RESUMO
Small-molecule inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6δ impairs KRas spatial organization and signaling in cells. However, despite potent binding inâ vitro (KD <10â nm), interference with Ras signaling and growth inhibition require 5-20â µm compound concentrations. We demonstrate that these findings can be explained by fast release of high-affinity inhibitors from PDE6δ by the release factor Arl2. This limitation is overcome by novel highly selective inhibitors that bind to PDE6δ with up to 7 hydrogen bonds, resulting in picomolar affinity. Their release by Arl2 is greatly decreased, and representative compounds selectively inhibit growth of KRas mutated and -dependent cells with the highest activity recorded yet. Our findings indicate that very potent inhibitors of the KRas-PDE6δ interaction may impair the growth of tumors driven by oncogenic KRas.
RESUMO
Woodward's reagent K (WRK) is a reactive heterocyclic compound that has been employed in protein chemistry to covalently and unspecifically label proteins at nucleophilic amino acids, notably at histidine and cysteine. We have developed a panel of WRK-derived activity-based probes and show that surprisingly and unexpectedly, these probes are fairly selective for a few proteins in the human proteome. The WRK-derived probes show unique reactivity towards the catalytic N-terminal proline in the macrophage migration inhibitory factor (MIF) and can be used to label and, if equipped with a fluorophore, to image MIF activities in living cells.
Assuntos
Isoxazóis/química , Fatores Inibidores da Migração de Macrófagos/metabolismo , Proteoma/química , Catálise , Cromatografia Líquida de Alta Pressão , Células HeLa , Humanos , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/química , Microscopia de Fluorescência , Prolina/química , Proteoma/análise , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The versatile thiol mercaptosuccinate has a wide range of applications, e.g. in quantum dot research or in bioimaging. Its metabolism is investigated in Variovorax paradoxus strain B4, which can utilize this compound as the sole source of carbon and sulfur. Proteomic studies of strain B4 resulted in the identification of a putative mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, possibly representing the key enzyme in the degradation of mercaptosuccinate. Therefore, the putative mercaptosuccinate dioxygenase was heterologously expressed, purified, and characterized in this study. The results clearly demonstrated that the enzyme utilizes mercaptosuccinate with concomitant consumption of oxygen. Thus, the enzyme is designated as mercaptosuccinate dioxygenase. Succinate and sulfite were verified as the final reaction products. The enzyme showed an apparent Km of 0.4 mM, and a specific activity (Vmax) of 20.0 µmol min(-1) mg(-1) corresponding to a kcat of 7.7 s(-1). Furthermore, the enzyme was highly specific for mercaptosuccinate, no activity was observed with cysteine, dithiothreitol, 2-mercaptoethanol, and 3-mercaptopropionate. These structurally related thiols did not have an inhibitory effect either. Fe(II) could clearly be identified as metal cofactor of the mercaptosuccinate dioxygenase with a content of 0.6 mol of Fe(II)/mol of enzyme. The recently proposed hypothesis for the degradation pathway of mercaptosuccinate based on proteome analyses could be strengthened in the present study. (i) Mercaptosuccinate is first converted to sulfinosuccinate by this mercaptosuccinate dioxygenase; (ii) sulfinosuccinate is spontaneously desulfinated to succinate and sulfite; and (iii) whereas succinate enters the central metabolism, sulfite is detoxified by the previously identified putative molybdopterin oxidoreductase.
Assuntos
Proteínas de Bactérias/química , Comamonadaceae/enzimologia , Cisteína Dioxigenase/química , Tiomalatos/química , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/isolamento & purificação , Cisteína Dioxigenase/antagonistas & inibidores , Cisteína Dioxigenase/isolamento & purificação , Inibidores Enzimáticos/química , Cinética , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
3-Sulfinopropionyl-coenzyme A (3SP-CoA) desulfinase (AcdDPN7; EC 3.13.1.4) was identified during investigation of the 3,3'-dithiodipropionic acid (DTDP) catabolic pathway in the betaproteobacterium Advenella mimigardefordensis strain DPN7(T). DTDP is an organic disulfide and a precursor for the synthesis of polythioesters (PTEs) in bacteria, and is of interest for biotechnological PTE production. AcdDPN7 catalyzes sulfur abstraction from 3SP-CoA, a key step during the catabolism of DTDP. Here, the crystal structures of apo AcdDPN7 at 1.89 Å resolution and of its complex with the CoA moiety from the substrate analogue succinyl-CoA at 2.30 Å resolution are presented. The apo structure shows that AcdDPN7 belongs to the acyl-CoA dehydrogenase superfamily fold and that it is a tetramer, with each subunit containing one flavin adenine dinucleotide (FAD) molecule. The enzyme does not show any dehydrogenase activity. Dehydrogenase activity would require a catalytic base (Glu or Asp residue) at either position 246 or position 366, where a glutamine and a glycine are instead found, respectively, in this desulfinase. The positioning of CoA in the crystal complex enabled the modelling of a substrate complex containing 3SP-CoA. This indicates that Arg84 is a key residue in the desulfination reaction. An Arg84Lys mutant showed a complete loss of enzymatic activity, suggesting that the guanidinium group of the arginine is essential for desulfination. AcdDPN7 is the first desulfinase with an acyl-CoA dehydrogenase fold to be reported, which underlines the versatility of this enzyme scaffold.
Assuntos
Acil-CoA Desidrogenase/química , Alcaligenaceae/enzimologia , Coenzima A/química , Enzimas/química , Propionatos/química , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de ProteínaRESUMO
In a previous study, the essential role of 3-sulfinopropionyl coenzyme A (3SP-CoA) desulfinase acyl-CoA dehydrogenase (Acd) in Advenella mimigardefordensis strain DPN7(T) (AcdDPN7) during degradation of 3,3'-dithiodipropionic acid (DTDP) was elucidated. DTDP is a sulfur-containing precursor substrate for biosynthesis of polythioesters (PTEs). AcdDPN7 showed high amino acid sequence similarity to acyl-CoA dehydrogenases but was unable to catalyze a dehydrogenation reaction. Hence, it was investigated in the present study whether 3SP-CoA desulfinase activity is an uncommon or a widespread property within the acyl-CoA dehydrogenase superfamily. Therefore, proteins of the acyl-CoA dehydrogenase superfamily from Advenella kashmirensis WT001, Bacillus cereus DSM31, Cupriavidus necator N-1, Escherichia coli BL21, Pseudomonas putida KT2440, Burkholderia xenovorans LB400, Ralstonia eutropha H16, Variovorax paradoxus B4, Variovorax paradoxus S110, and Variovorax paradoxus TBEA6 were expressed in E. coli strains. All purified acyl-CoA dehydrogenases appeared as homotetramers, as revealed by size exclusion chromatography. AcdS110, AcdB4, AcdH16, and AcdKT2440 were able to dehydrogenate isobutyryl-CoA. AcdKT2440 additionally dehydrogenated butyryl-CoA and valeryl-CoA, whereas AcdDSM31 dehydrogenated only butyryl-CoA and valeryl-CoA. No dehydrogenation reactions were observed with propionyl-CoA, isovaleryl-CoA, succinyl-CoA, and glutaryl-CoA for any of the investigated acyl-CoA dehydrogenases. Only AcdTBEA6, AcdN-1, and AcdLB400 desulfinated 3SP-CoA and were thus identified as 3SP-CoA desulfinases within the acyl-CoA dehydrogenase family, although none of these three Acds dehydrogenated any of the tested acyl-CoA thioesters. No appropriate substrates were identified for AcdBL21 and AcdWT001. Spectrophotometric assays provided apparent Km and Vmax values for active substrates and indicated the applicability of phylogenetic analyses to predict the substrate range of uncharacterized acyl-CoA dehydrogenases. Furthermore, C. necator N-1 was found to utilize 3SP as the sole source of carbon and energy.
Assuntos
Acil-CoA Desidrogenases/isolamento & purificação , Acil-CoA Desidrogenases/metabolismo , Bactérias/enzimologia , Acil Coenzima A/metabolismo , Acil-CoA Desidrogenases/genética , Bactérias/genética , Cromatografia em Gel , Clonagem Molecular , Expressão Gênica , Cinética , Oxirredução , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrofotometria , Especificidade por SubstratoRESUMO
Three succinate coenzyme A (succinate-CoA) ligases (SucCD) from Escherichia coli, Advenella mimigardefordensis DPN7(T), and Alcanivorax borkumensis SK2 were characterized regarding their substrate specificity concerning succinate analogues. Previous studies had suggested that SucCD enzymes might be promiscuous toward succinate analogues, such as itaconate and 3-sulfinopropionate (3SP). The latter is an intermediate of the degradation pathway of 3,3'-dithiodipropionate (DTDP), a precursor for the biotechnical production of polythioesters (PTEs) in bacteria. The sucCD genes were expressed in E. coli BL21(DE3)/pLysS. The SucCD enzymes of E. coli and A. mimigardefordensis DPN7(T) were purified in the native state using stepwise purification protocols, while SucCD from A. borkumensis SK2 was equipped with a C-terminal hexahistidine tag at the SucD subunit. Besides the preference for the physiological substrates succinate, itaconate, ATP, and CoA, high enzyme activity was additionally determined for both enantiomeric forms of malate, amounting to 10 to 21% of the activity with succinate. Km values ranged from 2.5 to 3.6 mM for l-malate and from 3.6 to 4.2 mM for d-malate for the SucCD enzymes investigated in this study. As l-malate-CoA ligase is present in the serine cycle for assimilation of C1 compounds in methylotrophs, structural comparison of these two enzymes as members of the same subsubclass suggested a strong resemblance of SucCD to l-malate-CoA ligase and gave rise to the speculation that malate-CoA ligases and succinate-CoA ligases have the same evolutionary origin. Although enzyme activities were very low for the additional substrates investigated, liquid chromatography/electrospray ionization-mass spectrometry analyses proved the ability of SucCD enzymes to form CoA-thioesters of adipate, glutarate, and fumarate. Since all SucCD enzymes were able to activate 3SP to 3SP-CoA, we consequently demonstrated that the activation of 3SP is not a unique characteristic of the SucCD from A. mimigardefordensis DPN7(T). The essential role of sucCD in the activation of 3SP in vivo was proved by genetic complementation.
Assuntos
Alcaligenaceae/enzimologia , Alcanivoraceae/enzimologia , Coenzima A/metabolismo , Escherichia coli/enzimologia , Malatos/metabolismo , Succinato-CoA Ligases/metabolismo , Compostos de Enxofre/metabolismo , Acil Coenzima A/metabolismo , Ésteres/metabolismo , Cinética , Especificidade por Substrato , Succinato-CoA Ligases/isolamento & purificaçãoRESUMO
In this study, a propionate CoA-transferase (H16_A2718; EC 2.8.3.1) from Ralstonia eutropha H16 (Pct(Re)) was characterized in detail. Glu342 was identified as catalytically active amino acid residue via site-directed mutagenesis. Activity of Pct(Re) was irreversibly lost after the treatment with NaBH4 in the presence of acetyl-CoA as it is shown for all CoA-transferases from class I, thereby confirming the formation of the covalent enzyme-CoA intermediate by Pct(Re). In addition to already known CoA acceptors for Pct Re such as 3-hydroxypropionate, 3-hydroxybutyrate, acrylate, succinate, lactate, butyrate, crotonate and 4-hydroxybutyrate, it was found that glycolate, chloropropionate, acetoacetate, valerate, trans-2,3-pentenoate, isovalerate, hexanoate, octanoate and trans-2,3-octenoate formed also corresponding CoA-thioesters after incubation with acetyl-CoA and Pct(Re). Isobutyrate was found to be preferentially used as CoA acceptor amongst other carboxylates tested in this study. In contrast, no products were detected with acetyl-CoA and formiate, bromopropionate, glycine, pyruvate, 2-hydroxybutyrate, malonate, fumarate, itaconate, ß-alanine, γ-aminobutyrate, levulate, glutarate or adipate as potential CoA acceptor. Amongst CoA donors, butyryl-CoA, crotonyl-CoA, 3-hydroxybutyryl-CoA, isobutyryl-CoA, succinyl-CoA and valeryl-CoA apart from already known propionyl-CoA and acetyl-CoA could also donate CoA to acetate. The highest rate of the reaction was observed with 3-hydroxybutyryl-CoA (2.5 µmol mg⻹ min⻹). K(m) values for propionyl-CoA, acetyl-CoA, acetate and 3-hydroxybutyrate were 0.3, 0.6, 4.5 and 4.3 mM, respectively. The rather broad substrate range might be a good starting point for enzyme engineering approaches and for the application of Pct(Re) in biotechnological polyester production.
Assuntos
Coenzima A-Transferases/metabolismo , Coenzima A/metabolismo , Cupriavidus necator/enzimologia , Substituição de Aminoácidos , Coenzima A-Transferases/genética , Cupriavidus necator/genética , Análise Mutacional de DNA , Cinética , Mutagênese Sítio-Dirigida , Especificidade por SubstratoRESUMO
3-Sulfinopropionyl coenzyme A (3SP-CoA) desulfinase (AcdDPN7) is a new desulfinase that catalyzes the sulfur abstraction from 3SP-CoA in the betaproteobacterium Advenella mimigardefordensis strain DPN7(T). During investigation of a Tn5::mob-induced mutant defective in growth on 3,3'-dithiodipropionate (DTDP) and also 3-sulfinopropionate (3SP), the transposon insertion was mapped to an open reading frame with the highest homology to an acyl-CoA dehydrogenase (Acd) from Burkholderia phenoliruptrix strain BR3459a (83% identical and 91% similar amino acids). An A. mimigardefordensis Δacd mutant was generated and verified the observed phenotype of the Tn5::mob-induced mutant. For enzymatic studies, AcdDPN7 was heterologously expressed in Escherichia coli BL21(DE3)/pLysS by using pET23a::acdDPN7. The purified protein is yellow and contains a noncovalently bound flavin adenine dinucleotide (FAD) cofactor, as verified by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) analyses. Size-exclusion chromatography revealed a native molecular mass of about 173 kDa, indicating a homotetrameric structure (theoretically 179 kDa), which is in accordance with other members of the acyl-CoA dehydrogenase superfamily. In vitro assays unequivocally demonstrated that the purified enzyme converted 3SP-CoA into propionyl-CoA and sulfite (SO3(2-)). Kinetic studies of AcdDPN7 revealed a Vmax of 4.19 µmol min(-1) mg(-1), an apparent Km of 0.013 mM, and a kcat/Km of 240.8 s(-1) mM(-1) for 3SP-CoA. However, AcdDPN7 is unable to perform a dehydrogenation, which is the usual reaction catalyzed by members of the acyl-CoA dehydrogenase superfamily. Comparison to other known desulfinases showed a comparably high catalytic efficiency of AcdDPN7 and indicated a novel reaction mechanism. Hence, AcdDPN7 encodes a new desulfinase based on an acyl-CoA dehydrogenase (EC 1.3.8.x) scaffold. Concomitantly, we identified the gene product that is responsible for the final desulfination step during catabolism of 3,3'-dithiodipropionate (DTDP), a sulfur-containing precursor substrate for biosynthesis of polythioesters.
Assuntos
Acil-CoA Desidrogenase/metabolismo , Alcaligenaceae/enzimologia , Alcaligenaceae/metabolismo , Propionatos/metabolismo , Acil-CoA Desidrogenase/química , Acil-CoA Desidrogenase/genética , Burkholderia/genética , Clonagem Molecular , Coenzimas/metabolismo , Elementos de DNA Transponíveis , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Expressão Gênica , Técnicas de Inativação de Genes , Cinética , Dados de Sequência Molecular , Peso Molecular , Mutagênese Insercional , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, Act(TBEA6) (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3'-thiodipropionate (TDP) degradation. In a previous study, accumulation of 3SP was observed in a Tn5::mob-induced mutant defective in growth on TDP. In contrast to the wild type and all other obtained mutants, this mutant showed no growth when 3SP was applied as the sole source of carbon and energy. The transposon Tn5::mob was inserted in a gene showing high homology to class III CoA-transferases. In the present study, analyses of the translation product clearly allocated Act(TBEA6) to this protein family. The predicted secondary structure indicates the lack of a C-terminal α-helix. Act(TBEA6) was heterologously expressed in Escherichia coli Lemo21(DE3) and was then purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography. Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent V(max) of 44.6 µmol min(-1) mg(-1) for succinyl-CoA, which corresponds to a turnover number of 36.0 s(-1) per subunit of Act(TBEA6). For 3SP, the apparent V(max) was determined as 46.8 µmol min(-1) mg(-1), which corresponds to a turnover number of 37.7 s(-1) per subunit of Act(TBEA6). The apparent K(m) values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. Nonetheless, the V. paradoxus Δact mutant did not reproduce the phenotype of the Tn5::mob-induced mutant. This defined deletion mutant was able to utilize TDP or 3SP as the sole carbon source, like the wild type. Complementation of the Tn5::mob-induced mutant with pBBR1MCS5::acdDPN7 partially restored growth on 3SP, which indicated a polar effect of the Tn5::mob transposon on acd(TBEA6), located downstream of act(TBEA6).
Assuntos
Coenzima A-Transferases/metabolismo , Comamonadaceae/enzimologia , Comamonadaceae/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Boroidretos , Clonagem Molecular , Coenzima A-Transferases/genética , Comamonadaceae/genética , Hidroxilamina , Dados de Sequência Molecular , Estrutura Molecular , Propionatos/química , Propionatos/metabolismoRESUMO
In this study, we have investigated a propionate CoA-transferase (Pct) homologue encoded in the genome of Ralstonia eutropha H16. The corresponding gene has been cloned into the vector pET-19b to yield a histidine-tagged enzyme which was expressed in Escherichia coli BL21 (DE3). After purification, high-performance liquid chromatography/mass spectrometry (HPLC/MS) analyses revealed that the enzyme exhibits a broad substrate specificity for carboxylic acids. The formation of the corresponding CoA-thioesters of acetate using propionyl-CoA as CoA donor, and of propionate, butyrate, 3-hydroxybutyrate, 3-hydroxypropionate, crotonate, acrylate, lactate, succinate and 4-hydroxybutyrate using acetyl-CoA as CoA donor could be shown. According to the substrate specificity, the enzyme can be allocated in the family I of CoA-transferases. The apparent molecular masses as determined by gel filtration and detected by SDS polyacrylamide gel electrophoresis were 228 and 64 kDa, respectively, and point to a quaternary structure of the native enzyme (α4). The enzyme exhibited similarities in sequence and structure to the well investigated Pct of Clostridium propionicum. It does not contain the typical conserved (S)ENG motif, but the derived motif sequence EXG with glutamate 342 to be, most likely, the catalytic residue. Due to the homo-oligomeric structure and the sequence differences with the subclasses IA-C of family I CoA-transferases, a fourth subclass of family I is proposed, comprising - amongst others - the Pcts of R. eutropha H16 and C. propionicum. A markerless precise-deletion mutant R. eutropha H16∆pct was generated. The growth and accumulation behaviour of this mutant on gluconate, gluconate plus 3,3'-dithiodipropionic acid (DTDP), acetate and propionate was investigated but resulted in no observable phenotype. Both, the wild type and the mutant showed the same growth and storage behaviour with these carbon sources. It is probable that R. eutropha H16 is upregulating other CoA-transferase(s) or CoA-synthetase(s), thereby compensating for the lacking Pct. The ability of R. eutropha H16 to substitute absent enzymes by isoenzymes has been already shown in different other studies in the past.
Assuntos
Acetilcoenzima A/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ácidos Carboxílicos/metabolismo , Coenzima A-Transferases/química , Coenzima A-Transferases/metabolismo , Cupriavidus necator/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Coenzima A-Transferases/genética , Cupriavidus necator/química , Cupriavidus necator/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
The sucCD gene of Advenella mimigardefordensis strain DPN7(T) encodes a succinyl coenzyme A (succinyl-CoA) synthetase homologue (EC 6.2.1.4 or EC 6.2.1.5) that recognizes, in addition to succinate, the structural analogues 3-sulfinopropionate (3SP) and itaconate as substrates. Accumulation of 3SP during 3,3'-dithiodipropionic acid (DTDP) degradation was observed in Tn5::mob-induced mutants of A. mimigardefordensis strain DPN7(T) disrupted in sucCD and in the defined deletion mutant A. mimigardefordensis ΔsucCD. These mutants were impaired in growth with DTDP and 3SP as the sole carbon source. Hence, it was proposed that the succinyl-CoA synthetase homologue in A. mimigardefordensis strain DPN7(T) activates 3SP to the corresponding CoA-thioester (3SP-CoA). The putative genes coding for A. mimigardefordensis succinyl-CoA synthetase (SucCD(Am)) were cloned and heterologously expressed in Escherichia coli BL21(DE3)/pLysS. Purification and characterization of the enzyme confirmed its involvement during degradation of DTDP. 3SP, the cleavage product of DTDP, was converted into 3SP-CoA by the purified enzyme, as demonstrated by in vitro enzyme assays. The structure of 3SP-CoA was verified by using liquid chromatography-electrospray ionization-mass spectrometry. SucCD(Am) is Mg²âº or Mn²âº dependent and unspecific regarding ATP or GTP. In kinetic studies the enzyme showed highest enzyme activity and substrate affinity with succinate (V(max) = 9.85 ± 0.14 µmol min⻹ mg⻹, K(m) = 0.143 ± 0.001 mM). In comparison to succinate, activity with 3SP was only ca. 1.2% (V(max) = 0.12 ± 0.01 µmol min⻹ mg⻹) and the affinity was 6-fold lower (K(m) = 0.818 ± 0.046 mM). Based on the present results, we conclude that SucCD(Am) is physiologically associated with the citric acid cycle but is mandatory for the catabolic pathway of DTDP and its degradation intermediate 3SP.
Assuntos
Betaproteobacteria/enzimologia , Propionatos/metabolismo , Succinato-CoA Ligases/metabolismo , Betaproteobacteria/classificação , Cromatografia Líquida de Alta Pressão , Passeio de Cromossomo , Cromatografia Gasosa-Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Propionatos/química , Succinato-CoA Ligases/genéticaRESUMO
3,3'-Thiodipropionic acid (TDP) is used as an additive in food and cosmetic industry and as precursor substrate for biotechnical polythioester production. Its catabolism was investigated in Variovorax paradoxus TBEA6 previous to this study. It was reported that the insertion of the transposon Tn5::mob into a gene showing high homology to flavin-dependent oxidoreductases (fox) resulted in impaired growth with TDP. Therefore, it was assumed that the initial cleavage of TDP is catalyzed by an FAD-dependent oxidoreductase (Fox, VPARA_05580). Accordingly, fox was heterologously expressed as a thioredoxin fusion protein. Analytical size exclusion chromatography revealed a homodimeric structure and the presence of the cofactor FAD. In vitro experiments showed that FoxTBEA6 is a D-2-hydroxy acid specific dehydrogenase and that its activity is enhanced in presence of either Ni2+, Co2+ or Zn2+. Cleavage of TDP by FoxTBEA6 was not observed. The findings are contrary to restricted growth with TDP of the transposon mutants and the previously published deletion mutant V. paradoxus TBEA6 Δfox. In this study, this contradiction was investigated by generation of additional deletion mutants, in which partial or complete deletion of fox did not affect utilization of TDP, and the mapping of single nucleotide polymorphisms (SNPs) in V. paradoxus TBEA6 Δfox.
Assuntos
Comamonadaceae/enzimologia , Comamonadaceae/genética , Flavinas/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Ativação Enzimática , Enzimas Imobilizadas , Expressão Gênica , Íons , Cinética , Metais , Oxirredutases/isolamento & purificação , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes de Fusão , Especificidade por SubstratoRESUMO
Monitoring how, when, and where small molecules engage their targets inside living cells is a critical step in chemical biology and pharmacological research, because it enables compound efficacy and confirmation of mode of action to be assessed. In this mini-review we summarize the currently available methodologies to detect and prove direct target engagement in cells and offer a critical view of their key advantages and disadvantages. As the interest of the field shifts toward discovery and validation of high-quality agents, we expect that efforts to develop and refine these types of methodologies will also intensify in the near future.