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1.
J Cell Mol Med ; 28(3): e18073, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38063077

RESUMO

Diabetic kidney disease (DKD) can lead to accumulation of glucose upstream metabolites due to dysfunctional glycolysis. But the effects of accumulated glycolysis metabolites on podocytes in DKD remain unknown. The present study examined the effect of dihydroxyacetone phosphate (DHAP) on high glucose induced podocyte pyroptosis. By metabolomics, levels of DHAP, GAP, glucose-6-phosphate and fructose 1, 6-bisphosphate were significantly increased in glomeruli of db/db mice. Furthermore, the expression of LDHA and PKM2 were decreased. mRNA sequencing showed upregulation of pyroptosis-related genes (Nlrp3, Casp1, etc.). Targeted metabolomics demonstrated higher level of DHAP in HG-treated podocytes. In vitro, ALDOB expression in HG-treated podocytes was significantly increased. siALDOB-transfected podocytes showed less DHAP level, mTORC1 activation, reactive oxygen species (ROS) production, and pyroptosis, while overexpression of ALDOB had opposite effects. Furthermore, GAP had no effect on mTORC1 activation, and mTORC1 inhibitor rapamycin alleviated ROS production and pyroptosis in HG-stimulated podocytes. Our findings demonstrate that DHAP represents a critical metabolic product for pyroptosis in HG-stimulated podocytes through regulation of mTORC1 pathway. In addition, the results provide evidence that podocyte injury in DKD may be treated by reducing DHAP.


Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Podócitos , Camundongos , Animais , Nefropatias Diabéticas/metabolismo , Podócitos/metabolismo , Fosfato de Di-Hidroxiacetona/metabolismo , Fosfato de Di-Hidroxiacetona/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Piroptose , Glucose/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Diabetes Mellitus/metabolismo
2.
Cell Signal ; 99: 110443, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35988808

RESUMO

Recent studies have reported that Angiotensin II (Ang II) contributes to podocyte injury by interfering with metabolism. Glycolysis is essential for podocytes and glycolysis abnormality is associated with glomerular injury in chronic kidney disease (CKD). Glycerol-3-phosphate (G-3-P) biosynthesis is a shunt pathway of glycolysis, in which cytosolic glycerol-3-phosphate dehydrogenase 1 (GPD1) catalyzes dihydroxyacetone phosphate (DHAP) to generate G-3-P in the presence of the NADH. G-3-P is not only a substrate in glycerophospholipids and glyceride synthesis but also can be oxidated by mitochondrial glycerol-3-phosphate dehydrogenase (GPD2) to regenerate DHAP in mitochondria. Since G-3-P biosynthesis links to glycolysis, mitochondrial metabolism and lipid synthesis, we speculate G-3-P biosynthesis abnormality is probably involved in podocyte injury. In this study, we demonstrated that Ang II upregulated GPD1 expression and increased G-3-P and glycerophospholipid syntheses in podocytes. GPD1 knockdown protected podocytes from Ang II-induced lipid accumulation and mitochondrial dysfunction. GPD1 overexpression exacerbated Ang II-induced podocyte injury. In addition, we proved that lipid accumulation and mitochondrial dysfunction were correlated with G-3-P content in podocytes. These results suggest that Ang II upregulates GPD1 and promotes G-3-P biosynthesis in podocytes, which promote lipid accumulation and mitochondrial dysfunction in podocytes.


Assuntos
Podócitos , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Fosfato de Di-Hidroxiacetona/metabolismo , Glicerídeos/metabolismo , Glicerol/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Glicerofosfolipídeos/metabolismo , Glicólise , Lipídeos , NAD/metabolismo , Fosfatos/metabolismo , Podócitos/metabolismo
3.
Biochemistry ; 61(10): 856-867, 2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35502876

RESUMO

The cationic K120 and K204 side chains lie close to the C-2 carbonyl group of substrate dihydroxyacetone phosphate (DHAP) at the active site of glycerol-3-phosphate dehydrogenase (GPDH), and the K120 side chain is also positioned to form a hydrogen bond to the C-1 hydroxyl of DHAP. The kinetic parameters for unactivated and phosphite dianion-activated GPDH-catalyzed reduction of glycolaldehyde and acetaldehyde (AcA) show that the transition state for the former reaction is stabilized by ca 5 kcal/mole by interactions of the C-1 hydroxyl group with the protein catalyst. The K120A and K204A substitutions at wild-type GPDH result in similar decreases in kcat, but Km is only affected by the K120A substitution. These results are consistent with 3 kcal/mol stabilizing interactions between the K120 or K204 side chains and a negative charge at the C-2 oxygen at the transition state for hydride transfer from NADH to DHAP. This stabilization resembles that observed at oxyanion holes for other enzymes. There is no detectable rescue of the K204A variant by ethylammonium cation (EtNH3+), compared with the efficient rescue of the K120A variant. This is consistent with a difference in the accessibility of the variant enzyme active sites to exogenous EtNH3+. The K120A/K204A substitutions cause a (6 × 106)-fold increase in the promiscuity of wild-type hlGPDH for catalysis of the reduction of AcA compared to DHAP. This may reflect conservation of the active site for an ancestral alcohol dehydrogenase, whose relative activity for catalysis of reduction of AcA increases with substitutions that reduce the activity for reduction of the specific substrate DHAP.


Assuntos
Glicerolfosfato Desidrogenase , Catálise , Domínio Catalítico , Fosfato de Di-Hidroxiacetona/química , Glicerolfosfato Desidrogenase/química , Cinética
4.
ACS Chem Biol ; 16(11): 2423-2433, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34609124

RESUMO

Quinolinate synthase, also called NadA, is a [4Fe-4S]-containing enzyme that uses what is probably the oldest pathway to generate quinolinic acid (QA), the universal precursor of the biologically essential cofactor nicotinamide adenine dinucleotide (NAD). Its synthesis comprises the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA), which involves dephosphorylation, isomerization, cyclization, and two dehydration steps. The convergence of the three homologous domains of NadA defines a narrow active site that contains a catalytically essential [4Fe-4S] cluster. A tunnel, which can be opened or closed depending on the nature (or absence) of the bound ligand, connects this cofactor to the protein surface. One outstanding riddle has been the observation that the so far characterized active site is too small to bind IA and DHAP simultaneously. Here, we have used site-directed mutagenesis, X-ray crystallography, functional analyses, and molecular dynamics simulations to propose a condensation mechanism that involves the transient formation of a second active site cavity to which one of the substrates can migrate before this reaction takes place.


Assuntos
Complexos Multienzimáticos/química , Ácido Quinolínico/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Fosfato de Di-Hidroxiacetona/química , Modelos Moleculares , Complexos Multienzimáticos/metabolismo , Conformação Proteica , Especificidade por Substrato
5.
ACS Synth Biol ; 10(9): 2252-2265, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34478281

RESUMO

The field of metabolic engineering has yielded remarkable accomplishments in using cells to produce valuable molecules, and cell-free expression (CFE) systems have the potential to push the field even further. However, CFE systems still face some outstanding challenges, including endogenous metabolic activity that is poorly understood yet has a significant impact on CFE productivity. Here, we use metabolomics to characterize the temporal metabolic changes in CFE systems and their constituent components, including significant metabolic activity in central carbon and amino acid metabolism. We find that while changing the reaction starting state via lysate preincubation impacts protein production, it has a comparatively small impact on metabolic state. We also demonstrate that changes to lysate preparation have a larger effect on protein yield and temporal metabolic profiles, though general metabolic trends are conserved. Finally, while we improve protein production through targeted supplementation of metabolic enzymes, we show that the endogenous metabolic activity is fairly resilient to these enzymatic perturbations. Overall, this work highlights the robust nature of CFE reaction metabolism as well as the importance of understanding the complex interdependence of metabolites and proteins in CFE systems to guide optimization efforts.


Assuntos
Escherichia coli/genética , Engenharia Metabólica/métodos , Metaboloma , Sistema Livre de Células , Fosfato de Di-Hidroxiacetona/metabolismo , Proteínas de Escherichia coli/genética , Cromatografia Gasosa-Espectrometria de Massas , Glicólise/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Análise de Componente Principal
6.
Microb Cell Fact ; 20(1): 123, 2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34187467

RESUMO

BACKGROUND: Klebsiella pneumoniae is a bacterium that can be used as producer for numerous chemicals. Glycerol can be catabolised by K. pneumoniae and dihydroxyacetone is an intermediate of this catabolism pathway. Here dihydroxyacetone and glycerol were produced from glucose by this bacterium based a redirected glycerol catabolism pathway. RESULTS: tpiA, encoding triosephosphate isomerase, was knocked out to block the further catabolism of dihydroxyacetone phosphate in the glycolysis. After overexpression of a Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase (hdpA), the engineered strain produced remarkable levels of dihydroxyacetone (7.0 g/L) and glycerol (2.5 g/L) from glucose. Further increase in product formation were obtained by knocking out gapA encoding an iosenzyme of glyceraldehyde 3-phosphate dehydrogenase. There are two dihydroxyacetone kinases in K. pneumoniae. They were both disrupted to prevent an inefficient reaction cycle between dihydroxyacetone phosphate and dihydroxyacetone, and the resulting strains had a distinct improvement in dihydroxyacetone and glycerol production. pH 6.0 and low air supplement were identified as the optimal conditions for dihydroxyacetone and glycerol production by K, pneumoniae ΔtpiA-ΔDHAK-hdpA. In fed batch fermentation 23.9 g/L of dihydroxyacetone and 10.8 g/L of glycerol were produced after 91 h of cultivation, with the total conversion ratio of 0.97 mol/mol glucose. CONCLUSIONS: This study provides a novel and highly efficient way of dihydroxyacetone and glycerol production from glucose.


Assuntos
Di-Hidroxiacetona/metabolismo , Klebsiella pneumoniae/metabolismo , Fosfato de Di-Hidroxiacetona/metabolismo , Ácidos Difosfoglicéricos/metabolismo , Fermentação , Genes Bacterianos , Glucose/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicerol/metabolismo , Concentração de Íons de Hidrogênio , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/crescimento & desenvolvimento , Engenharia Metabólica , Redes e Vias Metabólicas , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Termodinâmica
7.
Nat Metab ; 3(6): 859-875, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34140692

RESUMO

Global histone acetylation varies with changes in the nutrient and cell cycle phases; however, the mechanisms connecting these variations are not fully understood. Herein, we report that nutrient-related and cell-cycle-regulated nuclear acetate regulates global histone acetylation. Histone deacetylation-generated acetate accumulates in the nucleus and induces histone hyperacetylation. The nuclear acetate levels were controlled by glycolytic enzyme triosephosphate isomerase 1 (TPI1). Cyclin-dependent kinase 2 (CDK2), which is phosphorylated and activated by nutrient-activated mTORC1, phosphorylates TPI1 Ser 117 and promotes nuclear translocation of TPI1, decreases nuclear dihydroxyacetone phosphate (DHAP) and induces nuclear acetate accumulation because DHAP scavenges acetate via the formation of 1-acetyl-DHAP. CDK2 accumulates in the cytosol during the late G1/S phases. Inactivation or blockade of nuclear translocation of TPI1 abrogates nutrient-dependent and cell-cycle-dependent global histone acetylation, chromatin condensation, gene transcription and DNA replication. These results identify the mechanism of maintaining global histone acetylation by nutrient and cell cycle signals.


Assuntos
Ciclo Celular/fisiologia , Núcleo Celular/metabolismo , Fosfato de Di-Hidroxiacetona/metabolismo , Histonas/metabolismo , Nutrientes/metabolismo , Transdução de Sinais , Acetatos/metabolismo , Acetilação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Replicação do DNA , Humanos , Fosforilação , Transcrição Gênica
8.
Environ Mol Mutagen ; 62(3): 185-202, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33496975

RESUMO

Dihydroxyacetone (DHA) is a three-carbon sugar that is the active ingredient in sunless tanning products and a by-product of electronic cigarette (e-cigarette) combustion. Increased use of sunless tanning products and e-cigarettes has elevated exposures to DHA through inhalation and absorption. Studies have confirmed that DHA is rapidly absorbed into cells and can enter into metabolic pathways following phosphorylation to dihydroxyacetone phosphate (DHAP), a product of fructose metabolism. Recent reports have suggested metabolic imbalance and cellular stress results from DHA exposures. However, the impact of elevated exposure to DHA on human health is currently under-investigated. We propose that exogenous exposures to DHA increase DHAP levels in cells and mimic fructose exposures to produce oxidative stress, mitochondrial dysfunction, and gene and protein expression changes. Here, we review cell line and animal model exposures to fructose to highlight similarities in the effects produced by exogenous exposures to DHA. Given the long-term health consequences of fructose exposure, this review emphasizes the pressing need to further examine DHA exposures from sunless tanning products and e-cigarettes.


Assuntos
Fosfato de Di-Hidroxiacetona/metabolismo , Di-Hidroxiacetona/toxicidade , Mitocôndrias/genética , Estresse Oxidativo/efeitos dos fármacos , Di-Hidroxiacetona/metabolismo , Frutose/toxicidade , Humanos , Redes e Vias Metabólicas/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Estresse Oxidativo/genética , Fosforilação
9.
Biochemistry ; 59(51): 4856-4863, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33305938

RESUMO

K120 of glycerol 3-phosphate dehydrogenase (GPDH) lies close to the carbonyl group of the bound dihydroxyacetone phosphate (DHAP) dianion. pH rate (pH 4.6-9.0) profiles are reported for kcat and (kcat/Km)dianion for wild type and K120A GPDH-catalyzed reduction of DHAP by NADH, and for (kcat/KdKam) for activation of the variant-catalyzed reduction by CH3CH2NH3+, where Kam and Kd are apparent dissociation constants for CH3CH2NH3+ and DHAP, respectively. These profiles provide evidence that the K120 side chain cation, which is stabilized by an ion-pairing interaction with the D260 side chain, remains protonated between pH 4.6 and 9.0. The profiles for wild type and K120A variant GPDH show downward breaks at a similar pH value (7.6) that are attributed to protonation of the K204 side chain, which also lies close to the substrate carbonyl oxygen. The pH profiles for (kcat/Km)dianion and (kcat/KdKam) for the K120A variant show that the monoprotonated form of the variant is activated for catalysis by CH3CH2NH3+ but has no detectable activity, compared to the diprotonated variant, for unactivated reduction of DHAP. The pH profile for kcat shows that the monoprotonated K120A variant is active toward reduction of enzyme-bound DHAP, because of activation by a ligand-driven conformational change. Upward breaks in the pH profiles for kcat and (kcat/Km)dianion for K120A GPDH are attributed to protonation of D260. These breaks are consistent with the functional replacement of K120 by D260, and a plasticity in the catalytic roles of the active site side chains.


Assuntos
Fosfato de Di-Hidroxiacetona/química , Glicerolfosfato Desidrogenase/química , NAD/química , Biocatálise , Glicerolfosfato Desidrogenase/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Lisina/química , Mutação , Oxirredução
11.
Biochemistry ; 59(48): 4573-4580, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33231431

RESUMO

Non-typhoidal Salmonella are capable of colonizing livestock and humans, where they can progressively cause disease. Previously, a library of targeted single-gene deletion mutants of Salmonella enterica serotype Typhimurium was inoculated to ligated ileal loops in calves to identify genes under selection. Of those genes identified, a cluster of genes is related to carbohydrate metabolism and transportation. It is proposed that an incoming carbohydrate is first phosphorylated by a phosphoenolpyruvate-dependent phosphotransferase system. The metabolite is further phosphorylated by the kinase STM3781 and then cleaved by the aldolase STM3780. STM3780 is functionally annotated as a class II fructose-bisphosphate aldolase. The aldolase was purified to homogeneity, and its aldol condensation activity with a range of aldehydes was determined. In the condensation reaction, STM3780 was shown to catalyze the abstraction of the pro-S hydrogen from C3 of dihydroxyacetone and subsequent formation of a carbon-carbon bond with S stereochemistry at C3 and R stereochemistry at C4. The best aldehyde substrate was identified as l-threouronate. Surprisingly, STM3780 was also shown to catalyze the condensation of two molecules of dihydroxyacetone phosphate to form the branched carbohydrate dendroketose bisphosphate.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Genes Bacterianos , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Animais , Biocatálise , Metabolismo dos Carboidratos , Carboidratos/química , Bovinos , Doenças dos Bovinos/microbiologia , Medição da Troca de Deutério , Fosfato de Di-Hidroxiacetona/metabolismo , Humanos , Família Multigênica , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonelose Animal/microbiologia , Sorogrupo , Estereoisomerismo , Especificidade por Substrato
12.
Nat Metab ; 2(9): 893-901, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32719541

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1.


Assuntos
Fosfato de Di-Hidroxiacetona/fisiologia , Glucose/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Di-Hidroxiacetona/metabolismo , Fosfato de Di-Hidroxiacetona/biossíntese , Metabolismo Energético , Frutose-Bifosfato Aldolase/metabolismo , Glucose/deficiência , Glicólise , Células HEK293 , Humanos , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Serina-Treonina Quinases TOR/genética
13.
Biochemistry ; 59(16): 1582-1591, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32250105

RESUMO

A comparison of the values of kcat/Km for reduction of dihydroxyacetone phosphate (DHAP) by NADH catalyzed by wild type and K120A/R269A variant glycerol-3-phosphate dehydrogenase from human liver (hlGPDH) shows that the transition state for enzyme-catalyzed hydride transfer is stabilized by 12.0 kcal/mol by interactions with the cationic K120 and R269 side chains. The transition state for the K120A/R269A variant-catalyzed reduction of DHAP is stabilized by 1.0 and 3.8 kcal/mol for reactions in the presence of 1.0 M EtNH3+ and guanidinium cation (Gua+), respectively, and by 7.5 kcal/mol for reactions in the presence of a mixture of each cation at 1.0 M, so that the transition state stabilization by the ternary E·EtNH3+·Gua+ complex is 2.8 kcal/mol greater than the sum of stabilization by the respective binary complexes. This shows that there is cooperativity between the paired activators in transition state stabilization. The effective molarities (EMs) of ∼50 M determined for the K120A and R269A side chains are ≪106 M, the EM for entropically controlled reactions. The unusually efficient rescue of the activity of hlGPDH-catalyzed reactions by the HPi/Gua+ pair and by the Gua+/EtNH3+ activator pair is due to stabilizing interactions between the protein and the activator pieces that organize the K120 and R269 side chains at the active site. This "preorganization" of side chains promotes effective catalysis by hlGPDH and many other enzymes. The role of the highly conserved network of side chains, which include Q295, R269, N270, N205, T264, K204, D260, and K120, in catalysis is discussed.


Assuntos
Glicerolfosfato Desidrogenase/química , Catálise , Domínio Catalítico , Fosfato de Di-Hidroxiacetona/química , Ativadores de Enzimas/química , Etilaminas/química , Glicerolfosfato Desidrogenase/genética , Guanidina/química , Humanos , Cinética , Mutação , Oxirredução
14.
Biophys Chem ; 258: 106330, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31981743

RESUMO

The glycolytic pathway is present in most organisms and represents a central part of the energy production mechanism in a cell. For a general understanding of glycolysis, the investigation from a thermodynamic point of view is essential and allows realising thermodynamic feasibility analyses under in vivo conditions. However, available literature standard Gibbs energies of reaction, ΔRg'0, are calculated using equilibrium-molality ratios Km', which might lead to a misinterpretation of the glycolytic pathway. It was the aim of this work to thermodynamically investigate the triosephosphate isomerase (TPI) reaction to provide new activity-based reaction data. In vitro equilibrium experiments were performed, and activity coefficients were predicted with the equation of state electrolyte PC-SAFT (ePC-SAFT). The combination of experimental concentrations and predicted activity coefficients yielded the thermodynamic equilibrium constant Ka and a new value for ΔRg'0(298.15 K, pH 7) = 7.1 ± 0.3 kJ mol­1. The availability of the new ΔRg'0 value allowed predicting influences of the reaction medium on the reaction equilibrium of the TPI reaction. In this work, influences of the initial substrate concentration, pH and Mg2+ concentration on the reaction equilibrium were investigated and a method is presented to predict these influences. The higher the substrate concentration and the higher the temperature, the stronger the reaction equilibrium is shifted on the product side. While the pH did not have a significant influence on the reaction equilibrium, Mg2+ yielded a shift of the reaction equilibrium to the substrate side. All these effects were predicted correctly with ePC-SAFT. Based on the ePC-SAFT predictions we concluded that a charge-reduction of the product by complexation of the product with Mg2+ was responsible for the strong influence of Mg2+ on the reaction equilibrium. Finally, the standard enthalpy of reaction of ΔRh'0(pH 7) = 18 ± 7 kJ mol­1 was determined with the equilibrium constants Ka at 298.15 K, 304.15 K and 310.15 K using the van 't Hoff equation.


Assuntos
Termodinâmica , Triose-Fosfato Isomerase/metabolismo , Fosfato de Di-Hidroxiacetona/química , Fosfato de Di-Hidroxiacetona/metabolismo , Magnésio/análise , Magnésio/metabolismo , Modelos Estatísticos
15.
J Agric Food Chem ; 68(5): 1347-1353, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31961681

RESUMO

A facile approach is introduced here for the synthesis of rare ketoses from glycerol and d-/l-glyceraldehyde (d-/l-GA). The reactions were carried out in a one-pot multienzyme fashion in which the only carbon source is glycerol. In the enzymatic cascade, glycerol is phosphorylated and then oxidized at C2 to afford dihydroxyacetone phosphate (DHAP), the key donor for enzymatic aldol reaction. Meanwhile, the primary alcohol of glycerol is also oxidized to give the acceptor molecule GA in situ (d- or l-isomer could be formed stereospecifically with either alditol oxidase or horse liver alcohol dehydrogenase). Different DHAP-dependent aldolases were used to generate the aldol adducts (rare ketohexose phosphates) with various stereoconfigurations and diastereomeric ratios. It is worth noting that the enzyme that catalyzes the phosphorylation reaction in the first step could also help recycle the phosphate in the last step to provide free rare sugar molecules. This study provides a useful method for rare ketose synthesis on a 100 mg to g scale, starting from relatively inexpensive materials which solved the problem of supplying both glycerol 3-phosphate and GA in our previous work. It also demonstrates an example of green synthesis due to highly efficient carbon usage and recycling of cofactors.


Assuntos
Álcool Desidrogenase/química , Aldeído Liases/química , Glicerol/química , Cetoses/química , Animais , Biocatálise , Fosfato de Di-Hidroxiacetona/química , Cavalos , Fosforilação
16.
Mol Microbiol ; 113(5): 923-937, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31950558

RESUMO

S-adenosyl-l-methionine (SAM) is a necessary cosubstrate for numerous essential enzymatic reactions including protein and nucleotide methylations, secondary metabolite synthesis and radical-mediated processes. Radical SAM enzymes produce 5'-deoxyadenosine, and SAM-dependent enzymes for polyamine, neurotransmitter and quorum sensing compound synthesis produce 5'-methylthioadenosine as by-products. Both are inhibitory and must be addressed by all cells. This work establishes a bifunctional oxygen-independent salvage pathway for 5'-deoxyadenosine and 5'-methylthioadenosine in both Rhodospirillum rubrum and Extraintestinal Pathogenic Escherichia coli. Homologous genes for this pathway are widespread in bacteria, notably pathogenic strains within several families. A phosphorylase (Rhodospirillum rubrum) or separate nucleoside and kinase (Escherichia coli) followed by an isomerase and aldolase sequentially function to salvage these two wasteful and inhibitory compounds into adenine, dihydroxyacetone phosphate and acetaldehyde or (2-methylthio)acetaldehyde during both aerobic and anaerobic growth. Both SAM by-products are metabolized with equal affinity during aerobic and anaerobic growth conditions, suggesting that the dual-purpose salvage pathway plays a central role in numerous environments, notably the human body during infection. Our newly discovered bifunctional oxygen-independent pathway, widespread in bacteria, salvages at least two by-products of SAM-dependent enzymes for carbon and sulfur salvage, contributing to cell growth.


Assuntos
Proteínas de Bactérias/metabolismo , Desoxiadenosinas/metabolismo , Escherichia coli/metabolismo , Rhodospirillum rubrum/metabolismo , S-Adenosilmetionina/metabolismo , Tionucleosídeos/metabolismo , Proteínas de Bactérias/genética , Carbono/metabolismo , Fosfato de Di-Hidroxiacetona/metabolismo , Escherichia coli/genética , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Isomerases/genética , Isomerases/metabolismo , Redes e Vias Metabólicas/genética , Metionina/metabolismo , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Oxigênio/metabolismo , Fosforilases/genética , Fosforilases/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Rhodospirillum rubrum/genética
17.
J Biol Chem ; 295(7): 1867-1878, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31871051

RESUMO

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PPi-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PPi-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K½ value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.


Assuntos
Clostridiales/genética , Clostridium thermocellum/genética , Frutose-Bifosfato Aldolase/genética , Via de Pentose Fosfato/genética , Fosfofrutoquinase-1/genética , Clostridiales/enzimologia , Clostridium thermocellum/enzimologia , Fosfato de Di-Hidroxiacetona/genética , Fosfato de Di-Hidroxiacetona/metabolismo , Escherichia coli/enzimologia , Frutose-Bifosfato Aldolase/metabolismo , Frutosefosfatos/metabolismo , Cinética , Pentoses/biossíntese , Pentoses/metabolismo , Fosfofrutoquinase-1/metabolismo , Fosfotransferases/metabolismo , Ribose/biossíntese , Ribose/metabolismo , Fosfatos Açúcares/metabolismo , Transaldolase/genética , Transaldolase/metabolismo , Xilose/biossíntese , Xilose/metabolismo
18.
Plant J ; 102(1): 153-164, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31762135

RESUMO

Dunaliella has been extensively studied due to its intriguing adaptation to high salinity. Its di-domain glycerol-3-phosphate dehydrogenase (GPDH) isoform is likely to underlie the rapid production of the osmoprotectant glycerol. Here, we report the structure of the chimeric Dunaliella salina GPDH (DsGPDH) protein featuring a phosphoserine phosphatase-like domain fused to the canonical glycerol-3-phosphate (G3P) dehydrogenase domain. Biochemical assays confirm that DsGPDH can convert dihydroxyacetone phosphate (DHAP) directly to glycerol, whereas a separate phosphatase protein is required for this conversion process in most organisms. The structure of DsGPDH in complex with its substrate DHAP and co-factor nicotinamide adenine dinucleotide (NAD) allows the identification of the residues that form the active sites. Furthermore, the structure reveals an intriguing homotetramer form that likely contributes to the rapid biosynthesis of glycerol.


Assuntos
Clorofíceas/enzimologia , Fosfato de Di-Hidroxiacetona/metabolismo , Glicerol/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Domínio Catalítico , Clorofíceas/genética , Clorofíceas/metabolismo , Glicerolfosfato Desidrogenase/química , Glicerolfosfato Desidrogenase/genética , NAD/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência
19.
J Am Chem Soc ; 141(40): 16139-16150, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31508957

RESUMO

We report results of detailed empirical valence bond simulations that model the effect of several amino acid substitutions on the thermodynamic (ΔG°) and kinetic activation (ΔG⧧) barriers to deprotonation of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) bound to wild-type triosephosphate isomerase (TIM), as well as to the K12G, E97A, E97D, E97Q, K12G/E97A, I170A, L230A, I170A/L230A, and P166A variants of this enzyme. The EVB simulations model the observed effect of the P166A mutation on protein structure. The E97A, E97Q, and E97D mutations of the conserved E97 side chain result in ≤1.0 kcal mol-1 decreases in the activation barrier for substrate deprotonation. The agreement between experimental and computed activation barriers is within ±1 kcal mol-1, with a strong linear correlation between ΔG⧧ and ΔG° for all 11 variants, with slopes ß = 0.73 (R2 = 0.994) and ß = 0.74 (R2 = 0.995) for the deprotonation of DHAP and GAP, respectively. These Brønsted-type correlations show that the amino acid side chains examined in this study function to reduce the standard-state Gibbs free energy of reaction for deprotonation of the weak α-carbonyl carbon acid substrate to form the enediolate phosphate reaction intermediate. TIM utilizes the cationic side chain of K12 to provide direct electrostatic stabilization of the enolate oxyanion, and the nonpolar side chains of P166, I170, and L230 are utilized for the construction of an active-site cavity that provides optimal stabilization of the enediolate phosphate intermediate relative to the carbon acid substrate.


Assuntos
Fosfato de Di-Hidroxiacetona/química , Gliceraldeído 3-Fosfato/química , Prótons , Triose-Fosfato Isomerase/química , Substituição de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Catálise , Domínio Catalítico , Cinética , Modelos Moleculares , Mutação , Termodinâmica , Triose-Fosfato Isomerase/genética
20.
J Am Chem Soc ; 141(36): 14142-14151, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31390192

RESUMO

Quinolinic acid is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide and its derivatives in all organisms that synthesize the molecule de novo. In most prokaryotes, it is formed from the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA) by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique noncysteinyl-ligated iron ion (Fea), which is proposed to bind the hydroxyl group of an intermediate in its reaction to facilitate a dehydration step. However, direct evidence for this role in catalysis has yet to be provided, and the exact chemical mechanism that underlies this transformation remains elusive. Herein, we present a structure of NadA from Pyrococcus horikoshii (PhNadA) in complex with IA and show that a carboxylate group of the molecule is ligated to Fea of the iron-sulfur cluster, occupying the site to which DHAP has been proposed to bind during catalysis. When crystals of PhNadA in complex with IA are soaked briefly in DHAP before freezing, electron density for a new molecule is observed, which we suggest is related to an intermediate in the reaction. Similar, but slightly different, "intermediates" are observed when crystals of a PhNadA Glu198Gln variant are incubated with DHAP, oxaloacetate, and ammonium chloride, conditions under which IA is formed chemically. Continuous-wave and pulse electron paramagnetic resonance techniques are used to verify the binding mode of substrates and proposed intermediates in frozen solution.


Assuntos
Ácido Aspártico/análogos & derivados , Fosfato de Di-Hidroxiacetona/metabolismo , Complexos Multienzimáticos/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Biocatálise , Cristalografia por Raios X , Fosfato de Di-Hidroxiacetona/química , Modelos Moleculares , Estrutura Molecular , Complexos Multienzimáticos/química , Pyrococcus horikoshii/enzimologia
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