RESUMO
The cardiac potassium IKs current is carried by a channel complex formed from α-subunits encoded by KCNQ1 and ß-subunits encoded by KCNE1. Deleterious mutations in either gene are associated with hereditary long QT syndrome. Interactions between the transmembrane domains of the α- and ß-subunits determine the activation kinetics of IKs. A physical and functional interaction between COOH termini of the proteins has also been identified that impacts deactivation rate and voltage dependence of activation. We sought to explore the specific physical interactions between the COOH termini of the subunits that confer such control. Hydrogen/deuterium exchange coupled to mass spectrometry narrowed down the region of interaction to KCNQ1 residues 352-374 and KCNE1 residues 70-81, and provided evidence of secondary structure within these segments. Key mutations of residues in these regions tended to shift voltage dependence of activation toward more depolarizing voltages. Double-mutant cycle analysis then revealed energetic coupling between KCNQ1-I368 and KCNE1-D76 during channel activation. Our results suggest that the proximal COOH-terminal regions of KCNQ1 and KCNE1 participate in a physical and functional interaction during channel opening that is sensitive to perturbation and may explain the clustering of long QT mutations in the region.NEW & NOTEWORTHY Interacting ion channel subunits KCNQ1 and KCNE1 have received intense investigation due to their critical importance to human cardiovascular health. This work uses physical (hydrogen/deuterium exchange with mass spectrometry) and functional (double-mutant cycle analyses) studies to elucidate precise and important areas of interaction between the two proteins in an area that has eluded structural definition of the complex. It highlights the importance of pathogenic mutations in these regions.
Assuntos
Citoplasma/metabolismo , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Clonagem Molecular , Cricetinae , Deutério/metabolismo , Fenômenos Eletrofisiológicos , Células HEK293 , Humanos , Hidrogênio/metabolismo , Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Mutação , Plasmídeos/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genéticaRESUMO
Hereditary long QT syndrome is caused by deleterious mutation in one of several genetic loci, including locus LQT2 that contains the KCNH2 gene (or hERG, human ether-a-go-go related gene), causing faulty cardiac repolarization. Here, we describe and characterize a novel mutation, p.Asp219Val in the hERG channel, identified in an 11-year-old male with syncope and prolonged QT interval. Genetic sequencing showed a nonsynonymous variation in KCNH2 (c.656A>T: amino acid p.Asp219Val). p.Asp219Val resides in a region of the channel predicted to be unstructured and flexible, located between the PAS (Per-Arnt-Sim) domain and its interaction sites in the transmembrane domain. The p.Asp219Val hERG channel produced K(+) current that activated with modest changes in voltage dependence. Mutant channels were also slower to inactivate, recovered from inactivation more readily and demonstrated a significantly accelerated deactivation rate compared with the slow deactivation of wild-type channels. The intermediate nature of the biophysical perturbation is consistent with the degree of severity in the clinical phenotype. The findings of this study demonstrate a previously unknown role of the proximal N-terminus in deactivation and support the hypothesis that the proximal N-terminal domain is essential in maintaining slow hERG deactivation.
Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Síndrome do QT Longo/genética , Mutação , Criança , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Células HEK293 , Humanos , Síndrome do QT Longo/fisiopatologia , Masculino , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
The HERG (human ether-a-go-go related gene) potassium channel aids in the repolarization of the cardiomyocyte membrane at the end of each action potential. We have previously shown that sustained protein kinase A or C (PKA and PKC) activity specifically enhances channel synthesis over the course of hours to days in heterologous expression and cardiac myocytes. The kinase-mediated augmentation of the channel is post-transcriptional and occurs near or at the endoplasmic reticulum. Here we report our further investigations into the mechanisms of kinase-mediated augmentation of HERG channel protein. We show that HERG channel phosphorylation alone is not sufficient for the PKA-dependent increase to occur. In vitro translation studies indicate that an additional factor is required for the process. Pharmacologic inhibitors suggest that the channel augmentation is not due to kinase-mediated alteration in proteasome or lysosome activity. PKA activation had no effect on stability of HERG mRNA and polyribosomal profiling showed that kinase activity did not elevate translation from low to high rates. Transcriptional inhibition results suggest that the additional cellular factor is a PKA-regulated protein. Together, these findings suggest that PKA-mediated augmentation of HERG abundance is more complex than previously appreciated involving enhancement of already active translation rates, phosphorylation of the channel protein and at least one other cyclic-AMP/PKA-responsive protein. Further exploration of molecular components of this regulatory pathway will be necessary to determine exact mechanism and the biomedical impact of this process in vivo.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais de Potássio Éter-A-Go-Go/genética , Regulação da Expressão Gênica , Proteínas 14-3-3/metabolismo , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Dactinomicina/farmacologia , Canal de Potássio ERG1 , Ativação Enzimática , Canais de Potássio Éter-A-Go-Go/biossíntese , Células HEK293 , Humanos , Leupeptinas/farmacologia , Fosforilação , Inibidores de Proteassoma/farmacologia , Processamento de Proteína Pós-Traducional , Inibidores da Síntese de Proteínas/farmacologia , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: The hereditary Long QT Syndrome is a common cardiac disorder where ventricular repolarization is delayed, abnormally prolonging the QTc interval on electrocardiograms. LQTS is linked to various genetic loci, including the KCNH2 (HERG) gene that encodes the α-subunit of the cardiac potassium channel that carries I(Kr). Here, we report and characterize a novel pathologic missense mutation, G816V HERG, in a patient with sudden cardiac death. METHODS: Autopsy-derived tissue sample was used for DNA extraction and sequencing from an unexpected sudden death victim. The G816V HERG mutation was studied using heterologous expression in mammalian cell culture, whole cell patch clamp, confocal immunofluorescence, and immunochemical analyses. RESULTS: The mutant G816V HERG channel has reduced protein expression and shows a trafficking defective phenotype that is incapable of carrying current when expressed at physiological temperatures. The mutant channel showed reduced cell surface localization compared to wild-type HERG (WT HERG) but the mutant and wild-type subunits are capable of interacting. Expression studies at reduced temperatures enabled partial rescue of the trafficking defect with appearance of potassium currents, albeit with reduced current density and altered voltage-dependent activation. Lastly, we examined a potential role for hypokalemia as a contributory factor to the patient's lethal arrhythmia by possible low-potassium-induced degradation of WT HERG and haplo-insufficiency of G816V HERG. CONCLUSION: The G816V mutation in HERG causes a trafficking defect that acts in a partially dominant negative manner. This intermediate severity defect agrees with the mild clinical presentation in other family members harboring the same mutation. Possible hypokalemia in the proband induced WT HERG degradation combined with haplo-insufficiency may have further compromised repolarization reserve and contributed to the lethal arrhythmia.
Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Predisposição Genética para Doença/genética , Síndrome do QT Longo/genética , Adulto , Canal de Potássio ERG1 , Feminino , Humanos , Mutação/genética , FenótipoRESUMO
Ion channel subunits encoded by KCNQ1 and KCNE1 produce the slowly activating K+ current (IKs) that plays a central role in myocardial repolarization. The KCNQ1 alpha-subunit and the KCNE1 beta-subunit assemble with their membrane-spanning segments interacting, resulting in transformation of channel activation kinetics. We recently reported a functional interaction involving C-terminal portions of the two subunits with ensuing regulation of channel deactivation. In the present study, we provide evidence characterizing a physical interaction between the KCNQ1-CT (KCNE1 C-terminus) and the KCNE1-CT (KCNE1 C-terminus). When expressed in cultured cells, the KCNE1-CT co-localized with KCNQ1, co-immunoprecipitated with KCNQ1 and perturbed deactivation kinetics of the KCNQ1 currents. Purified KCNQ1-CT and KCNE1-CT physically interacted in pull-down experiments, indicating a direct association. Deletion analysis of KCNQ1-CT indicated that the KCNE1-CT binds to a KCNQ1 region just after the last transmembrane segment, but N-terminal to the tetramerization domain. SPR (surface plasmon resonance) corroborated the pull-down results, showing that the most proximal region (KCNQ1 amino acids 349-438) contributed most to the bimolecular interaction with a dissociation constant of approximately 4 microM. LQT (long QT) mutants of the KCNE1-CT, D76N and W87F, retained binding to the KCNQ1-CT with comparable affinity, indicating that these disease-causing mutations do not alter channel behaviour by disruption of the association. Several LQT mutations involving the KCNQ1-CT, however, showed various effects on KCNQ1/KCNE1 association. Our results indicate that the KCNQ1-CT and the KCNE1-CT comprise an independent interaction domain that may play a role in IKs channel regulation that is potentially affected in some LQTS (LQT syndrome) mutations.
Assuntos
Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Citoplasma/metabolismo , Humanos , Ativação do Canal Iônico , Dados de Sequência Molecular , TransfecçãoRESUMO
We previously reported a transgenic rabbit model of long QT syndrome based on overexpression of pore mutants of repolarizing K(+) channels KvLQT1 (LQT1) and HERG (LQT2).The transgenes in these rabbits eliminated the slow and fast components of the delayed rectifier K(+) current (I(Ks) and I(Kr), respectively), as expected. Interestingly, the expressed pore mutants of HERG and KvLQT1 downregulated the remaining reciprocal repolarizing currents, I(Ks) and I(Kr), without affecting the steady-state levels of the native polypeptides. Here, we sought to further explore the functional interactions between HERG and KvLQT1 in heterologous expression systems. Stable Chinese hamster ovary (CHO) cell lines expressing KvLQT1-minK or HERG were transiently transfected with expression vectors coding for mutant or wild-type HERG or KvLQT1. Transiently expressed pore mutant or wild-type KvLQT1 downregulated I(Kr) in HERG stable CHO cell lines by 70% and 44%, respectively. Immunostaining revealed a severalfold lower surface expression of HERG, which could account for the reduction in I(Kr) upon KvLQT1 expression. Deletion of the KvLQT1 NH(2)-terminus did not abolish the downregulation, suggesting that the interactions between the two channels are mediated through their COOH-termini. Similarly, transiently expressed HERG reduced I(Ks) in KvLQT1-minK stable cells. Coimmunoprecipitations indicated a direct interaction between HERG and KvLQT1, and surface plasmon resonance analysis demonstrated a specific, physical association between the COOH-termini of KvLQT1 and HERG. Here, we present an in vitro model system consistent with the in vivo reciprocal downregulation of repolarizing currents seen in transgenic rabbit models, illustrating the importance of the transfection method when studying heterologous ion channel expression and trafficking. Moreover, our data suggest that interactions between KvLQT1 and HERG are mediated through COOH-termini.
Assuntos
Potenciais de Ação/fisiologia , Regulação para Baixo/fisiologia , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/fisiologia , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/fisiologia , Mutação/genética , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Regulação para Baixo/genética , Canal de Potássio ERG1 , Eletrofisiologia , Feminino , Deleção de Genes , Humanos , Rim/citologia , Rim/fisiologia , Ovário/citologia , Ovário/fisiologia , Técnicas de Patch-Clamp , TransfecçãoRESUMO
Neonatal calvarial bone has been widely used for investigating the biological behaviour of intramembranous bones. This work evaluated the bone formation of neonatal calvarial bone by microcomputed tomography (micro-CT) and histomorphometry. Moreover, the viability of neonatal calvarial bone and the effect of micro-CT radiation exposure on neonatal calvarial bone viability were investigated. The calvarial bones of 4-day-old CD-1 mice were cultured in Dulbecco's modified Eagle's medium (DMEM) or osteogenic medium (OM) for 23 days. Micro-CT scanning and histological analysis were performed on days 2, 9, 16 and 23. An "OM-control" group was scanned only on days 2 and 23 to evaluate the effect of a single micro-CT radiation dose on calvarial bones. Histomorphometric measurements revealed that the number of osteoblasts per unit bone surface area (N. Ob/BS, /mm2) (days 9, 16 and 23) and the number of osteoclasts per unit bone surface area (N. Oc/BS, /mm2) (days 9 and 16) were higher and lower, respectively, in the OM group than in the DMEM group. Moreover, the calvarial bone survived for at least 16 days in vitro, as indicated by tartrate-resistant acid phosphatase (TRAP)-positive staining. Micro-CT assessment revealed that the bone surface (BS), bone volume (BV), bone surface density (BS/TV(Tissue volume)) and percent bone volume (BV/TV) were greater in the OM group than in the DMEM group except at baseline on day 2. All bone parameters of calvariae cultured in OM and OM-control conditions were not significantly different on days 2 and 23. Thus, the radiation dose from micro-CT in our study design had no perceptible effect on the formation of mouse calvarial bone in vitro.
Assuntos
Desenvolvimento Ósseo/fisiologia , Osso e Ossos/metabolismo , Osteoblastos/citologia , Osteogênese/fisiologia , Crânio/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Camundongos , Microtomografia por Raio-X/métodosRESUMO
BACKGROUND: Deleterious mutations in KCNQ1 may lead to an autosomal dominant form of long QT syndrome (LQTS) (Romano-Ward) or autosomal recessive form (Jervell and Lange-Nielsen). Both are associated with severe ventricular tachyarrhythmias due to the reduction of the slowly activating delayed rectifier K+ current (IKs). Our objective was to investigate the functional consequences of KCNQ1-R562S mutation in an atypical form of KCNQ1-linked LQTS. METHODS: Mutant KCNQ1-R562S was analyzed via confocal imaging, surface biotinylation assays, co-immunoprecipitation, phosphatidylinositol-4,5-bisphosphate pulldown test, whole-cell patch clamp, and computational intrinsic disorder analyses. RESULTS: Protein expression, assembly with KCNE1, and trafficking to the surface membrane of KCNQ1-R562S were comparable with wild-type channels. The most significant functional effect of the R562S mutation was a depolarizing shift in the voltage dependence of activation that was dependent on association with KCNE1. The biophysical abnormality was only partially dominant over coexpressed wild-type channels. R562S mutation impaired C-terminal association with membrane phosphatidylinositol-4,5-bisphosphate. These changes led to compromised rate-related accumulation of repolarizing current that is an important property of normal IKs. CONCLUSIONS: KCNQ1-R562S mutation reduces effective IKs due to channel gating alteration with a mild clinical expression in the heterozygous state due to minimal dominant phenotype. In the homozygous state, it is exhibited with a moderately severe LQTS phenotype due to the incomplete absence of IKs.
Assuntos
Canal de Potássio KCNQ1/genética , Síndrome de Romano-Ward/genética , Biotinilação/métodos , Heterozigoto , Homozigoto , Humanos , Imunoprecipitação/métodos , Microscopia Confocal/métodos , Mutação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Índice de Gravidade de DoençaRESUMO
When grown in culture Mycobacterium smegmatis metabolized S-nitrosoglutathione to oxidized glutathione and nitrate, which suggested a possible involvement of an S-nitrosothiol reductase and mycobacterial haemoglobin. The mycothiol-dependent formaldehyde dehydrogenase from M. smegmatis was purified by a combination of Ni2+-IMAC (immobilized metal ion affinity chromatography), hydrophobic interaction, anion-exchange and affinity chromatography. The enzyme had a subunit molecular mass of 38263 kDa. Steady-state kinetic studies indicated that the enzyme catalyses the NAD+-dependent conversion of S-hydroxymethylmycothiol into formic acid and mycothiol by a rapid-equilibrium ordered mechanism. The enzyme also catalysed an NADH-dependent decomposition of S-nitrosomycothiol (MSNO) by a sequential mechanism and with an equimolar stoichiometry of NADH:MSNO, which indicated that the enzyme reduces the nitroso group to the oxidation level of nitroxyl. Vmax for the MSNO reductase reaction indicated a turnover per subunit of approx. 116700 min(-1), which was 76-fold faster than the formaldehyde dehydrogenase activity. A gene, Rv2259, annotated as a class III alcohol dehydrogenase in the Mycobacterium tuberculosis genome was cloned and expressed in M. smegmatis as the C-terminally His6-tagged product. The purified recombinant enzyme from M. tuberculosis also catalysed both activities. M. smegmatis S-nitrosomycothiol reductase converted MSNO into the N -hydroxysulphenamide, which readily rearranged to mycothiolsulphinamide. In the presence of MSNO reductase, M. tuberculosis HbN (haemoglobin N) was converted with low efficiency into metHbN [HbN(Fe3+)] and this conversion was dependent on turnover of MSNO reductase. These observations suggest a possible route in vivo for the dissimilation of S-nitrosoglutathione.
Assuntos
Dissacarídeos/metabolismo , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Pirazóis/metabolismo , S-Nitrosoglutationa/metabolismo , Compostos de Sulfidrila/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/isolamento & purificação , Aldeído Oxirredutases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Carboidratos , Clonagem Molecular , Cisteína , Estabilidade Enzimática , Glicopeptídeos , Concentração de Íons de Hidrogênio , Inositol , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5'-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5'-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5'-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5'-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket.
Assuntos
Metiltioinosina/análogos & derivados , Plasmodium falciparum/enzimologia , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cinética , Metiltioinosina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/genética , Pirimidinonas/metabolismo , Pirimidinonas/farmacologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified a Trypanosoma cruzi homologue of the MAPK family that we have called TcMAPK2. Sequence analyses demonstrates TcMAPK2 has high homology with lower eukaryotic ERK2 but has significant differences from mammalian ERK2. Enzymatic assays of both recombinant TcMAPK2 and native protein obtained by immunoprecipitation using anti-TcMAPK2 demonstrated that both preparations of TcMAPK2 were catalytically active. Immunofluorescence analysis of the subcellular localization of TcMAPK2 determined it is mainly cytoplasmic in epimastigotes, along the flagella in trypomastigotes and on the plasma membrane of intracellular amastigotes. Phosphorylated TcMAPK2 was highest in trypomastigotes and lowest in amastigotes. Recombinant TcMAPK2 was able to phosphorylate the recombinant protein of a cAMP specific phosphodiesterase. Overexpression of TcMAPK2 in epimastigotes inhibited growth and development leading to death. TcMAPK2 has an important role in the stress response of the parasite and may be important in regulating proliferation and differentiation.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/genética , Dados de Sequência Molecular , Fosforilação , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
The KCNE1 gene product (minK protein) associates with the cardiac KvLQT1 potassium channel (encoded by KCNQ1) to create the cardiac slowly activating delayed rectifier, I(Ks). Mutations throughout both genes are linked to the hereditary cardiac arrhythmias in the Long QT Syndrome (LQTS). KCNE1 exerts its specific regulation of KCNQ1 activation via interactions between membrane-spanning segments of the two proteins. Less detailed attention has been focused on the role of the KCNE1 C-terminus in regulating channel behavior. We analyzed the effects of an LQT5 point mutation (D76N) and the truncation of the entire C-terminus (Delta70) on channel regulation, assembly and interaction. Both mutations significantly shifted voltage dependence of activation in the depolarizing direction and decreased I(Ks) current density. They also accelerated rates of channel deactivation but notably, did not affect activation kinetics. Truncation of the C-terminus reduced the apparent affinity of KCNE1 for KCNQ1, resulting in impaired channel formation and presentation of KCNQ1/KCNE1 complexes to the surface. Complete saturation of KCNQ1 channels with KCNE1-Delta70 could be achieved by relative over-expression of the KCNE subunit. Rate-dependent facilitation of K(+) conductance, a key property of I(Ks) that enables action potential shortening at higher heart rates, was defective for both KCNE1 C-terminal mutations, and may contribute to the clinical phenotype of arrhythmias triggered by heart rate elevations during exercise in LQTS mutations. These results support several roles for KCNE1 C-terminus interaction with KCNQ1: regulation of channel assembly, open-state destabilization, and kinetics of channel deactivation.
Assuntos
Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Humanos , Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Ligação ProteicaRESUMO
Streptococcus pneumoniae 5'-methylthioadenosine/S-adenosylhomocysteine hydrolase (MTAN) catalyzes the hydrolytic deadenylation of its substrates to form adenine and 5-methylthioribose or S-ribosylhomocysteine (SRH). MTAN is not found in mammals but is involved in bacterial quorum sensing. MTAN gene disruption affects the growth and pathogenicity of bacteria, making it a target for antibiotic design. Kinetic isotope effects and computational studies have established a dissociative S(N)1 transition state for Escherichia coli MTAN, and transition state analogues resembling the transition state are powerful inhibitors of the enzyme [Singh, V., Lee, J. L., Núñez, S., Howell, P. L., and Schramm, V. L. (2005) Biochemistry 44, 11647-11659]. The sequence of MTAN from S. pneumoniae is 40% identical to that of E. coli MTAN, but S. pneumoniae MTAN exhibits remarkably distinct kinetic and inhibitory properties. 5'-Methylthio-Immucillin-A (MT-ImmA) is a transition state analogue resembling an early S(N)1 transition state. It is a weak inhibitor of S. pneumoniae MTAN with a K(i) of 1.0 microM. The X-ray structure of S. pneumoniae MTAN with MT-ImmA indicates a dimer with the methylthio group in a flexible hydrophobic pocket. Replacing the methyl group with phenyl (PhT-ImmA), tolyl (p-TolT-ImmA), or ethyl (EtT-ImmA) groups increases the affinity to give K(i) values of 335, 60, and 40 nM, respectively. DADMe-Immucillins are geometric and electrostatic mimics of a fully dissociated transition state and bind more tightly than Immucillins. MT-DADMe-Immucillin-A inhibits with a K(i) value of 24 nM, and replacing the 5'-methyl group with p-Cl-phenyl (p-Cl-PhT-DADMe-ImmA) gave a K(i) value of 0.36 nM. The inhibitory potential of DADMe-Immucillins relative to the Immucillins supports a fully dissociated transition state structure for S. pneumoniae MTAN. Comparison of active site contacts in the X-ray crystal structures of E. coli and S. pneumoniae MTAN with MT-ImmA would predict equal binding, yet most analogues bind 10(3)-10(4)-fold more tightly to the E. coli enzyme. Catalytic site efficiency is primarily responsible for this difference since k(cat)/K(m) for S. pneumoniae MTAN is decreased 845-fold relative to that of E. coli MTAN.
Assuntos
N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Streptococcus pneumoniae/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Catálise/efeitos dos fármacos , Cristalografia por Raios X/métodos , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , N-Glicosil Hidrolases/genética , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ribitol/análogos & derivados , Ribitol/farmacologia , Análise de Sequência de Proteína , Transdução de Sinais/efeitos dos fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Ketopantoate reductase (EC 1.1.1.169), an enzyme in the pantothenate biosynthetic pathway, catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate. The enzyme exhibits high specificity for ketopantoate, with V and V/K for ketopantoate being 5- and 365-fold higher than those values for alpha-ketoisovalerate and 20- and 648-fold higher than those values for alpha-keto-beta-methyl-n-valerate, respectively. For pyridine nucleotides, V/K for beta-NADPH is 3-500-fold higher than that for other nucleotide substrates. The magnitude of the primary deuterium kinetic isotope effects on V and V/K varied substantially when different ketoacid and pyridine nucleotide substrates were used. The small primary deuterium kinetic isotope effects observed using NADPH and NHDPH suggest that the chemical step is not rate-limiting, while larger primary deuterium isotope effects were observed for poor ketoacid and pyridine nucleotide substrates, indicating that the chemical reaction has become partially or completely rate-limiting. The pH dependence of (D)V using ketopantoate was observed to vary from a value of 1.1 at low pH to a value of 2.5 at high pH, while the magnitude of (D)V/K(NADPH) and (D)V/K(KP) were pH-independent. The value of (D)V is large and pH-independent when alpha-keto-beta-methyl-n-valerate was used as the ketoacid substrate. Solvent kinetic isotope effects of 2.2 and 1.2 on V and V/K, respectively, were observed with alpha-keto-beta-methyl-n-valerate. Rapid reaction analysis of NADPH oxidation using ketopantoate showed no "burst" phase, suggesting that product-release steps are not rate-limiting and the cause of the small observed kinetic isotope effects with this substrate pair. Large primary deuterium isotope effects on V and V/K using 3-APADPH in steady-state experiments, equivalent to the isotope effect observed in single turnover studies, suggests that chemistry is rate-limiting for this poorer reductant. These results are discussed in terms of a kinetic and chemical mechanism for the enzyme.
Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Escherichia coli/enzimologia , Calorimetria/métodos , Catálise , Deutério , Concentração de Íons de Hidrogênio , Isótopos , Cetoácidos/metabolismo , Cinética , NADP/análogos & derivados , NADP/metabolismo , Oxirredução , Ácido Pantotênico/análogos & derivados , Ácido Pantotênico/biossíntese , Especificidade por Substrato , TermodinâmicaRESUMO
Dihydrodipicolinate reductase (DHPR) catalyzes the reduced pyridine nucleotide-dependent reduction of the alpha,beta-unsaturated cyclic imine, dihydrodipicolinate, to generate tetrahydrodipicolinate. This enzyme catalyzes the second step in the bacterial biosynthetic pathway that generates meso-diaminopimelate, a component of bacterial cell walls, and the amino acid L-lysine. The Mycobacterium tuberculosis dapB-encoded DHPR has been cloned, expressed, purified, and crystallized in two ternary complexes with NADH or NADPH and the inhibitor 2,6-pyridinedicarboxylate (2,6-PDC). The structures have been solved using molecular replacement strategies, and the DHPR-NADH-2,6-PDC and DHPR-NADPH-2,6-PDC complexes have been refined against data to 2.3 and 2.5 A, respectively. The M. tuberculosis DHPR is a tetramer of identical subunits, with each subunit composed of two domains connected by two flexible hinge regions. The N-terminal domain binds pyridine nucleotide, while the C-terminal domain is involved in both tetramer formation and substrate/inhibitor binding. The M. tuberculosis DHPR uses NADH and NADPH with nearly equal efficiency based on V/K values. To probe the nature of this substrate specificity, we have generated two mutants, K9A and K11A, residues that are close to the 2'-phosphate of NADPH. These two mutants exhibit decreased specificity for NADPH by factors of 6- and 30-fold, respectively, but the K11A mutant exhibits 270% of WT activity using NADH. The highly conserved structure of the nucleotide fold may permit other enzyme's nucleotide specificity to be altered using similar mutagenic strategies.
Assuntos
Inibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimologia , NADP/química , NADP/metabolismo , NAD/química , NAD/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/química , Oxirredutases/metabolismo , Ácidos Picolínicos/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Di-Hidrodipicolinato Redutase , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Oxirredutases/genética , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The panB gene that encodes ketopantoate hydroxymethyltransferase has been cloned from Mycobacterium tuberculosis, expressed, and purified to homogeneity. 1H NMR spectroscopy was used to determine the rate of (i) tetrahydrofolate-independent hydroxymethyltransferase chemistry between formaldehyde and alpha-ketoisovalerate and (ii) deuterium exchange in the methylenetetrahydrofolate-independent enolization of alpha-ketoisovalerate and other alpha-keto acids, catalyzed by PanB. These studies have demonstrated that substrate enolization by PanB is divalent metal-dependent with a preference of Mg2+ > Zn2+ > Co2+ > Ni2+ > Ca2+. The rate of enolization is pH-dependent with optimal activity in the range of 7.0-7.5. The pH profile was bell-shaped, depending on the ionization state of two ionizable groups with apparent pK values of 6.2 and 8.3. Enolization and isotope exchange occurs with some alpha-keto acids (e.g., pyruvate and alpha-ketobutyrate), resulting in the complete exchange of all beta-hydrogens. Enzyme-catalyzed enolization and isotope exchange occur with other long-chain and branched alpha-keto acids, resulting in the stereospecific exchange of only one of the beta-hydrogen atoms. These results are discussed in the context of steric restrictions present in the enzyme active site and the stereochemistry of base-catalyzed isotope exchange.
Assuntos
Proteínas de Bactérias/química , Hidroximetil e Formil Transferases/química , Cetoácidos/química , Mycobacterium tuberculosis/enzimologia , Tetra-Hidrofolatos/química , Aminoácidos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Catálise , Deutério/química , Ativação Enzimática , Hemiterpenos , Concentração de Íons de Hidrogênio , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/isolamento & purificação , Metais/química , Conformação Molecular , Mycobacterium tuberculosis/genética , Ressonância Magnética Nuclear Biomolecular , Ácido Pantotênico/química , Prótons , Ácido Pirúvico/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrofotometria , Especificidade por SubstratoRESUMO
Pantothenate synthetase (EC 6.3.2.1) catalyzes the formation of pantothenate from ATP, D-pantoate, and beta-alanine in bacteria, yeast, and plants. The three-dimensional structural determination of pantothenate synthetase from Mycobacterium tuberculosis has indicated specific roles for His44, His47, Asn69, Gln72, Lys160, and Gln164 residues in the binding of substrates and the pantoyl adenylate intermediate. To evaluate the functional roles of these strictly conserved residues, we constructed six Ala mutants and determined their catalytic properties. The substitution of alanine for H44, H47, N69, Q72, and K160 residues in M. tuberculosis pantothenate synthetase caused a greater than 1000-fold reduction in enzyme activity, while the Q164A mutant exhibited 50-fold less activity. The rate of the isolated adenylation reaction in single turnover studies was also reduced 40-1000-fold by the replacement of one of these six amino acids with alanine, suggesting that these residues are essential for the formation of the pantoyl adenylate intermediate. The rate of pantothenate formation from the adenylate and beta-alanine in the second half reaction could not be measured for the H44A, H47A, N69A, Q72A, and K160A mutants and was reduced 40-fold in the Q164A mutants. The activity of the K160C mutant enzyme was markedly enhanced by the alkylation of cysteine with bromoethylamine, further supporting the critical role of the K160 residue in pantoyl adenylate formation. Isothermal titration microcalorimetry analysis demonstrated that the substitution of either H47 or K160 for Ala resulted in a decreased affinity of the enzyme for ATP. These results indicate that the highly conserved His44, His47, Asn69, Gln72, Lys160 and residues are essential for the formation and stabilization of pantoyl adenylate intermediate in the pantothenate synthetase reaction.
Assuntos
Monofosfato de Adenosina/metabolismo , Aminoácidos/química , Mycobacterium tuberculosis/enzimologia , Peptídeo Sintases/química , Sítios de Ligação , Sequência Conservada , Ácido Pantotênico/metabolismo , Peptídeo Sintases/metabolismoRESUMO
The aacA29b gene, which confers an atypical aminoglycoside resistance pattern to Escherichia coli, was identified on a class 1 integron from a multidrug-resistant isolate of Pseudomonas aeruginosa. On the basis of amino acid sequence homology, it was proposed that the gene encoded a 6'-N-acetyltransferase. The resistance gene was cloned into the pET23a(+) vector, and overexpression conferred high-level resistance to the usual substrates of the aminoglycoside N-acetyltransferase AAC(6')-I, except netilmicin. The level of resistance conferred by aacA29b correlated perfectly with the level of expression of the gene. The corresponding C-terminal six-His-tagged AAC(6')-29b protein was purified and found to exist as a dimer in solution. With a spectrophotometric assay, an extremely feeble AAC activity was detected with acetyl coenzyme A (acetyl-CoA) as an acetyl donor. Fluorescence titrations of the protein with aminoglycosides demonstrated the very tight binding of tobramycin, dibekacin, kanamycin A, sisomicin (K(d), =1 micro M) and a weaker affinity for amikacin (K(d), approximately 60 micro M). The binding of netilmicin and acetyl-CoA could not be detected by either fluorescence spectroscopy or isothermal titration calorimetry. The inability of AAC(6')-29b to efficiently bind acetyl-CoA is supported by an alignment analysis of its amino acid sequence compared with those of other AAC(6')-I family members. AAC(6')-29b lacks a number of residues involved in acetyl-CoA binding. These results lead to the conclusion that AAC(6')-29b is able to confer aminoglycoside resistance by sequestering the drug as a result of tight binding.
Assuntos
Acetiltransferases/genética , Acetiltransferases/metabolismo , Antibacterianos/farmacologia , Acetiltransferases/química , Sequência de Aminoácidos , Aminoglicosídeos , Antibacterianos/metabolismo , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Dados de Sequência MolecularRESUMO
Pantothenate synthetase from Mycobacterium tuberculosis catalyzes the formation of pantothenate from ATP, D-pantoate, and beta-alanine. The formation of a kinetically competent pantoyl-adenylate intermediate was established by the observation of a positional isotope exchange (PIX) reaction within (18)O-labeled ATP in the presence of d-pantoate. When [betagamma-(18)O(6)]-ATP was incubated with pantothenate synthetase in the presence of d-pantoate, an (18)O label gradually appeared in the alphabeta-bridge position from both the beta- and the gamma-nonbridge positions. The rates of these two PIX reactions were followed by (31)P NMR spectroscopy and found to be identical. These results are consistent with the formation of enzyme-bound pantoyl-adenylate and pyrophosphate upon the mixing of ATP, D-pantoate, and enzyme. In addition, these results require the complete torsional scrambling of the two phosphoryl groups of the labeled pyrophosphate product. The rate of the PIX reaction increased as the D-pantoate concentration was elevated and then decreased to zero at saturating levels of D-pantoate. These inhibition results support the ordered binding of ATP and D-pantoate to the enzyme active site. The PIX reaction was abolished with the addition of pyrophosphatase; thus, PP(i) must be free to dissociate from the active site upon formation of the pantoyl-adenylate intermediate. The PIX reaction rate diminished when the concentrations of ATP and D-pantoate were held constant and the concentration of the third substrate, beta-alanine, was increased. This observation is consistent with a kinetic mechanism that requires the binding of beta-alanine after the release of pyrophosphate from the active site of pantothenate synthetase. Positional isotope exchange reactions have therefore demonstrated that pantothenate synthetase catalyzes the formation of a pantoyl-adenylate intermediate upon the ordered addition of ATP and pantoate.