RESUMO
The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the bilayer and enclose it in the active site. Here, we show that the positively charged helix-turn-helix motif in the carboxy terminus (CTD) is responsible for interacting with the negatively charged phosphatidylglycerol (PG) bilayer. Super-resolution microscopy of Staphylococcus aureus cells expressing green fluorescent protein fused to OhyA or the CTD sequence shows subcellular localization along the cellular boundary, indicating OhyA is membrane-associated and the CTD sequence is sufficient for membrane recruitment. Using cryo-electron microscopy, we solved the OhyA dimer structure and conducted 3D variability analysis of the reconstructions to assess CTD flexibility. Our surface plasmon resonance experiments corroborated that OhyA binds the PG bilayer with nanomolar affinity and we found the CTD sequence has intrinsic PG binding properties. We determined that the nuclear magnetic resonance structure of a peptide containing the CTD sequence resembles the OhyA crystal structure. We observed intermolecular NOE from PG liposome protons next to the phosphate group to the CTD peptide. The addition of paramagnetic MnCl2 indicated the CTD peptide binds the PG surface but does not insert into the bilayer. Molecular dynamics simulations, supported by site-directed mutagenesis experiments, identify key residues in the helix-turn-helix that drive membrane association. The data show that the OhyA CTD binds the phosphate layer of the PG surface to obtain bilayer-embedded unsaturated fatty acids.
Assuntos
Ácido Oleico , Peptídeos , Staphylococcus aureus , Microscopia Crioeletrônica , Ácidos Graxos Insaturados , Bicamadas Lipídicas/metabolismo , Fosfatos , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genéticaRESUMO
Histone acetyltransferases (HATs) are implicated as both oncogene and nononcogene dependencies in diverse human cancers. Acetyl-CoA-competitive HAT inhibitors have emerged as potential cancer therapeutics and the first clinical trial for this class of drugs is ongoing (NCT04606446). Despite these developments, the potential mechanisms of therapeutic response and evolved drug resistance remain poorly understood. Having discovered that multiple regulators of de novo coenzyme A (CoA) biosynthesis can modulate sensitivity to CBP/p300 HAT inhibition (PANK3, PANK4 and SLC5A6), we determined that elevated acetyl-CoA concentrations can outcompete drug-target engagement to elicit acquired drug resistance. This not only affects structurally diverse CBP/p300 HAT inhibitors, but also agents related to an investigational KAT6A/B HAT inhibitor that is currently in Phase 1 clinical trials. Altogether, this work uncovers CoA metabolism as an unexpected liability of anticancer HAT inhibitors and will therefore buoy future efforts to optimize the efficacy of this new form of targeted therapy.
Assuntos
Histona Acetiltransferases , Neoplasias , Humanos , Histona Acetiltransferases/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilcoenzima A/metabolismo , Ligação ProteicaRESUMO
Sulfonolipids are unusual lipids found in the outer membranes of Gram-negative bacteria in the phylum Bacteroidetes. Sulfonolipid and its deacylated derivative, capnine, are sulfur analogs of ceramide-1-phosphate and sphingosine-1-phosphate, respectively; thus, sulfonolipid biosynthesis is postulated to be similar to the sphingolipid biosynthetic pathway. Here, we identify the first enzyme in sulfonolipid synthesis in Alistipes finegoldii as the product of the alfi_1224 gene, cysteate acyl-acyl carrier protein (ACP) transferase (SulA). We show SulA catalyzes the condensation of acyl-ACP and cysteate (3-sulfo-alanine) to form 3-ketocapnine. Acyl-CoA is a poor substrate. We show SulA has a bound pyridoxal phosphate (PLP) cofactor that undergoes a spectral redshift in the presence of cysteate, consistent with the transition of the lysine-aldimine complex to a substrate-aldimine complex. Furthermore, the SulA crystal structure shows the same prototypical fold found in bacterial serine palmitoyltransferases (Spts), enveloping the PLP cofactor bound to Lys251. We observed the SulA and Spt active sites are identical except for Lys281 in SulA, which is an alanine in Spt. Additionally, SulA(K281A) is catalytically inactive but binds cysteate and forms the external aldimine normally, highlighting the structural role of the Lys281 side chain in walling off the active site from bulk solvent. Finally, the electropositive groove on the protein surface adjacent to the active site entrance provides a landing pad for the electronegative acyl-ACP surface. Taken together, these data identify the substrates, products, and mechanism of SulA, the PLP-dependent condensing enzyme that catalyzes the first step in sulfonolipid synthesis in a gut commensal bacterium.
Assuntos
Bacteroidetes , Ácido Cisteico , Proteína de Transporte de Acila , Alanina/metabolismo , Bacteroidetes/metabolismo , Lipídeos , Fosfato de Piridoxal/metabolismoRESUMO
Staphylococcus aureus is an important pathogen that relies on a variety of mechanisms to evade and counteract the immune system. We show that S. aureus uses oleate hydratase (OhyA) to convert host cis-9 unsaturated fatty acids to their 10-hydroxy derivatives in human serum and at the infection site in a mouse neutropenic thigh model. Wild-type and ΔohyA strains were equally infective in the neutropenic thigh model, but recovery of the ΔohyA strain was 2 orders of magnitude lower in the immunocompetent skin infection model. Despite the lower bacterial abundance at the infection site, the levels of interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), IL-1ß, and tumor necrosis factor alpha (TNF-α) elicited by the ΔohyA strain were as robust as those of either the wild-type or the complemented strain, indicating that the immune system was more highly activated by the ΔohyA strain. Thus, OhyA functions to promote S. aureus virulence. IMPORTANCE The oleate hydratase protein family was discovered in commensal bacteria that utilize host unsaturated fatty acids as the substrates to produce a spectrum of hydroxylated products. These hydroxy fatty acids are thought to act as signaling molecules that suppress the inflammatory response to create a more tolerant environment for the microbiome. S. aureus is a significant human pathogen, and defining the mechanisms used to evade the immune response is critical to understanding pathogenesis. S. aureus expresses an OhyA that produces at least three 10-hydroxy fatty acids from host unsaturated fatty acids at the infection site, and an S. aureus strain lacking the ohyA gene has compromised virulence in an immunocompetent infection model. These data suggest that OhyA plays a role in immune modulation in S. aureus pathogenesis similar to that in commensal bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Ácido Oleico/metabolismo , Staphylococcus aureus/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Ácidos Graxos , Ácidos Graxos Insaturados/metabolismo , Camundongos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Fator de Necrose Tumoral alfa , Virulência , Fatores de Virulência/genéticaRESUMO
Pantothenate kinase (PANK) is a metabolic enzyme that regulates cellular coenzyme A (CoA) levels. There are three human PANK genes, and inactivating mutations in PANK2 lead to pantothenate kinase associated neurodegeneration (PKAN). Here we performed a library screen followed by chemical optimization to produce PZ-2891, an allosteric PANK activator that crosses the blood brain barrier. PZ-2891 occupies the pantothenate pocket and engages the dimer interface to form a PANKâ¢ATPâ¢Mg2+â¢PZ-2891 complex. The binding of PZ-2891 to one protomer locks the opposite protomer in a catalytically active conformation that is refractory to acetyl-CoA inhibition. Oral administration of PZ-2891 increases CoA levels in mouse liver and brain. A knockout mouse model of brain CoA deficiency exhibited weight loss, severe locomotor impairment and early death. Knockout mice on PZ-2891 therapy gain weight, and have improved locomotor activity and life span establishing pantazines as novel therapeutics for the treatment of PKAN.
Assuntos
Neurodegeneração Associada a Pantotenato-Quinase/terapia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Animais , Células Cultivadas , Coenzima A/deficiência , Coenzima A/metabolismo , Modelos Animais de Doenças , Estabilidade Enzimática , Feminino , Ligantes , Magnésio/metabolismo , Masculino , Camundongos Knockout , Neurônios/metabolismo , Especificidade de Órgãos , Neurodegeneração Associada a Pantotenato-Quinase/patologia , Conformação Proteica , Multimerização ProteicaRESUMO
The G protein-coupled receptor Smoothened (Smo) is the signal transducer of the Sonic Hedgehog (Shh) pathway. Smo signals through G protein-dependent and -independent routes, with G protein-independent canonical signaling to Gli effectors requiring Smo accumulation in the primary cilium. The mechanisms controlling Smo activation and trafficking are not yet clear but likely entail small-molecule binding to pockets in its extracellular cysteine-rich domain (CRD) and/or transmembrane bundle. Here, we demonstrate that the cytosolic phospholipase cPLA2α is activated through Gßγ downstream of Smo to release arachidonic acid. Arachidonic acid binds Smo and synergizes with CRD-binding agonists, promoting Smo ciliary trafficking and high-level signaling. Chemical or genetic cPLA2α inhibition dampens Smo signaling to Gli, revealing an unexpected contribution of G protein-dependent signaling to canonical pathway activity. Arachidonic acid displaces the Smo transmembrane domain inhibitor cyclopamine to rescue CRD agonist-induced signaling, suggesting that arachidonic acid may target the transmembrane bundle to allosterically enhance signaling by CRD agonist-bound Smo.
Assuntos
Fosfolipases A2 do Grupo IV/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Receptor Smoothened/metabolismo , Animais , Cílios/genética , Cílios/metabolismo , Ativação Enzimática/genética , Fosfolipases A2 do Grupo IV/genética , Proteínas Hedgehog/genética , Camundongos , Células NIH 3T3 , Transporte Proteico/genética , Receptor Smoothened/genéticaRESUMO
Serious bacterial infections in immunocompromised patients require highly effective antibacterial therapy for cure, and thus, this setting may reveal novel mechanisms by which bacteria circumvent antibiotics in the absence of immune pressure. Here, an infant with leukemia developed vancomycin-resistant Enterococcus faecium (VRE) bacteremia that persisted for 26 days despite appropriate antibiotic therapy. Sequencing of 22 consecutive VRE isolates identified the emergence of a single missense mutation (L152F) in relA, which constitutively activated the stringent response, resulting in elevated baseline levels of the alarmone guanosine tetraphosphate (ppGpp). Although the mutant remained susceptible to both linezolid and daptomycin in clinical MIC testing and during planktonic growth, it demonstrated tolerance to high doses of both antibiotics when growing in a biofilm. This biofilm-specific gain in resistance was reflected in the broad shift in transcript levels caused by the mutation. Only an experimental biofilm-targeting ClpP-activating antibiotic was able to kill the mutant strain in an established biofilm. The relA mutation was associated with a fitness trade-off, forming smaller and less-well-populated biofilms on biological surfaces. We conclude that clinically relevant relA mutations can emerge during prolonged VRE infection, causing baseline activation of the stringent response, subsequent antibiotic tolerance, and delayed eradication in an immunocompromised state. IMPORTANCE: The increasing prevalence of antibiotic-resistant bacterial pathogens is a major challenge currently facing the medical community. Such pathogens are of particular importance in immunocompromised patients as these individuals may favor emergence of novel resistance determinants due to lack of innate immune defenses and intensive antibiotic exposure. During the course of chemotherapy, a patient developed prolonged bacteremia with vancomycin-resistant Enterococcus faecium that failed to clear despite multiple front-line antibiotics. The consecutive bloodstream isolates were sequenced, and a single missense mutation identified in the relA gene, the mediator of the stringent response. Strains harboring the mutation had elevated baseline levels of the alarmone and displayed heightened resistance to the bactericidal activity of multiple antibiotics, particularly in a biofilm. Using a new class of compounds that modulate ClpP activity, the biofilms were successfully eradicated. These data represent the first clinical emergence of mutations in the stringent response in vancomycin-resistant entereococci.
Assuntos
Antibacterianos/farmacologia , Tolerância a Medicamentos , Enterococcus faecium/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/microbiologia , Hospedeiro Imunocomprometido , Ligases/genética , Proteínas Mutantes/genética , Bacteriemia/microbiologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Enterococcus faecium/genética , Enterococcus faecium/isolamento & purificação , Enterococcus faecium/fisiologia , Feminino , Guanosina Tetrafosfato/metabolismo , Humanos , Lactente , Testes de Sensibilidade Microbiana , Mutação de Sentido Incorreto , Análise de Sequência de DNA , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/genética , Enterococos Resistentes à Vancomicina/isolamento & purificaçãoRESUMO
Modulation of T cell proliferation and function by immunoregulatory myeloid cells are an essential means of preventing self-reactivity and restoring tissue homeostasis. Consumption of amino acids such as arginine and tryptophan by immunoregulatory macrophages is one pathway that suppresses local T cell proliferation. Using a reduced complexity in vitro macrophage-T cell co-culture system, we show that macrophage arginase-1 is the only factor required by M2 macrophages to block T cells in G1, and this effect is mediated by l-arginine elimination rather than metabolite generation. Tracking how T cells adjust their metabolism when deprived of arginine revealed the significance of macrophage-mediated arginine deprivation to T cells. We found mTORC1 activity was unaffected in the initial G1 block. After 2 days of arginine deprivation, mTORC1 activity declined paralleling a selective down-regulation of SREBP target gene expression, whereas mRNAs involved in glycolysis, gluconeogenesis, and T cell activation were unaffected. Cell cycle arrest was reversible at any point by exogenous arginine, suggesting starved T cells remain poised awaiting nutrients. Arginine deprivation-induced cell cycle arrest was mediated in part by Rictor/mTORC2, providing evidence that this nutrient recognition pathway is a central component of how T cells measure environmental arginine.
Assuntos
Arginina/metabolismo , Proteínas de Transporte/metabolismo , Pontos de Checagem do Ciclo Celular/imunologia , Proliferação de Células , Complexos Multiproteicos/metabolismo , Células Mieloides/imunologia , Linfócitos T/imunologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Tolerância Imunológica , Terapia de Imunossupressão , Ativação Linfocitária , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Linfócitos T/metabolismoRESUMO
This study examines the alteration in Staphylococcus aureus gene expression following treatment with the type 2 fatty acid synthesis inhibitor AFN-1252. An Affymetrix array study showed that AFN-1252 rapidly increased the expression of fatty acid synthetic genes and repressed the expression of virulence genes controlled by the SaeRS 2-component regulator in exponentially growing cells. AFN-1252 did not alter virulence mRNA levels in a saeR deletion strain or in strain Newman expressing a constitutively active SaeS kinase. AFN-1252 caused a more pronounced increase in fabH mRNA levels in cells entering stationary phase, whereas the depression of virulence factor transcription was attenuated. The effect of AFN-1252 on gene expression in vivo was determined using a mouse subcutaneous granuloma infection model. AFN-1252 was therapeutically effective, and the exposure (area under the concentration-time curve from 0 to 48 h [AUC(0-48)]) of AFN-1252 in the pouch fluid was comparable to the plasma levels in orally dosed animals. The inhibition of fatty acid biosynthesis by AFN-1252 in the infected pouches was signified by the substantial and sustained increase in fabH mRNA levels in pouch-associated bacteria, whereas depression of virulence factor mRNA levels in the AFN-1252-treated pouch bacteria was not as evident as it was in exponentially growing cells in vitro. The trends in fabH and virulence factor gene expression in the animal were similar to those in slower-growing bacteria in vitro. These data indicate that the effects of AFN-1252 on virulence factor gene expression depend on the physiological state of the bacteria.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Benzofuranos/farmacologia , Enoil-(Proteína de Transporte de Acila) Redutase (NADPH, B-Específica)/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Pironas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Antibacterianos/farmacocinética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzofuranos/farmacocinética , Enoil-(Proteína de Transporte de Acila) Redutase (NADPH, B-Específica)/genética , Enoil-(Proteína de Transporte de Acila) Redutase (NADPH, B-Específica)/metabolismo , Inibidores Enzimáticos/farmacocinética , Ácidos Graxos/metabolismo , Perfilação da Expressão Gênica , Granuloma/tratamento farmacológico , Granuloma/microbiologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Pironas/farmacocinética , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Nitric oxide (NO) defends against intracellular pathogens, but its synthesis must be regulated due to cell and tissue toxicity. During infection, macrophages import extracellular arginine to synthesize NO, generating the byproduct citrulline. Accumulated intracellular citrulline is thought to fuel arginine synthesis catalyzed by argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl), which would lead to abundant NO production. Instead, we find that citrulline is exported from macrophages during early stages of NO production with <2% retained for recycling via the Ass1-Asl pathway. Later, extracellular arginine is depleted, and Ass1 expression allows macrophages to synthesize arginine from imported citrulline to sustain NO output. Ass1-deficient macrophages fail to salvage citrulline in arginine-scarce conditions, leading to their inability to control mycobacteria infection. Thus, extracellular arginine fuels rapid NO production in activated macrophages, and citrulline recycling via Ass1 and Asl is a fail-safe system that sustains optimum NO production.
Assuntos
Argininossuccinato Sintase/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Mycobacterium bovis/imunologia , Óxido Nítrico/metabolismo , Animais , Arginina/metabolismo , Argininossuccinato Sintase/genética , Células Cultivadas , Citrulina/metabolismo , CamundongosRESUMO
Isocitrate dehydrogenase (IDH) is a reversible enzyme that catalyzes the NADP(+)-dependent oxidative decarboxylation of isocitrate (ICT) to α-ketoglutarate (αKG) and the NADPH/CO(2)-dependent reductive carboxylation of αKG to ICT. Reductive carboxylation by IDH1 was potently inhibited by NADP(+) and, to a lesser extent, by ICT. IDH1 and IDH2 with cancer-associated mutations at the active site arginines were unable to carry out the reductive carboxylation of αKG. These mutants were also defective in ICT decarboxylation and converted αKG to 2-hydroxyglutarate using NADPH. These mutant proteins were thus defective in both of the normal reactions of IDH. Biochemical analysis of heterodimers between wild-type and mutant IDH1 subunits showed that the mutant subunit did not inactivate reductive carboxylation by the wild-type subunit. Cells expressing the mutant IDH are thus deficient in their capacity for reductive carboxylation and may be compromised in their ability to produce acetyl-CoA under hypoxia or when mitochondrial function is otherwise impaired.
Assuntos
Isocitrato Desidrogenase , Proteínas Mitocondriais , NADP/metabolismo , Proteínas de Neoplasias , Neoplasias , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Hipóxia Celular/genética , Linhagem Celular Tumoral , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , NADP/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Neoplasias/genética , OxirreduçãoRESUMO
Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin-like protein (UBL) Atg8 during autophagy. Here we report that the unique N-terminal domain of Atg7 (Atg7(NTD)) recruits a unique "flexible region" from Atg3 (Atg3(FR)). The structure of an Atg7(NTD)-Atg3(FR) complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, important for Atg8 conjugation. We also report the structure of the homodimeric Atg7 C-terminal domain, which is homologous to canonical E1s and bacterial antecedents. The structures, SAXS, and crosslinking data allow modeling of a full-length, dimeric (Atg7~Atg8-Atg3)(2) complex. The model and biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic cysteine of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a distinctive E1~UBL-E2 architecture for enzymes mediating autophagy.
Assuntos
Autofagia , Proteínas de Transporte/química , Fibroblastos/enzimologia , Proteínas Associadas aos Microtúbulos/química , Enzimas de Conjugação de Ubiquitina/química , Sequência de Aminoácidos , Animais , Proteína 7 Relacionada à Autofagia , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Cristalografia por Raios X , Fibroblastos/patologia , Interações Hidrofóbicas e Hidrofílicas , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos , Mutação , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Transfecção , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Pantothenate kinase (PanK) catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. PanK3 is stringently regulated by acetyl-CoA and uses an ordered kinetic mechanism with ATP as the leading substrate. Biochemical analysis of site-directed mutants indicates that pantothenate binds in a tunnel adjacent to the active site that is occupied by the pantothenate moiety of the acetyl-CoA regulator in the PanK3acetyl-CoA binary complex. A high-throughput screen for PanK3 inhibitors and activators was applied to a bioactive compound library. Thiazolidinediones, sulfonylureas and steroids were inhibitors, and fatty acyl-amides and tamoxifen were activators. The PanK3 activators and inhibitors either stimulated or repressed CoA biosynthesis in HepG2/C3A cells. The flexible allosteric acetyl-CoA regulatory domain of PanK3 also binds the substrates, pantothenate and pantetheine, and small molecule inhibitors and activators to modulate PanK3 activity.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Acetilcoenzima A/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação , Coenzima A/biossíntese , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Células Hep G2 , Ensaios de Triagem em Larga Escala , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato , Compostos de Sulfonilureia/metabolismo , Tiazolidinedionas/metabolismoRESUMO
DgkB is a soluble diacylglycerol (DAG) kinase that is essential for membrane lipid homeostasis in many Gram-positive pathogens. Anionic phospholipids, like phosphatidylglycerol (PtdGro), were required for DgkB to recognize diacylglycerol embedded in a phospholipid bilayer. An activity-independent vesicle binding assay was used to determine the role of specific residues in DgkB-PtdGro interactions. Lys15 and Lys165 were required for DgkB to dock with PtdGro vesicles and flank the entrance to the DgkB active site. Mg2+ was required for vesicle binding. The compromised vesicle binding by mutants in the key asparate residues forming the structural Mg2+-aspartate-water network within the substrate binding domain revealed that interfacial binding of DgkB required a Mg2+-dependent conformational change. DgkB interaction with phospholipid vesicles was not influenced by the presence of ATP, but anionic vesicles decreased the Km of the enzyme for ATP. Arg100 and Lys15 are two surface residues in the ATP binding domain that were necessary for high affinity ATP binding. The key residues responsible for the structural Mg2+ binding site, the conformational changes that increase ATP affinity, and interfacial recognition of anionic phospholipids were identical in DgkB and the mammalian diacylglycerol kinase catalytic cores. This sequence conservation suggests that the mammalian enzymes also require a structural divalent cation and surface positively charged residues to bind phospholipid bilayers and trigger conformational changes that accelerate catalysis.
Assuntos
Proteínas de Bactérias/química , Membrana Celular/química , Diacilglicerol Quinase/química , Bicamadas Lipídicas/química , Fosfatidilgliceróis/química , Staphylococcus aureus/enzimologia , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico/fisiologia , Membrana Celular/genética , Membrana Celular/metabolismo , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Bicamadas Lipídicas/metabolismo , Magnésio/química , Magnésio/metabolismo , Mutação , Fosfatidilgliceróis/genética , Fosfatidilgliceróis/metabolismo , Estrutura Terciária de Proteína/fisiologia , Staphylococcus aureus/genéticaRESUMO
2,4-Dihydroxyquinoline (DHQ) is an abundant extracellular metabolite of the opportunistic pathogen Pseudomonas aeruginosa that is secreted into growth medium in stationary phase to concentrations comparable with those of the Pseudomonas quinolone signal. Using a combination of biochemical and genetic approaches, we show that PqsD, a condensing enzyme in the pqs operon that is essential for Pseudomonas quinolone signal synthesis, accounts for DHQ formation in vivo. First, the anthraniloyl moiety is transferred to the active-site Cys of PqsD to form an anthraniloyl-PqsD intermediate, which then condenses with either malonyl-CoA or malonyl-acyl carrier protein to produce 3-(2-aminophenyl)-3-oxopropanoyl-CoA. This short-lived intermediate undergoes an intramolecular rearrangement to form DHQ. DHQ was produced by Escherichia coli coexpressing PqsA and PqsD, illustrating that these two proteins are the only factors necessary for DHQ synthesis. Thus, PqsD is responsible for the production of DHQ in P. aeruginosa.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/fisiologia , Proteínas de Bactérias/química , Pseudomonas aeruginosa/metabolismo , Quinolinas/química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Domínio Catalítico , Cisteína/química , Fibrose Cística/microbiologia , Cinética , Camundongos , Modelos Químicos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
Soluble diacylglycerol (DAG) kinases function as regulators of diacylglycerol metabolism in cell signaling and intermediary metabolism. We report the structure of a DAG kinase, DgkB from Staphylococcus aureus, both as the free enzyme and in complex with ADP. The molecule is a tight homodimer, and each monomer comprises two domains with the catalytic center located within the interdomain cleft. Two distinctive features of DkgB are a structural Mg2+ site and an associated Asp*water*Mg2+ network that extends toward the active site locale. Site-directed mutagenesis revealed that these features play important roles in the catalytic mechanism. The key active site residues and the components of the Asp*water*Mg2+ network are conserved in the catalytic cores of the mammalian signaling DAG kinases, indicating that these enzymes use the same mechanism and have similar structures as DgkB.
Assuntos
Proteínas de Bactérias/química , Diacilglicerol Quinase/química , Staphylococcus aureus/enzimologia , Difosfato de Adenosina/química , Sequência de Aminoácidos , Ácido Aspártico/química , Proteínas de Bactérias/genética , Sítios de Ligação , Catálise , Cátions Bivalentes/química , Diacilglicerol Quinase/genética , Dimerização , Humanos , Magnésio/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , SolubilidadeRESUMO
The most widely distributed biosynthetic pathway to initiate phosphatidic acid formation in bacterial membrane phospholipid biosynthesis involves the conversion of acyl-acyl carrier protein to acylphosphate by PlsX and the transfer of the acyl group from acylphosphate to glycerol 3-phosphate by an integral membrane protein, PlsY. The membrane topology of Streptococcus pneumoniae PlsY was determined using the substituted cysteine accessibility method. PlsY has five membrane-spanning segments with the amino terminus and two short loops located on the external face of the membrane. Each of the three larger cytoplasmic domains contains a highly conserved sequence motif. Site-directed mutagenesis revealed that each conserved domain was critical for PlsY catalysis. Motif 1 had an essential serine and arginine residue. Motif 2 had the characteristics of a phosphate-binding loop. Mutations of the conserved glycines in motif 2 to alanines resulted in a Km defect for glycerol 3-phosphate binding leading to the conclusion that this motif corresponded to the glycerol 3-phosphate binding site. Motif 3 contained a conserved histidine and asparagine that were important for activity and a glutamate that was critical to the structural integrity of PlsY. PlsY was noncompetitively inhibited by palmitoyl-CoA. These data define the membrane architecture and the critical active site residues in the PlsY family of bacterial acyltransferases.
Assuntos
Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Streptococcus pneumoniae/enzimologia , Motivos de Aminoácidos , Sítios de Ligação , Membrana Celular/metabolismo , Sequência Conservada , Inibidores Enzimáticos/farmacologia , Glicerol-3-Fosfato O-Aciltransferase/antagonistas & inibidores , Glicerol-3-Fosfato O-Aciltransferase/genética , Cinética , Dados de Sequência Molecular , Mutação/genética , Streptococcus pneumoniae/genéticaRESUMO
Three distinct isoforms of pantothenate kinase (CoaA) in bacteria catalyze the first step in coenzyme A biosynthesis. The structures of the type II (Staphylococcus aureus, SaCoaA) and type III (Pseudomonas aeruginosa, PaCoaA) enzymes reveal that they assemble nearly identical subunits with actin-like folds into dimers that exhibit distinct biochemical properties. PaCoaA has a fully enclosed pantothenate binding pocket and requires a monovalent cation to weakly bind ATP in an open cavity that does not interact with the adenine nucleotide. Pantothenate binds to an open pocket in SaCoaA that strongly binds ATP by using a classical P loop architecture coupled with specific interactions with the adenine moiety. The PaCoaA*Pan binary complex explains the resistance of bacteria possessing this isoform to the pantothenamide antibiotics, and the similarity between SaCoaA and human pantothenate kinase 2 explains the molecular basis for the development of the neurodegenerative phenotype in three mutations in the human protein.
Assuntos
Modelos Moleculares , Fosfotransferases (Aceptor do Grupo Álcool)/química , Pseudomonas aeruginosa/química , Staphylococcus aureus/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Dimerização , Humanos , Isoenzimas/química , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Alinhamento de Sequência , Especificidade da EspécieRESUMO
beta-Ketoacyl-ACP synthases catalyze the condensation steps in fatty acid and polyketide synthesis and are targets for the development of novel antibiotics and anti-obesity and anti-cancer agents. The roles of the active site residues in Streptococcus pneumoniae FabF (beta-ketoacyl-ACP synthase II; SpFabF) were investigated to clarify the mechanism for this enzyme superfamily. The nucleophilic cysteine of the active site triad was required for acyl-enzyme formation and the overall condensation activity. The two active site histidines in the elongation condensing enzyme have different electronic states and functions. His337 is essential for condensation activity, and its protonated Nepsilon stabilizes the negative charge developed on the malonyl thioester carbonyl in the transition state. The Nepsilon of His303 accelerated catalysis by deprotonating a structured active site water for nucleophilic attack on the C3 of malonate, releasing bicarbonate. Lys332 controls the electronic state of His303 and also plays a critical role in the positioning of His337. Phe396 functions as a gatekeeper that controls the order of substrate addition. These data assign specific roles for each active site residue and lead to a revised general mechanism for this important class of enzymes.
Assuntos
Fenilalanina/química , Streptococcus pneumoniae/metabolismo , Água/química , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Ácido Graxo Sintases/metabolismo , Histidina/química , Lisina/química , Malonatos/química , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Pantothenate kinase catalyzes a key regulatory step in coenzyme A biosynthesis, and there are four mammalian genes that encode isoforms of this enzyme. Pantothenate kinase isoform PanK3 is highly related to the previously characterized PanK1beta isoform (79% identical, 91% similar), and these two almost identical proteins are expressed most highly in the same tissues. PanK1beta and PanK3 had very similar molecular sizes, oligomeric form, cytoplasmic cellular location, and kinetic constants for ATP and pantothenate. However, these two PanK isoforms possessed distinct regulatory properties. PanK3 was significantly more sensitive to feedback regulation by acetyl-CoA (IC50 = 1 microm) than PanK1beta (IC50 = 10 microm), and PanK3 was stringently regulated by long-chain acyl-CoA (IC50 = 2 microm), whereas PanK1beta was not. Domain swapping experiments localized the difference in the two proteins to a 48-amino-acid domain, where they are the most divergent. Consistent with these more stringent regulatory properties, metabolic labeling experiments showed that coenzyme A (CoA) levels in cells overexpressing PanK3 were lower than in cells overexpressing an equivalent amount of PanK1beta. Thus, the distinct regulatory properties exhibited by the family of the pantothenate kinases allowed the rate of CoA biosynthesis to be controlled by regulatory signals from CoA thioesters involved in different branches of intermediary metabolism.