ABSTRACT
Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The de novo biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP. Here we present high-resolution crystal structures of an HMPK from Acinetobacter baumannii (AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC50 of 768 µM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present in A. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin.
ABSTRACT
Human uridine 5'-monophosphate synthase (HsUMPS) is a bifunctional enzyme that catalyzes the final two steps in de novo pyrimidine biosynthesis. The individual orotate phosphoribosyl transferase and orotidine monophosphate domains have been well characterized, but little is known about the overall structure of the protein and how the organization of domains impacts function. Using a combination of chromatography, electron microscopy, and complementary biophysical methods, we report herein that HsUMPS can be observed in two structurally distinct states, an enzymatically active dimeric form and a nonactive multimeric form. These two states readily interconvert to reach an equilibrium that is sensitive to perturbations of the active site and the presence of substrate. We determined that the smaller molecular weight form of HsUMPS is an S-shaped dimer that can self-assemble into relatively well-ordered globular condensates. Our analysis suggests that the transition between dimer and multimer is driven primarily by oligomerization of the orotate phosphoribosyl transferase domain. While the cellular distribution of HsUMPS is unaffected, quantification by mass spectrometry revealed that de novo pyrimidine biosynthesis is dysregulated when this protein is unable to assemble into inactive condensates. Taken together, our data suggest that HsUMPS self-assembles into biomolecular condensates as a means to store metabolic potential for the regulation of metabolic rates.
Subject(s)
Biomolecular Condensates , Orotate Phosphoribosyltransferase , Orotidine-5'-Phosphate Decarboxylase , Uridine Monophosphate , Humans , Orotate Phosphoribosyltransferase/metabolism , Orotidine-5'-Phosphate Decarboxylase/metabolism , Pyrimidines/biosynthesis , Uridine , Uridine Monophosphate/metabolismABSTRACT
Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N5-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N5-CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC50 values. Several of these compounds, including the α1-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.
Subject(s)
Intramolecular Transferases , Ribonucleotides , Humans , Ribonucleotides/chemistry , Escherichia coli/metabolism , Intramolecular Transferases/metabolism , Purine Nucleotides/metabolismABSTRACT
The suppressor of TCR signaling (Sts) proteins, Sts-1 and Sts-2, are a pair of closely related signaling molecules that belong to the histidine phosphatase (HP) family of enzymes by virtue of an evolutionarily conserved C-terminal phosphatase domain. HPs derive their name from a conserved histidine that is important for catalytic activity and the current evidence indicates that the Sts HP domain plays a critical functional role. Sts-1HP has been shown to possess a readily measurable protein tyrosine phosphatase activity that regulates a number of important tyrosine-kinase-mediated signaling pathways. The in vitro catalytic activity of Sts-2HP is significantly lower than that of Sts-1HP, and its signaling role is less characterized. The highly conserved unique structure of the Sts proteins, in which additional domains, including one that exhibits a novel phosphodiesterase activity, are juxtaposed together with the phosphatase domain, suggesting that Sts-1 and -2 occupy a specialized intracellular signaling niche. To date, the analysis of Sts function has centered predominately around the role of Sts-1 and -2 in regulating host immunity and other responses associated with cells of hematopoietic origin. This includes their negative regulatory role in T cells, platelets, mast cells and other cell types, as well as their less defined roles in regulating host responses to microbial infection. Regarding the latter, the use of a mouse model lacking Sts expression has been used to demonstrate that Sts contributes non-redundantly to the regulation of host immunity toward a fungal pathogen (C. albicans) and a Gram-negative bacterial pathogen (F. tularensis). In particular, Sts-/- animals demonstrate significant resistance to lethal infections of both pathogens, a phenotype that is correlated with some heightened anti-microbial responses of phagocytes derived from mutant mice. Altogether, the past several years have seen steady progress in our understanding of Sts biology.
Subject(s)
Histidine , Protein Tyrosine Phosphatases , Animals , Mice , Protein Tyrosine Phosphatases/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/physiology , T-Lymphocytes/metabolismABSTRACT
The suppressor of T-cell receptor (TCR) signaling (Sts) proteins Sts-1 and Sts-2 suppress receptor-mediated signaling pathways in various immune cells, including the TCR pathway in T cells and the Dectin-1 signaling pathway in phagocytes. As multidomain enzymes, they contain an N-terminal ubiquitin-association domain, a central Src homology 3 domain, and a C-terminal histidine phosphatase domain. Recently, a 2-histidine (2H) phosphoesterase motif was identified within the N-terminal portion of Sts. The 2H phosphoesterase motif defines an evolutionarily ancient protein domain present in several enzymes that hydrolyze cyclic phosphate bonds on different substrates, including cyclic nucleotides. It is characterized by two invariant histidine residues that play a critical role in catalytic activity. Consistent with its assignment as a phosphoesterase, we demonstrate here that the Sts-1 2H phosphoesterase domain displays catalytic, saturable phosphodiesterase activity toward the dinucleotide 2',3'-cyclic NADP. The enzyme exhibited a high degree of substrate specificity and selectively generated the 3'-nucleotide as the sole product. Sts-1 also had phosphodiesterase catalytic activity toward a 5-mer RNA oligonucleotide containing a 2',3'-cyclic phosphate group at its 3' terminus. To investigate the functional significance of Sts-1 2H phosphoesterase activity, we generated His-to-Ala variants and examined their ability to negatively regulate cellular signaling pathways. Substitution of either conserved histidine compromised the ability of Sts-1 to suppress signaling pathways downstream of both the TCR and the Dectin-1 receptor. Our results identify a heretofore unknown cellular enzyme activity associated with Sts-1 and indicate that this catalytic activity is linked to specific cell-signaling outcomes.
Subject(s)
Protein Tyrosine Phosphatases/metabolism , Signal Transduction/physiology , Amino Acid Motifs , Animals , Catalytic Domain , Humans , Interferon-gamma/metabolism , Kinetics , Lectins, C-Type/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutagenesis, Site-Directed , NADP/analogs & derivatives , NADP/metabolism , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/genetics , Receptors, Antigen, T-Cell/metabolism , Sequence Alignment , Substrate Specificity , T-Lymphocytes/cytology , T-Lymphocytes/metabolismABSTRACT
[This corrects the article DOI: 10.1371/journal.ppat.1006843.].
ABSTRACT
Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in orf75A exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host.
Subject(s)
Gammaherpesvirinae/physiology , Herpesviridae Infections/virology , Lung/virology , Macrophages/virology , Open Reading Frames , Spleen/virology , Viral Proteins/metabolism , Animals , Bone Marrow Cells/cytology , Cells, Cultured , Codon, Nonsense , DNA, Recombinant/metabolism , DNA, Viral/metabolism , Embryo, Mammalian/cytology , Gammaherpesvirinae/growth & development , Gammaherpesvirinae/pathogenicity , Herpesviridae Infections/immunology , Herpesviridae Infections/pathology , Lung/immunology , Lung/pathology , Macrophages/immunology , Macrophages/pathology , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Phylogeny , Spleen/immunology , Spleen/pathology , Viral Load , Viral Proteins/genetics , Virus Latency , Virus ReplicationABSTRACT
Guanine deaminase is a metabolic enzyme, found in all forms of life, which catalyzes the conversion of guanine to xanthine. Despite the availability of several crystal structures, the molecular determinants of substrate orientation and mechanism remain to be elucidated for the amidohydrolase family of guanine deaminase enzymes. Here, we report the crystal structures of Escherichia coli and Saccharomyces cerevisiae guanine deaminase enzymes (EcGuaD and Gud1, respectively), both members of the amidohydrolase superfamily. EcGuaD and Gud1 retain the overall TIM barrel tertiary structure conserved among amidohydrolase enzymes. Both proteins also possess a single zinc cation with trigonal bipyrimidal coordination geometry within their active sites. We also determined a liganded structure of Gud1 bound to the product, xanthine. Analysis of this structure, along with kinetic data of native and site-directed mutants of EcGuaD, identifies several key residues that are responsible for substrate recognition and catalysis. In addition, after a small library of compounds had been screened, two guanine derivatives, 8-azaguanine and 1-methylguanine, were identified as EcGuaD substrates. Interestingly, both EcGuaD and Gud1 also exhibit secondary ammeline deaminase activity. Overall, this work details key structural features of substrate recognition and catalysis of the amidohydrolase family of guanine deaminase enzymes in support of our long-term goal to engineer these enzymes with altered activity and substrate specificity.
Subject(s)
Amidohydrolases/chemistry , Escherichia coli Proteins/chemistry , Guanine Deaminase/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Amidohydrolases/metabolism , Catalytic Domain/physiology , Escherichia coli Proteins/metabolism , Guanine Deaminase/metabolism , Protein Binding/physiology , Protein Structure, Secondary , Saccharomyces cerevisiae Proteins/metabolism , Substrate Specificity/physiologyABSTRACT
N-Acyl sulfamoyladenosines (acyl-AMS) have been used extensively to inhibit adenylate-forming enzymes that are involved in a wide range of biological processes. These acyl-AMS inhibitors are nonhydrolyzable mimics of the cognate acyl adenylate intermediates that are bound tightly by adenylate-forming enzymes. However, the anionic acyl sulfamate moiety presents a pharmacological liability that may be detrimental to cell permeability and pharmacokinetic profiles. We have previously developed the acyl sulfamate OSB-AMS (1) as a potent inhibitor of the adenylate-forming enzyme MenE, an o-succinylbenzoate-CoA (OSB-CoA) synthetase that is required for bacterial menaquinone biosynthesis. Herein, we report the use of computational docking to develop novel, non-acyl sulfamate inhibitors of MenE. A m-phenyl ether-linked analogue (5) was found to be the most potent inhibitor (IC50 = 8 µM; Kd = 244 nM), and its X-ray co-crystal structure was determined to characterize its binding mode in comparison to the computational prediction. This work provides a framework for the development of potent non-acyl sulfamate inhibitors of other adenylate-forming enzymes in the future.
Subject(s)
Enzyme Inhibitors/pharmacology , Escherichia coli Proteins/antagonists & inhibitors , Succinate-CoA Ligases/antagonists & inhibitors , Vitamin K 2/metabolism , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Kinetics , Microbial Sensitivity Tests , Models, Chemical , Molecular Docking Simulation , Molecular Structure , Mutation , Protein Conformation , Succinate-CoA Ligases/chemistry , Succinate-CoA Ligases/metabolism , Sulfonic Acids/chemistry , Sulfonic Acids/pharmacologyABSTRACT
BACKGROUND: Ribose-phosphate pyrophosphokinase (EC 2.7.6.1) is an enzyme that catalyzes the ATP-dependent conversion of ribose-5-phosphate to phosphoribosyl pyrophosphate. The reaction product is a key precursor for the biosynthesis of purine and pyrimidine nucleotides. RESULTS: We report the 2.2 Å crystal structure of the E. coli ribose-phosphate pyrophosphobinase (EcKPRS). The protein has two type I phosphoribosyltransferase folds, related by 2-fold pseudosymmetry. The propeller-shaped homohexameric structure of KPRS is composed of a trimer of dimers, with the C-terminal domains forming the dimeric blades of the propeller and the N-terminal domains forming the hexameric core. The key, conserved active site residues are well-defined in the structure and positioned appropriately to bind substrates, adenosine monophosphate and ribose-5-phosphate. The allosteric site is also relatively well conserved but, in the EcKPRS structure, several residues from a flexible loop occupy the site where the allosteric modulator, adenosine diphosphate, is predicted to bind. The presence of the loop in the allosteric site may be an additional level of regulation, whereby low affinity molecules are precluded from binding. CONCLUSIONS: Overall, this study details key structural features of an enzyme that catalyzes a critical step in nucleotide metabolism. This work provides a framework for future studies of this important protein and, as nucleotides are critical for viability, may serve as a foundation for the development of novel anti-bacterial drugs.
Subject(s)
Escherichia coli/enzymology , Ribose-Phosphate Pyrophosphokinase/chemistry , Ribose-Phosphate Pyrophosphokinase/metabolism , Adenosine Diphosphate/pharmacology , Allosteric Site , Crystallography, X-Ray , Escherichia coli/chemistry , Escherichia coli Proteins/chemistry , Models, Molecular , Protein Binding , Protein Conformation , Protein Folding , Protein MultimerizationABSTRACT
The light, oxygen, voltage (LOV) domain proteins are blue light photoreceptors that utilize a noncovalently bound flavin mononucleotide (FMN) cofactor as the chromophore. The modular nature of these proteins has led to their wide adoption in the emerging fields of optogenetics and optobiology, where the LOV domain has been fused to a variety of output domains leading to novel light-controlled applications. In this work, we extend our studies of the subpicosecond to several hundred microsecond transient infrared spectroscopy of the isolated LOV domain AsLOV2 to three full-length photoreceptors in which the LOV domain is fused to an output domain: the LOV-STAS protein, YtvA, the LOV-HTH transcription factor, EL222, and the LOV-histidine kinase, LovK. Despite differences in tertiary structure, the overall pathway leading to cysteine adduct formation from the FMN triplet state is highly conserved, although there are slight variations in rate. However, significant differences are observed in the vibrational spectra and kinetics after adduct formation, which are directly linked to the specific output function of the LOV domain. While the rate of adduct formation varies by only 3.6-fold among the proteins, the subsequent large-scale structural changes in the full-length LOV photoreceptors occur over the micro- to submillisecond time scales and vary by orders of magnitude depending on the different output function of each LOV domain.
Subject(s)
Photoreceptors, Microbial/radiation effects , Photoreceptors, Plant/radiation effects , Spectroscopy, Fourier Transform Infrared/methods , Binding Sites , Crystallography, X-Ray , Cysteine/chemistry , Flavin Mononucleotide/chemistry , Hydrogen Bonding , Models, Molecular , Photobleaching , Photochemistry , Photoreceptors, Microbial/chemistry , Photoreceptors, Plant/chemistry , Protein Conformation , Protein Domains , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/radiation effects , Subtraction TechniqueABSTRACT
The interactions between pairs of cells and within multicellular assemblies are critical to many biological processes such as intercellular communication, tissue and organ formation, immunological reactions, and cancer metastasis. The ability to precisely control the position of cells relative to one another and within larger cellular assemblies will enable the investigation and characterization of phenomena not currently accessible by conventional in vitro methods. We present a versatile surface acoustic wave technique that is capable of controlling the intercellular distance and spatial arrangement of cells with micrometer level resolution. This technique is, to our knowledge, among the first of its kind to marry high precision and high throughput into a single extremely versatile and wholly biocompatible technology. We demonstrated the capabilities of the system to precisely control intercellular distance, assemble cells with defined geometries, maintain cellular assemblies in suspension, and translate these suspended assemblies to adherent states, all in a contactless, biocompatible manner. As an example of the power of this system, this technology was used to quantitatively investigate the gap junctional intercellular communication in several homotypic and heterotypic populations by visualizing the transfer of fluorescent dye between cells.
Subject(s)
Cell Communication , Endothelial Cells/cytology , Sound , Cell Adhesion , Coloring Agents/metabolism , Endothelial Cells/metabolism , Gap Junctions/metabolism , HEK293 Cells , Humans , Surface PropertiesABSTRACT
The suppressor of T cell signaling (Sts) proteins, Sts-1 and Sts-2, are homologous phosphatases that negatively regulate signaling pathways downstream of the T cell receptor. Functional inactivation of Sts-1 and Sts-2 in a murine model leads to resistance to systemic infection by the opportunistic pathogen, Candida albicans. This suggests that modulation of the host immune response by inhibiting Sts function may be a viable strategy for treating these deadly fungal pathogen infections. To better understand the molecular determinants of function and structure, we characterized the structure and steady-state kinetics of the histidine phosphatase domains of human Sts-1 (Sts-1HP) and Sts-2 (Sts-2HP). We determined the X-ray crystal structures of unliganded Sts-1HP and Sts-1HP in complex with sulfate to 2.5 and 1.9 Å, respectively, and the structure of Sts-2HP with sulfate to 2.4 Å. The steady-state kinetic analysis shows, as expected, that Sts-1HP has a phosphatase activity significantly higher than that of Sts-2HP and that the human and mouse proteins behave similarly. In addition, comparison of the phosphatase activity of full-length Sts-1 protein to Sts-1HP reveals similar kinetics, indicating that Sts-1HP is a functional surrogate for the native protein. We also tested known phosphatase inhibitors and determined that the SHP-1 inhibitor, PHPS1, is a potent inhibitor of Sts-1 (Ki = 1.05 ± 0.15 µM). Finally, we demonstrated that human Sts-1 has robust phosphatase activity against the substrate, Zap-70, in a cell-based assay. Collectively, these data suggest that the human Sts proteins are druggable targets and provide a structural basis for future drug development efforts.
Subject(s)
Carrier Proteins/chemistry , Phosphoric Monoester Hydrolases/metabolism , Protein Tyrosine Phosphatases/chemistry , Catalytic Domain , Cloning, Molecular , Humans , Membrane Proteins , Models, Molecular , Phosphoric Monoester Hydrolases/chemistry , Protein Conformation , Protein DomainsABSTRACT
Campylobacter jejuni is the most common bacterial cause of gastroenteritis and a major contributor to infant mortality in the developing world. The increasing incidence of antibiotic-resistant C. jejuni only adds to the urgency to develop effective therapies. Because of the essential role that polyamines play, particularly in protection from oxidative stress, enzymes involved in the biosynthesis of these metabolites are emerging as promising antibiotic targets. The recent description of an alternative pathway for polyamine synthesis, distinct from that in human cells, in C. jejuni suggests this pathway could be a target for novel therapies. To that end, we determined X-ray crystal structures of C. jejuni agmatine deiminase (CjADI) and demonstrated that loss of CjADI function contributes to antibiotic sensitivity, likely because of polyamine starvation. The structures provide details of key molecular features of the active site of this protein. Comparison of the unliganded structure (2.1 Å resolution) to that of the CjADI-agmatine complex (2.5 Å) reveals significant structural rearrangements that occur upon substrate binding. The shift of two helical regions of the protein and a large conformational change in a loop near the active site generate a narrow binding pocket around the bound substrate. This change optimally positions the substrate for catalysis. In addition, kinetic analysis of this enzyme demonstrates that CjADI is an iminohydrolase that effectively deiminates agmatine. Our data suggest that C. jejuni agmatine deiminase is a potentially important target for combatting antibiotic resistance, and these results provide a valuable framework for guiding future drug development.
Subject(s)
Campylobacter jejuni/enzymology , Drug Resistance, Bacterial/drug effects , Hydrolases/antagonists & inhibitors , Aminoglycosides/pharmacology , Campylobacter jejuni/drug effects , Catalytic Domain , Crystallography, X-Ray , Hydrolases/chemistry , Hydrolases/genetics , Hydrolases/metabolism , Kinetics , Protein ConformationABSTRACT
The recent discovery of several forms of higher order protein structures in cells has shifted the paradigm of how we think about protein organization and metabolic regulation. These dynamic and controllable protein assemblies, which are composed of dozens or hundreds of copies of an enzyme or related enzymes, have emerged as important players in myriad cellular processes. We are only beginning to appreciate the breadth of function of these types of macromolecular assemblies. These higher order structures, which can be assembled in response to varied cellular stimuli including changing metabolite concentrations or signaling cascades, give the cell the capacity to modulate levels of biomolecules both temporally and spatially. This provides an added level of control with distinct kinetics and unique features that can be harnessed as a subtle, yet powerful regulatory mechanism. Due, in large part, to advances in structural methods, such as crystallography and cryo-electron microscopy, and the advent of super-resolution microscopy techniques, a rapidly increasing number of these higher order structures are being identified and characterized. In this review, we detail what is known about the structure, function and control mechanisms of these mesoscale protein assemblies, with a particular focus on those involved in purine and pyrimidine metabolism. These structures have important implications both for our understanding of fundamental cellular processes and as fertile ground for new targets for drug discovery and development.
Subject(s)
Purines/metabolism , Pyrimidines/metabolism , Animals , Carbon-Nitrogen Ligases/metabolism , Cryoelectron Microscopy , Humans , Macromolecular SubstancesABSTRACT
The flavin chromophore in blue-light-using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppABLUF by the introduction of fluorotyrosine (F-Tyr) analogues that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark- to light-adapted form) photoreaction was observed, the change in Y21 pKa led to a 4000-fold increase in the rate of dark-state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppABLUF, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton-coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppABLUF, despite their sharing highly conserved FAD binding architectures.
Subject(s)
Bacterial Proteins/metabolism , Bacterial Proteins/radiation effects , Flavoproteins/metabolism , Flavoproteins/radiation effects , Fluorine/metabolism , Light , Photoreceptors, Microbial/metabolism , Photoreceptors, Microbial/radiation effects , Tyrosine/metabolism , Binding Sites , Color , Electron Transport , Flavin-Adenine Dinucleotide/metabolism , Hydrogen Bonding , Hydrogen-Ion Concentration , Protein Domains , Protons , Synechocystis/chemistryABSTRACT
Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated that purinosome assembly responds to purine levels in culture medium. Purine-depleted medium or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) treatment stimulates the purinosome assembly in HeLa cells. Here, several metabolomic technologies were applied to quantify the static cellular levels of purine nucleotides and measure the de novo biosynthesis rate of IMP, AMP, and GMP. Direct comparison of purinosome-rich cells (cultured in purine-depleted medium) and normal cells showed a 3-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP and ATP/ADP for both. In addition, a higher level of IMP was also observed in HeLa cells treated with DMAT. Furthermore, increases in the de novo IMP/AMP/GMP biosynthetic flux rate under purine-depleted condition were observed. The synthetic enzymes, adenylosuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH), downstream of IMP were also shown to be part of the purinosome. Collectively, these results provide further evidence that purinosome assembly is directly related to activated de novo purine biosynthesis, consistent with the functionality of the purinosome.
Subject(s)
Metabolomics/methods , Multienzyme Complexes/metabolism , Purine Nucleotides/metabolism , Purines/metabolism , Adenylosuccinate Synthase/analysis , Adenylosuccinate Synthase/metabolism , Benzimidazoles/metabolism , Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/analysis , Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/metabolism , HeLa Cells , Humans , IMP Dehydrogenase/analysis , IMP Dehydrogenase/metabolism , Magnetic Resonance Spectroscopy , Multienzyme Complexes/analysis , Purine Nucleotides/analysis , Purines/analysis , Spectrometry, Mass, Electrospray IonizationABSTRACT
The de novo biosynthesis of purines is carried out by a highly conserved metabolic pathway that includes several validated targets for anticancer, immunosuppressant, and anti-inflammatory chemotherapeutics. The six enzymes in humans that catalyze the 10 chemical steps from phosphoribosylpyrophosphate to inosine monophosphate were recently shown to associate into a dynamic multiprotein complex called the purinosome. Here, we demonstrate that heat shock protein 90 (Hsp90), heat shock protein 70 (Hsp70), and several cochaperones functionally colocalize with this protein complex. Knockdown of expression levels of the identified cochaperones leads to disruption of purinosomes. In addition, small molecule inhibitors of Hsp90 and Hsp70 reversibly disrupt purinosomes and are shown to have a synergistic effect with methotrexate, an anticancer agent that targets purine biosynthesis. These data implicate the Hsp90/Hsp70 chaperone machinery in the assembly of the purinosome and provide a strategy for the development of improved anticancer therapies that disrupt purine biosynthesis.
Subject(s)
Biosynthetic Pathways/physiology , HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Multiprotein Complexes/metabolism , Purines/biosynthesis , Formazans , HeLa Cells , Humans , Immunoprecipitation , Luciferases , Methotrexate , Molecular Structure , Tetrazolium SaltsABSTRACT
With the rapidly growing wealth of genomic data, experimental inquiries on the functional significance of important divergence sites in protein evolution are becoming more accessible. Here we trace the evolution of dihydrofolate reductase (DHFR) and identify multiple key divergence sites among 233 species between humans and bacteria. We connect these sites, experimentally and computationally, to changes in the enzyme's binding properties and catalytic efficiency. One of the identified evolutionarily important sites is the N23PP modification (â¼mid-Devonian, 415-385 Mya), which alters the conformational states of the active site loop in Escherichia coli dihydrofolate reductase and negatively impacts catalysis. This enzyme activity was restored with the inclusion of an evolutionarily significant lid domain (G51PEKN in E. coli enzyme; â¼2.4 Gya). Guided by this evolutionary genomic analysis, we generated a human-like E. coli dihydrofolate reductase variant through three simple mutations despite only 26% sequence identity between native human and E. coli DHFRs. Molecular dynamics simulations indicate that the overall conformational motions of the protein within a common scaffold are retained throughout evolution, although subtle changes to the equilibrium conformational sampling altered the free energy barrier of the enzymatic reaction in some cases. The data presented here provide a glimpse into the evolutionary trajectory of functional DHFR through its protein sequence space that lead to the diverged binding and catalytic properties of the E. coli and human enzymes.
Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli/genetics , Evolution, Molecular , Phylogeny , Tetrahydrofolate Dehydrogenase/genetics , Amino Acid Sequence , Animals , Enzyme Activation/physiology , Escherichia coli/enzymology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Fishes , Humans , Mammals , Molecular Sequence Data , Mutagenesis/physiology , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Sea Urchins , Species Specificity , Structure-Activity Relationship , Tetrahydrofolate Dehydrogenase/chemistry , Tetrahydrofolate Dehydrogenase/metabolism , UrochordataABSTRACT
MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1), which has an IC50 value of ≤25 nM for Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in Staphylococcus aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is â¼1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure-activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future.