RESUMO
Iron-sulfur clusters are protein cofactors with an ancient evolutionary origin. These clusters are best known for their roles in redox proteins such as ferredoxins, but some iron-sulfur clusters have nonredox roles in the active sites of enzymes. Such clusters are often prone to oxidative degradation, making the enzymes difficult to characterize. Here we report a structural and functional characterization of dihydroxyacid dehydratase (DHAD) from Mycobacterium tuberculosis (Mtb), an essential enzyme in the biosynthesis of branched-chain amino acids. Conducting this analysis under fully anaerobic conditions, we solved the DHAD crystal structure, at 1.88 Å resolution, revealing a 2Fe-2S cluster in which one iron ligand is a potentially exchangeable water molecule or hydroxide. UV and EPR spectroscopy both suggested that the substrate binds directly to the cluster or very close to it. Kinetic analysis implicated two ionizable groups in the catalytic mechanism, which we postulate to be Ser-491 and the iron-bound water/hydroxide. Site-directed mutagenesis showed that Ser-491 is essential for activity, and substrate docking indicated that this residue is perfectly placed for proton abstraction. We found that a bound Mg2+ ion 6.5 Å from the 2Fe-2S cluster plays a key role in substrate binding. We also identified a putative entry channel that enables access to the cluster and show that Mtb-DHAD is inhibited by a recently discovered herbicide, aspterric acid, that, given the essentiality of DHAD for Mtb survival, is a potential lead compound for the design of novel anti-TB drugs.
Assuntos
Aminoácidos de Cadeia Ramificada/biossíntese , Hidroliases/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mycobacterium tuberculosis/química , Aminoácidos de Cadeia Ramificada/química , Sítios de Ligação , Hidroliases/química , Proteínas Ferro-Enxofre/química , Modelos Moleculares , Conformação Molecular , Mycobacterium tuberculosis/metabolismoRESUMO
The biosynthesis of branched-chain amino acids or BCAAs (l-isoleucine, l-leucine, and l-valine) is essential in eubacteria, but mammals are branched-chain amino acid auxotrophs, making the enzymes in the pathway excellent targets for antibacterial drug development. The biosynthesis of l-isoleucine, l-leucine, and l-valine is very efficient, requiring only eight enzymes. Threonine dehydratase (TD), a pyridoxal 5'-phosphate (PLP)-dependent enzyme encoded by the ilvA gene, is the enzyme responsible for the conversion of l-threonine (l-Thr) to α-ketobutyrate, ammonia, and water, which is the first step in the biosynthesis of l-isoleucine. We have cloned, expressed, and biochemically characterized the reaction catalyzed by Mycobacterium smegmatis TD (abbreviated as MsIlvA) using steady-state kinetics and kinetic isotope effects. We show here that in addition to l-threonine, l-allo-threonine and l-serine are also used as substrates by TD, and all exhibit sigmoidal, non-Michaelis-Menten kinetics. Curiously, ß-chloro-l-alanine was also a substrate rather than an inhibitor as expected. The enzymatic activity of TD is sensitive to the presence of allosteric regulators, including the activator l-valine or the end product feedback inhibitor of the BCAA pathway in which TD is involved, l-isoleucine. Primary deuterium kinetic isotopes are small, suggesting Cα proton abstraction is only partially rate-limiting. Solvent kinetic isotopes were significantly larger, indicating that a proton transfer occurring during the reaction is also partially rate-limiting. Finally, we demonstrate that l-cycloserine, a general inhibitor of PLP-dependent enzymes, is an excellent inhibitor of threonine deaminase.
Assuntos
Proteínas de Bactérias/química , Mycobacterium smegmatis/enzimologia , Treonina Desidratase/química , Regulação Alostérica/fisiologia , Proteínas de Bactérias/metabolismo , Catálise , Cinética , Especificidade por Substrato/fisiologia , Treonina Desidratase/metabolismoRESUMO
l-Aspartate oxidase, encoded by the nadB gene, is the first enzyme in the de novo synthesis of NAD+ in bacteria. This FAD-dependent enzyme catalyzes the oxidation of l-aspartate to generate iminoaspartate and reduced flavin. Distinct from most amino acid oxidases, it can use either molecular oxygen or fumarate to reoxidize the reduced enzyme. Sequence alignments and the three-dimensional crystal structure have revealed that the overall fold and catalytic residues of NadB closely resemble those of the succinate dehydrogenase/fumarate reductase family rather than those of the prototypical d-amino acid oxidases. This suggests that the enzyme can catalyze amino acid oxidation via typical amino acid oxidase chemistry, involving the removal of protons from the α-amino group and the transfer of the hydride from C2, or potentially deprotonation at C3 followed by transfer of the hydride from C2, similar to chemistry occurring during succinate oxidation. We have investigated this potential mechanistic ambiguity using a combination of primary, solvent, and multiple deuterium kinetic isotope effects in steady state experiments. Our results indicate that the chemistry is similar to that of typical amino acid oxidases in which the transfer of the hydride from C2 of l-aspartate to FAD is rate-limiting and occurs in a concerted manner with respect to deprotonation of the α-amine. Together with previous kinetic and structural data, we propose that NadB has structurally evolved from succinate dehydrogenase/fumarate reductase-type enzymes to gain the new functionality of oxidizing amino acids while retaining the ability to reduce fumarate.
Assuntos
Aminoácido Oxirredutases/metabolismo , Ácido Aspártico/metabolismo , Coenzimas/metabolismo , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Modelos Moleculares , Algoritmos , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/genética , Animais , Ácido Aspártico/química , Sítios de Ligação , Biocatálise , Domínio Catalítico , Coenzimas/química , Medição da Troca de Deutério , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Flavina-Adenina Dinucleotídeo/química , Concentração de Íons de Hidrogênio , Cinética , Malato Desidrogenase/metabolismo , Oxirredução , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sus scrofaRESUMO
The eight enzymes responsible for the biosynthesis of the three branched-chain amino acids (l-isoleucine, l-leucine, and l-valine) were identified decades ago using classical genetic approaches based on amino acid auxotrophy. This review will highlight the recent progress in the determination of the three-dimensional structures of these enzymes, their chemical mechanisms, and insights into their suitability as targets for the development of antibacterial agents. Given the enormous rise in bacterial drug resistance to every major class of antibacterial compound, there is a clear and present need for the identification of new antibacterial compounds with nonoverlapping targets to currently used antibacterials that target cell wall, protein, mRNA, and DNA synthesis.
Assuntos
Aminoácidos de Cadeia Ramificada/biossíntese , Antibacterianos/farmacologia , Enzimas/efeitos dos fármacos , Antibacterianos/química , Enzimas/química , Conformação MolecularRESUMO
Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests thatMtbrelies mainly on fatty acid catabolism in the host. However,Mtbalso maintains a functional glycolytic pathway and its role in the cellular metabolism ofMtbhas yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and theMtbgenome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show thatpykAencodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion ofpykApreventsMtbgrowth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism inMtb.
Assuntos
Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Glicólise/genética , Mycobacterium tuberculosis/metabolismo , Piruvato Quinase/metabolismo , Ácido Aconítico/metabolismo , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Regulação Alostérica , Animais , Proteínas de Bactérias/genética , Ácido Cítrico/metabolismo , Meios de Cultura/química , Ativação Enzimática , Ácidos Graxos Voláteis/farmacologia , Feminino , Deleção de Genes , Expressão Gênica , Glucose/metabolismo , Glucose-6-Fosfato/metabolismo , Glucose-6-Fosfato/farmacologia , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Glicólise/efeitos dos fármacos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Camundongos , Camundongos SCID , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Fosfoenolpiruvato/metabolismo , Aldeído Pirúvico/metabolismo , Piruvato Quinase/genética , Análise de Sobrevida , Tuberculose/microbiologia , Tuberculose/mortalidadeRESUMO
Iron is an essential element for life, but its soluble form is scarce in the environment and is rarer in the human body. Mtb (Mycobacterium tuberculosis) produces two aryl-capped siderophores, mycobactin (MBT) and carboxymycobactin (cMBT), to chelate intracellular iron. The adenylating enzyme MbtA catalyzes the first step of mycobactin biosynthesis in two half-reactions: activation of the salicylic acid as an acyl-adenylate and ligation onto the acyl carrier protein (ACP) domain of MbtB to form covalently salicylated MbtB-ACP. We report the first apo-MbtA structure from Mycobacterium smegmatis at 2.3 Å. We demonstrate here that MbtA activity can be reversibly, post-translationally regulated by acetylation. Indeed the mycobacterial Pat (protein lysine acetyltransferase), Rv0998, specifically acetylates MbtA on lysine 546, in a cAMP-dependent manner, leading to enzyme inhibition. MbtA acetylation can be reversed by the NAD+-dependent DAc (deacetyltransferase), Rv1151c. Deletion of Pat and DAc genes in Mtb revealed distinct phenotypes for strains lacking one or the other gene at low pH and limiting iron conditions. This study establishes a direct connection between the reversible acetylation system Pat/DAc and the ability of Mtb to adapt in limited iron conditions, which is critical for mycobacterial infection.
Assuntos
Ligases/metabolismo , Mycobacterium tuberculosis/enzimologia , Oxazóis/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Sideróforos/biossíntese , Acetilação , Catálise , Humanos , Ligases/genética , Lisina Acetiltransferases/genética , Lisina Acetiltransferases/metabolismo , Mycobacterium tuberculosis/genética , Domínios Proteicos , Sideróforos/genéticaRESUMO
Tuberculosis (TB) due to Mycobacterium tuberculosis remains a major global infectious disease problem, and a more efficacious vaccine is urgently needed for the control and prevention of disease caused by this organism. We previously reported that a genetically modified strain of Mycobacterium smegmatis called IKEPLUS is a promising TB vaccine candidate. Since protective immunity induced by IKEPLUS is dependent on antigen-specific CD4+ T cell memory, we hypothesized that the specificity of the CD4+ T cell response was a critical feature of this protection. Using in vitro assays of interferon gamma production (enzyme-linked immunosorbent spot [ELISPOT] assays) by splenocytes from IKEPLUS-immunized C57BL/6J mice, we identified an immunogenic peptide within the mycobacterial ribosomal large subunit protein RplJ, encoded by the Rv0651 gene. In a complementary approach, we generated major histocompatibility complex (MHC) class II-restricted T cell hybridomas from IKEPLUS-immunized mice. Screening of these T cell hybridomas against IKEPLUS and ribosomes enriched from IKEPLUS suggested that the CD4+ T cell response in IKEPLUS-immunized mice was dominated by the recognition of multiple components of the mycobacterial ribosome. Importantly, CD4+ T cells specific for mycobacterial ribosomes accumulate to significant levels in the lungs of IKEPLUS-immunized mice following aerosol challenge with virulent M. tuberculosis, consistent with a role for these T cells in protective host immunity in TB. The identification of CD4+ T cell responses to defined ribosomal protein epitopes expands the range of antigenic targets for adaptive immune responses to M. tuberculosis and may help to inform the design of more effective vaccines against tuberculosis.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Mycobacterium/imunologia , Tuberculose/imunologia , Tuberculose/microbiologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Linfócitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos de Linfócito T/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Imunização , Camundongos , Mycobacterium/patogenicidade , Peptídeos/química , Peptídeos/imunologia , Proteínas Ribossômicas/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Tuberculose/mortalidade , VirulênciaRESUMO
The GCN5-related N-acetyltransferases family (GNAT) is an important family of proteins that includes more than 100000 members among eukaryotes and prokaryotes. Acetylation appears as a major regulatory post-translational modification and is as widespread as phosphorylation. N-Acetyltransferases transfer an acetyl group from acetyl-CoA to a large array of substrates, from small molecules such as aminoglycoside antibiotics to macromolecules. Acetylation of proteins can occur at two different positions, either at the amino-terminal end (αN-acetylation) or at the ε-amino group (εN-acetylation) of an internal lysine residue. GNAT members have been classified into different groups on the basis of their substrate specificity, and in spite of a very low primary sequence identity, GNAT proteins display a common and conserved fold. This Current Topic reviews the different classes of bacterial GNAT proteins, their functions, their structural characteristics, and their mechanism of action.
Assuntos
Acetiltransferases/metabolismo , Aminoglicosídeos/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Farmacorresistência Bacteriana , Modelos Moleculares , Acetilação , Acetiltransferases/química , Acetiltransferases/classificação , Aminoaciltransferases/química , Aminoaciltransferases/classificação , Aminoaciltransferases/metabolismo , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Acetiltransferases N-Terminal/química , Acetiltransferases N-Terminal/classificação , Acetiltransferases N-Terminal/metabolismo , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
The biosynthetic pathway of the branched-chain amino acids is essential for Mycobacterium tuberculosis growth and survival. We report here the kinetic and chemical mechanism of the pyridoxal 5'-phosphate (PLP)-dependent branched-chain aminotransferase, IlvE, from M. tuberculosis (MtIlvE). This enzyme is responsible for the final step of the synthesis of the branched-chain amino acids isoleucine, leucine, and valine. As seen in other aminotransferases, MtIlvE displays a ping-pong kinetic mechanism. pK values were identified from the pH dependence on V as well as V/K, indicating that the phosphate ester of the PLP cofactor, and the α-amino group from l-glutamate and the active site Lys204, play roles in acid-base catalysis and binding, respectively. An intrinsic primary kinetic isotope effect was identified for the α-C-H bond cleavage of l-glutamate. Large solvent kinetic isotope effect values for the ping and pong half-reactions were also identified. The absence of a quininoid intermediate in combination with the Dkobs in our multiple kinetic isotope effects under single-turnover conditions suggests a concerted type of mechanism. The deprotonation of C2 of l-glutamate and the protonation of C4' of the PLP cofactor happen synchronously in the ping half-reaction. A chemical mechanism is proposed on the basis of the results obtained here.
Assuntos
Proteínas de Bactérias/química , Mycobacterium tuberculosis/enzimologia , Conformação Proteica , Transaminases/química , Proteínas de Bactérias/metabolismo , Biocatálise , Vias Biossintéticas , Domínio Catalítico , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Isoleucina/química , Isoleucina/metabolismo , Cinética , Leucina/química , Leucina/metabolismo , Lisina/química , Lisina/metabolismo , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Transaminases/metabolismo , Valina/química , Valina/metabolismoRESUMO
Mycobacterium tuberculosis is intrinsically resistant to most ß-lactam antibiotics because of the constitutive expression of the blaC-encoded ß-lactamase. This enzyme has extremely high activity against penicillins and cephalosporins, but weaker activity against carbapenems. The enzyme can be inhibited by clavulanate, avibactam, and boronic acids. In this study, we investigated the ability of 6-methylidene ß-lactams to inhibit BlaC. One such compound, penem 2, inhibited BlaC more than 70 times more efficiently than clavulanate. The compound forms a covalent complex with BlaC as shown by mass spectrometry. Crystallization of the complex revealed that the bound inhibitor was covalently attached via the Ser70 active site residue and that the covalently, acylated form of the inhibitor had undergone additional chemistry yielding a 4,7-thiazepine ring in place of the ß-lactam and a thiazapyroline ring generated as a result of ß-lactam ring opening. The stereochemistry of the product of the 7-endo-trig cyclization was the opposite of that observed previously for class A and D ß-lactamases. Addition of penem 2 greatly synergized the antibacterial properties of both ampicillin and meropenem against a growing culture of M. tuberculosis. Strikingly, penem 2 alone showed significant growth inhibition, suggesting that in addition to its capability of efficiently inhibiting BlaC, it also inhibited the peptidoglycan cross-linking transpeptidases.
Assuntos
Antituberculosos/química , Proteínas de Bactérias/química , Cefalosporinas/química , Mycobacterium tuberculosis/enzimologia , beta-Lactamases/química , Ampicilina/química , Ampicilina/farmacologia , Antituberculosos/farmacologia , Cefalosporinas/farmacologia , Sinergismo Farmacológico , Cinética , Meropeném , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Tienamicinas/química , Tienamicinas/farmacologiaRESUMO
The genome of Mycobacterium tuberculosis contains a gene, blaC, which encodes a highly active ß-lactamase (BlaC). We have previously shown that BlaC has an extremely broad spectrum of activity against penicillins and cephalosporins but weak activity against newer carbapenems. We have shown that carbapenems such as meropenem, doripenem, and ertapenem react with the enzyme to form enzyme-drug covalent complexes that are hydrolyzed extremely slowly. In the current study, we have determined apparent Km and kcat values of 0.8 µM and 0.03 min(-1), respectively, for tebipenem, a novel carbapenem whose prodrug form, the pivalyl ester, is orally available. Tebipenem exhibits slow tight-binding inhibition at low micromolar concentrations versus the chromogenic substrate nitrocefin. FT-ICR mass spectrometry demonstrated that the tebipenem acyl-enzyme complex remains stable for greater than 90 min and exists as mixture of the covalently bound drug and the bound retro-aldol cleavage product. We have also determined the high-resolution crystal structures of the BlaC-tebipenem covalent acylated adduct (1.9 Å) with wild-type BlaC and the BlaC-tebipenem Michaelis-Menten complex (1.75 Å) with the K73A BlaC variant. These structures are compared to each other and to other carbapenem-BlaC structures.
Assuntos
Carbapenêmicos/metabolismo , Mycobacterium tuberculosis/metabolismo , Inibidores de beta-Lactamases , beta-Lactamases/metabolismo , Carbapenêmicos/química , Carbapenêmicos/farmacologia , Cristalização , Cristalografia por Raios X , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologiaRESUMO
Mycobacterial siderophores are critical components for bacterial virulence in the host. Pathogenic mycobacteria synthesize iron chelating siderophores named mycobactin and carboxymycobactin to extract intracellular macrophage iron. The two siderophores differ in structure only by a lipophilic aliphatic chain attached on the ε-amino group of the lysine mycobactin core, which is transferred by MbtK. Prior to acyl chain transfer, the lipophilic chain requires activation by a specific fatty acyl-AMP ligase FadD33 (also known as MbtM) and is then loaded onto phosphopantetheinylated acyl carrier protein (holo-MbtL) to form covalently acylated MbtL. We demonstrate that FadD33 prefers long chain saturated lipids and initial velocity studies showed that FadD33 proceeds via a Bi Uni Uni Bi ping-pong mechanism. Inhibition experiments suggest that, during the first half-reaction (adenylation), fatty acid binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), holo-MbtL binds to the enzyme followed by the release of products AMP and acylated MbtL. In addition, we characterized a post-translational regulation mechanism of FadD33 by the mycobacterial protein lysine acetyltransferase in a cAMP-dependent manner. FadD33 acetylation leads to enzyme inhibition, which can be reversed by the NAD(+)-dependent deacetylase, MSMEG_5175 (DAc1). To the best of our knowledge, this is the first time that bacterial siderophore synthesis has been shown to be regulated via post-translational protein acetylation.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Ligases/metabolismo , Regulação Enzimológica da Expressão Gênica , Oxazóis/metabolismo , Acetilação , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Adenilato Quinase/metabolismo , Clonagem Molecular , Inibidores Enzimáticos/química , Família Multigênica , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Processamento de Proteína Pós-Traducional , Sideróforos/química , Especificidade por SubstratoRESUMO
Acetyl-CoA synthetase (ACS) catalyzes the formation of AcCoA from acetate, ATP and Coenzyme A, allowing the organism to grow on acetate as the sole carbon source. ACS was the first enzyme in Mycobacterium tuberculosis shown to be regulated by posttranslational acetylation by the cAMP-dependent protein acetyltransferase. This modification results in the inactivation of the enzyme and can be reversed in the presence of NAD(+) and a mycobacterial sirtuin-like deacetylase. In this study we characterize the kinetic mechanism of MtACS, where the overall reaction can be divided into two half-reactions: the acetyl-adenylate forming reaction and the thiol-ligation reaction. We also provide evidence for the existence of the acetyl-adenylate intermediate via (31)P NMR spectroscopy. Furthermore, we dissect the regulatory role of K617 acetylation and show that acetylation inhibits only the first, adenylation half-reaction while leaving the second half reaction unchanged. Finally, we demonstrate that the chemical mechanism of the enzyme relies on a conformational change which is controlled by the protonation state of aspartate 525. Together with our earlier results, this suggests a degree of regulation of enzyme activity that is appropriate for the role of the enzyme in central carbon metabolism.
Assuntos
Acetato-CoA Ligase/metabolismo , Acetilcoenzima A/metabolismo , Acetiltransferases/metabolismo , Monofosfato de Adenosina/análogos & derivados , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/química , Acetato-CoA Ligase/genética , Ácido Acético/metabolismo , Acetilação , Acetiltransferases/genética , Monofosfato de Adenosina/metabolismo , Ácido Aspártico/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/enzimologia , Conformação Proteica , Processamento de Proteína Pós-Traducional , Prótons , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The monofunctional proline dehydrogenase (ProDH) from Mycobacterium tuberculosis performs the flavin-dependent oxidation of l-proline to Δ(1)-pyrroline-5-carboxylate in the proline catabolic pathway. The ProDH gene, prub, was cloned into the pYUB1062 vector, and the C-terminal His-tagged 37 kDa protein was expressed and purified by nickel affinity chromatography. A steady-state kinetic analysis revealed a ping-pong mechanism with an overall kcat of 33 ± 2 s(-1) and Km values of 5.7 ± 0.8 mM and 3.4 ± 0.3 µM for l-proline and 2,6-dichlorophenolindophenol (DCPIP), respectively. The pH dependence of kcat revealed that one enzyme group exhibiting a pK value of 6.8 must be deprotonated for optimal catalytic activity. Site-directed mutagenesis suggests that this group is Lys110. The primary kinetic isotope effects on V/KPro and V of 5.5 and 1.1, respectively, suggest that the transfer of hydride from l-proline to FAD is rate-limiting for the reductive half-reaction, but that FAD reoxidation is the rate-limiting step in the overall reaction. Solvent and multiple kinetic isotope effects suggest that l-proline oxidation occurs in a stepwise rather than concerted mechanism. Pre-steady-state kinetics reveal an overall kred of 88.5 ± 0.7 s(-1), and this rate is subject to a primary kinetic isotope effect of 5.2. These data confirm that the overall reaction is limited by reduced flavin reoxidation in the second half-reaction.
Assuntos
Mycobacterium tuberculosis/enzimologia , Prolina Oxidase/metabolismo , 2,6-Dicloroindofenol/química , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Concentração de Íons de Hidrogênio , Isótopos , Cinética , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Prolina Oxidase/química , Prolina Oxidase/genética , Homologia de Sequência de AminoácidosRESUMO
The constitutively expressed, chromosomally encoded ß-lactamase (BlaC) is the enzyme responsible for the intrinsic resistance to ß-lactam antibiotics in Mycobacterium tuberculosis. Previous studies from this laboratory have shown that the enzyme exhibits an extended-spectrum phenotype, with very high levels of penicillinase and cephalosporinase activity, as well as weak carbapenemase activity [Tremblay, L. W., et al. (2008) Biochemistry 47, 5312-5316]. In this report, we have determined the pH dependence of the kinetic parameters, revealing that the maximal velocity depends on the ionization state of two groups: a general base exhibiting a pK value of 4.5 and a general acid exhibiting a pK value of 7.8. Having defined a region where the kinetic parameters are pH-independent (pH 6.5), we determined solvent kinetic isotope effects (SKIEs) for three substrates whose kcat values differ by 5.5 orders of magnitude. Nitrocefin is a highly activated, chromogenic cephalosporin derivative that exhibits steady-state solvent kinetic isotope effects of 1.4 on both V and V/K. Cefoxitin is a slower cephalosporin derivative that exhibits a large SKIE on V of 3.9 but a small SKIE of 1.8 on V/K in steady-state experiments. Pre-steady-state, stopped-flow experiments with cefoxitin revealed a burst of ß-lactam ring opening with associated SKIE values of 1.6 on the acylation step and 3.4 on the deacylation step. Meropenem is an extremely slow substrate for BlaC and exhibits burst kinetics in the steady-state experiments. SKIE determinations with meropenem revealed large SKIEs on both the acylation and deacylation steps of 3.8 and 4.0, respectively. Proton inventories in all cases were linear, indicating the participation of a single solvent-derived proton in the chemical step responsible for the SKIE. The rate-limiting steps for ß-lactam hydrolysis of these substrates are analyzed, and the chemical steps responsible for the observed SKIE are discussed.
Assuntos
Proteínas de Bactérias/química , Cefoxitina/química , Cefalosporinas/química , Mycobacterium tuberculosis/enzimologia , Tienamicinas/química , beta-Lactamases/química , Acilação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Meropeném , Solventes/química , Resistência beta-LactâmicaRESUMO
The breakdown and recycling of peptidoglycan, an essential polymeric cell structure, occur in a number of bacterial species. A key enzyme in the recycling pathway of one of the components of the peptidoglycan layer, N-acetylmuramic acid (MurNAc), is MurNAc 6-phosphate hydrolase (MurQ). This enzyme catalyzes the cofactor-independent cleavage of a relatively nonlabile ether bond and presents an interesting target for mechanistic studies. Open chain product and substrate analogues were synthesized and tested as competitive inhibitors (K(is) values of 1.1 ± 0.3 and 0.23 ± 0.02 mM, respectively) of the MurNAc 6P hydrolase from Escherichia coli (MurQ-EC). To identify the roles of active site residues that are important for catalysis, the substrate analogue was cocrystallized with the MurNAc 6P hydrolase from Haemophilus influenzae (MurQ-HI) that was amenable to crystallographic studies. The cocrystal structure of MurQ-HI with the substrate analogue showed that Glu89 was located in the proximity of both the C2 atom and the oxygen at the C3 position of the bound inhibitor and that no other potential acid/base residue that could act as an active site acid/base was located in the vicinity. The conserved residues Glu120 and Lys239 were found within hydrogen bonding distance of the C5 hydroxyl group and C6 phosphate group, suggesting that they play a role in substrate binding and ring opening. Combining these results with previous biochemical data, we propose a one-base mechanism of action in which Glu89 functions to both deprotonate at the C2 position and assist in the departure of the lactyl ether at the C3 position. This same residue would serve to deprotonate the incoming water and reprotonate the enolate in the second half of the catalytic cycle.
Assuntos
Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/química , Haemophilus influenzae/enzimologia , Modelos Moleculares , Ácidos Murâmicos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ligação Competitiva , Biocatálise , Domínio Catalítico/efeitos dos fármacos , Sequência Conservada , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Glutâmico/química , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Ligação de Hidrogênio , Hidrólise/efeitos dos fármacos , Lisina/química , Conformação Molecular/efeitos dos fármacos , Ácidos Murâmicos/química , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Isocitrate lyase (ICL) catalyzes the reversible retro-aldol cleavage of isocitrate to generate glyoxylate and succinate. ICL is the first enzyme of the glyoxylate shunt, which allows for the anaplerosis of citric acid cycle intermediates under nutrient limiting conditions. In Mycobacterium tuberculosis, the source of ICL for these studies, ICL is vital for the persistence phase of the bacterium's life cycle. Solvent kinetic isotope effects (KIEs) in the direction of isocitrate cleavage ((D2O)V = 2.0 ± 0.1, and (D2O)[V/K(isocitrate)] = 2.2 ± 0.3) arise from the initial deprotonation of the C2 hydroxyl group of isocitrate or the protonation of the aci-acid of the succinate product of the isocitrate aldol cleavage by a solvent-derived proton. This KIE suggested that an equilibrium mixture of all protiated isocitrate, glyoxylate, and succinate prepared in D2O would undergo transient changes in equilibrium concentrations as a result of the solvent KIE and solvent-derived deuterium incorporation into both succinate and isocitrate. No change in the isotopic composition of glyoxylate was expected or observed. We have directly monitored the changing concentrations of all isotopic species of all reactants and products using a combination of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Continuous monitoring of glyoxylate by ¹H NMR spectroscopy shows a clear equilibrium perturbation in D2O. The final equilibrium isotopic composition of reactants in D2O revealed dideuterated succinate, protiated glyoxylate, and monodeuterated isocitrate, with the transient appearance and disappearance of monodeuterated succinate. A model for the equilibrium perturbation of substrate species and their time-dependent isotopic composition is presented.
Assuntos
Proteínas de Bactérias/metabolismo , Isocitrato Liase/metabolismo , Isocitratos/metabolismo , Modelos Moleculares , Água/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Simulação por Computador , Medição da Troca de Deutério , Glioxilatos/química , Glioxilatos/metabolismo , Hidrólise , Isocitrato Liase/química , Isocitrato Liase/genética , Isocitratos/química , Cinética , Espectrometria de Massas , Mycobacterium tuberculosis/enzimologia , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ácido Succínico/química , Ácido Succínico/metabolismoRESUMO
Mycobacterium tuberculosis (Mtb) is the leading cause of death due to a bacterial infection. The success of the Mtb pathogen has largely been attributed to the nonreplicating, persistence phase of the life cycle, for which the glyoxylate shunt is required. In Escherichia coli, flux through the shunt is controlled by regulation of isocitrate dehydrogenase (ICDH). In Mtb, the mechanism of regulation is unknown, and currently, there is no mechanistic or structural information about ICDH. We optimized expression and purification to a yield sufficiently high to perform the first detailed kinetic and structural studies of Mtb ICDH-1. A large solvent kinetic isotope effect [(D2O)V = 3.0 ± 0.2, and (D2O)(V/Kisocitrate) = 1.5 ± 0.3] and a smaller primary kinetic isotope effect [(D)V = 1.3 ± 0.1, and (D)(V/K[2R-(2)H]isocitrate) = 1.5 ± 0.2] allowed us to perform the first multiple kinetic isotope effect studies on any ICDH and suggest a chemical mechanism. In this mechanism, protonation of the enolate to form product α-ketoglutarate is the rate-limiting step. We report the first structure of Mtb ICDH-1 to 2.18 Å by X-ray crystallography with NADPH and Mn(2+) bound. It is a homodimer in which each subunit has a Rossmann fold, and a common top domain of interlocking ß sheets. Mtb ICDH-1 is most structurally similar to the R132H mutant human ICDH found in glioblastomas. Similar to human R132H ICDH, Mtb ICDH-1 also catalyzes the formation of α-hydroxyglutarate. Our data suggest that regulation of Mtb ICDH-1 is novel.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/metabolismo , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/genética , Cristalografia por Raios X , Escherichia coli/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Isocitrato Desidrogenase/genética , Cinética , Ligantes , Modelos Moleculares , Mycobacterium tuberculosis/genética , Estrutura Quaternária de Proteína , Eletricidade Estática , Homologia Estrutural de ProteínaRESUMO
The current emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for novel treatment strategies. Recently, BlaC, the principal ß-lactamase of Mycobacterium tuberculosis, was recognized as a potential therapeutic target. The combination of meropenem and clavulanic acid, which inhibits BlaC, was found to be effective against even extensively drug-resistant M. tuberculosis strains when tested in vitro. Yet there is significant concern that drug resistance against this combination will also emerge. To investigate the potential of BlaC to evolve variants resistant to clavulanic acid, we introduced substitutions at important amino acid residues of M. tuberculosis BlaC (R220, A244, S130, and T237). Whereas the substitutions clearly led to in vitro clavulanic acid resistance in enzymatic assays but at the expense of catalytic activity, transformation of variant BlaCs into an M. tuberculosis H37Rv background revealed that impaired inhibition of BlaC did not affect inhibition of growth in the presence of ampicillin and clavulanate. From these data we propose that resistance to ß-lactam-ß-lactamase inhibitor combinations will likely not arise from structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be part of a successful treatment regimen against M. tuberculosis.
Assuntos
Antituberculosos/farmacologia , Ácido Clavulânico/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tienamicinas/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genética , Substituição de Aminoácidos , Quimioterapia Combinada , Escherichia coli/genética , Escherichia coli/metabolismo , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Expressão Gênica , Humanos , Meropeném , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Lactamases/metabolismoRESUMO
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic protein responsible for the conversion of glyceraldehyde 3-phosphate (G3P), inorganic phosphate and nicotinamide adenine dinucleotide (NAD(+)) to 1,3-bisphosphoglycerate (1,3-BPG) and the reduced form of nicotinamide adenine dinucleotide (NADH). Here we report the characterization of GAPDH from Mycobacterium tuberculosis (Mtb). This enzyme exhibits a kinetic mechanism in which first NAD(+), then G3P bind to the active site resulting in the formation of a covalently bound thiohemiacetal intermediate. After oxidation of the thiohemiacetal and subsequent nucleotide exchange (NADH off, NAD(+) on), the binding of inorganic phosphate and phosphorolysis yields the product 1,3-BPG. Mutagenesis and iodoacetamide (IAM) inactivation studies reveal the conserved C158 to be responsible for nucleophilic catalysis and that the conserved H185 to act as a catalytic base. Primary, solvent and multiple kinetic isotope effects revealed that the first half-reaction is rate limiting and utilizes a step-wise mechanism for thiohemiacetal oxidation via a transient alkoxide to promote hydride transfer and thioester formation.