glycerol, mannitol, mannose, N-acetylglucosamine, N-acetylmuramic acid, sorbitol, salicin, sucrose, and trehalose were PTS-dependent on translocation and coupled to phosphorylation. The role of each putative PTS was investigated and six PTS-deletion variants could not grow on arbutin, mannitol, N-acetylglucosamine, sorbitol, and trehalose as the main carbon source, or showed diminished growth on N-acetylmuramic acid. We concluded that PTS is a pivotal factor in the sugar metabolism of P. thermoglucosidasius and established six PTS variants important for the translocation of specific carbohydrates. This study lays the groundwork for engineering efforts with P. thermoglucosidasius towards efficient utilization of diverse carbon substrates for whole-cell biocatalysis.
Topics: Acetylglucosamine; Arbutin; Trehalose; Phosphotransferases; Carbohydrates; Bacteria; Mannitol; Sorbitol; Phosphoenolpyruvate Sugar Phosphotransferase System; Bacterial Proteins
PubMed: 37130962
DOI: 10.1038/s41598-023-33918-1 -
International Journal of Molecular... Apr 2021Cataracts are the major cause of blindness worldwide, largely resulting from aging and diabetes mellitus. Advanced glycation end products (AGEs) have been identified as...
Cataracts are the major cause of blindness worldwide, largely resulting from aging and diabetes mellitus. Advanced glycation end products (AGEs) have been identified as major contributors in cataract formation because they alter lens protein structure and stability and induce covalent cross-linking, aggregation, and insolubilization of lens crystallins. We investigated the potential of the deglycating enzyme fructosamine-3-kinase (FN3K) in the disruption of AGEs in cataractous lenses. Macroscopic changes of equine lenses were evaluated after ex vivo intravitreal FN3K injection. The mechanical properties of an equine lens pair were evaluated after treatment with saline and FN3K. AGE-type autofluorescence (AF) was measured to assess the time-dependent effects of FN3K on glycolaldehyde-induced AGE-modified porcine lens fragments and to evaluate its actions on intact lenses after in vivo intravitreal FN3K injection of murine eyes. A potential immune response after injection was evaluated by analysis of IL-2, TNFα, and IFNγ using an ELISA kit. Dose- and time-dependent AF kinetics were analyzed on pooled human lens fragments. Furthermore, AF measurements and a time-lapse of macroscopic changes were performed on intact cataractous human eye lenses after incubation with an FN3K solution. At last, AF measurements were performed on cataractous human eyes after crossover topical treatment with either saline- or FN3K-containing drops. While the lenses of the equine FN3K-treated eyes appeared to be clear, the saline-treated lenses had a yellowish-brown color. Following FN3K treatment, color restoration could be observed within 30 min. The extension rate of the equine FN3K-treated lens was more than twice the extension rate of the saline-treated lens. FN3K treatment induced significant time-dependent decreases in AGE-related AF values in the AGE-modified porcine lens fragments. Furthermore, in vivo intravitreal FN3K injection of murine eyes significantly reduced AF values of the lenses. Treatment did not provoke a systemic immune response in mice. AF kinetics of FN3K-treated cataractous human lens suspensions revealed dose- and time-dependent decreases. Incubation of cataractous human eye lenses with FN3K resulted in a macroscopic lighter color of the cortex and a decrease in AF values. At last, crossover topical treatment of intact human eyes revealed a decrease in AF values during FN3K treatment, while showing no notable changes with saline. Our study suggests, for the first time, a potential additional role of FN3K as an alternative treatment for AGE-related cataracts.
Topics: Animals; Cataract; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Eye; Glycation End Products, Advanced; Horses; Humans; Immunohistochemistry; Intravitreal Injections; Lens, Crystalline; Mice; Phosphotransferases (Alcohol Group Acceptor)
PubMed: 33917258
DOI: 10.3390/ijms22083841 -
Topics in Current Chemistry (Cham) Apr 2017The phosphoryl group, PO, is the dynamic structural unit in the biological chemistry of phosphorus. Its transfer from a donor to an acceptor atom, with oxygen much more... (Review)
Review
The phosphoryl group, PO, is the dynamic structural unit in the biological chemistry of phosphorus. Its transfer from a donor to an acceptor atom, with oxygen much more prevalent than nitrogen, carbon, or sulfur, is at the core of a great majority of enzyme-catalyzed reactions involving phosphate esters, anhydrides, amidates, and phosphorothioates. The serendipitous discovery that the phosphoryl group could be labeled by "nuclear mutation," by substitution of PO by MgF or AlF, has underpinned the application of metal fluoride (MF ) complexes to mimic transition states for enzymatic phosphoryl transfer reactions, with sufficient stability for experimental analysis. Protein crystallography in the solid state and F NMR in solution have enabled direct observation of ternary and quaternary protein complexes embracing MF transition state models with precision. These studies have underpinned a radically new mechanistic approach to enzyme catalysis for a huge range of phosphoryl transfer processes, as varied as kinases, phosphatases, phosphomutases, and phosphohydrolases. The results, without exception, have endorsed trigonal bipyramidal geometry (tbp) for concerted, "in-line" stereochemistry of phosphoryl transfer. QM computations have established the validity of tbp MF complexes as reliable models for true transition states, delivering similar bond lengths, coordination to essential metal ions, and virtually identical hydrogen bond networks. The emergence of protein control of reactant orbital overlap between bond-forming species within enzyme transition states is a new challenging theme for wider exploration.
Topics: Aluminum Compounds; Fluorides; Magnesium Compounds; Molecular Structure; Phosphines; Phosphoric Monoester Hydrolases; Phosphotransferases; Phosphotransferases (Phosphomutases)
PubMed: 28299727
DOI: 10.1007/s41061-017-0130-y -
The Journal of Biological Chemistry Mar 2022GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in...
GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are α2β2γ2 hexamers in which the core catalytic α- and β-subunits are derived from the GNPTAB (N-acetylglucosamine-1-phosphate transferase subunits alpha and beta) gene. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. We identified four conserved regions located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase. Comparison with UDP-glucose glycoprotein glucosyltransferase also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, we show that DmGNPTAB forms a homodimer that is evolutionarily conserved and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-1-phosphotransferase function and related diseases.
Topics: Animals; Cryoelectron Microscopy; Drosophila melanogaster; Lysosomes; Mammals; Mucolipidoses; Proteins; Structure-Activity Relationship; Transferases (Other Substituted Phosphate Groups)
PubMed: 35148990
DOI: 10.1016/j.jbc.2022.101702 -
International Journal of Molecular... Mar 2022Plant hormones are critical chemicals that participate in almost all aspects of plant life by triggering cellular response cascades. FERONIA is one of the most well... (Review)
Review
Plant hormones are critical chemicals that participate in almost all aspects of plant life by triggering cellular response cascades. FERONIA is one of the most well studied members in the subfamily of receptor-like kinase1-like (RLK1Ls) hormones. It has been proved to be involved in many different processes with the discovery of its ligands, interacting partners, and downstream signaling components. A growing body of evidence shows that FERONIA serves as a hub to integrate inter- and intracellular signals in response to internal and external cues. Here, we summarize the recent advances of FERONIA in regulating plant growth, development, and immunity through interactions with multiple plant hormone signaling pathways.
Topics: Arabidopsis Proteins; Catharanthus; Hormones; Phosphotransferases; Plant Development; Plant Growth Regulators
PubMed: 35409090
DOI: 10.3390/ijms23073730 -
Nature Dec 2021The human microbiome encodes a large repertoire of biochemical enzymes and pathways, most of which remain uncharacterized. Here, using a metagenomics-based search...
The human microbiome encodes a large repertoire of biochemical enzymes and pathways, most of which remain uncharacterized. Here, using a metagenomics-based search strategy, we discovered that bacterial members of the human gut and oral microbiome encode enzymes that selectively phosphorylate a clinically used antidiabetic drug, acarbose, resulting in its inactivation. Acarbose is an inhibitor of both human and bacterial α-glucosidases, limiting the ability of the target organism to metabolize complex carbohydrates. Using biochemical assays, X-ray crystallography and metagenomic analyses, we show that microbiome-derived acarbose kinases are specific for acarbose, provide their harbouring organism with a protective advantage against the activity of acarbose, and are widespread in the microbiomes of western and non-western human populations. These results provide an example of widespread microbiome resistance to a non-antibiotic drug, and suggest that acarbose resistance has disseminated in the human microbiome as a defensive strategy against a potential endogenous producer of a closely related molecule.
Topics: Acarbose; Amylases; Animals; Drug Resistance, Bacterial; Gastrointestinal Microbiome; Humans; Hypoglycemic Agents; Inactivation, Metabolic; Metagenome; Models, Molecular; Mouth; Phosphotransferases (Alcohol Group Acceptor)
PubMed: 34819672
DOI: 10.1038/s41586-021-04091-0