RESUMO
N-myristoyltransferase (NMT) is a promising antimalarial drug target. Despite biochemical similarities between Plasmodium vivax and human NMTs, our recent research demonstrated that high selectivity is achievable. Herein, we report PvNMT-inhibiting compounds aimed at identifying novel mechanisms of selectivity. Various functional groups are appended to a pyrazole moiety in the inhibitor to target a pocket formed beneath the peptide binding cleft. The inhibitor core group polarity, lipophilicity, and size are also varied to probe the water structure near a channel. Selectivity index values range from 0.8 to 125.3. Cocrystal structures of two selective compounds, determined at 1.97 and 2.43 Å, show that extensions bind the targeted pocket but with different stabilities. A bulky naphthalene moiety introduced into the core binds next to instead of displacing protein-bound waters, causing a shift in the inhibitor position and expanding the binding site. Our structure-activity data provide a conceptual foundation for guiding future inhibitor optimizations.
Assuntos
Aciltransferases , Antimaláricos , Inibidores Enzimáticos , Plasmodium vivax , Pirazóis , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/síntese química , Plasmodium vivax/enzimologia , Plasmodium vivax/efeitos dos fármacos , Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Aciltransferases/química , Relação Estrutura-Atividade , Antimaláricos/química , Antimaláricos/farmacologia , Antimaláricos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Sítios de LigaçãoRESUMO
Drugs targeting multiple stages of the Plasmodium vivax life cycle are needed to reduce the health and economic burdens caused by malaria worldwide. N-myristoyltransferase (NMT) is an essential eukaryotic enzyme and a validated drug target for combating malaria. However, previous PvNMT inhibitors have failed due to their low selectivity over human NMTs. Herein, we apply a structure-guided hybridization approach combining chemical moieties of previously reported NMT inhibitors to develop the next generation of PvNMT inhibitors. A high-resolution crystal structure of PvNMT bound to a representative selective hybrid compound reveals a unique binding site architecture that includes a selective conformation of a key tyrosine residue. The hybridized compounds significantly decrease P. falciparum blood-stage parasite load and consistently exhibit dose-dependent inhibition of P. vivax liver stage schizonts and hypnozoites. Our data demonstrate that hybridized NMT inhibitors can be multistage antimalarials, targeting dormant and developing forms of liver and blood stage.
Assuntos
Malária Falciparum , Malária Vivax , Humanos , Animais , Plasmodium vivax , Esquizontes , Fígado , AciltransferasesRESUMO
Each year, approximately 50,000 children under 5 die as a result of diarrhea caused by Cryptosporidium parvum, a protozoan parasite. There are currently no effective drugs or vaccines available to cure or prevent Cryptosporidium infection, and there are limited tools for identifying and validating targets for drug or vaccine development. We previously reported a high throughput screening (HTS) of a large compound library against Plasmodium N-myristoyltransferase (NMT), a validated drug target in multiple protozoan parasite species. To identify molecules that could be effective against Cryptosporidium, we counter-screened hits from the Plasmodium NMT HTS against Cryptosporidium NMT. We identified two potential hit compounds and validated them against CpNMT to determine if NMT might be an attractive drug target also for Cryptosporidium. We tested the compounds against Cryptosporidium using both cell-based and NMT enzymatic assays. We then determined the crystal structure of CpNMT bound to Myristoyl-Coenzyme A (MyrCoA) and structures of ternary complexes with MyrCoA and the hit compounds to identify the ligand binding modes. The binding site architectures display different conformational states in the presence of the two inhibitors and provide a basis for rational design of selective inhibitors.
Assuntos
Criptosporidiose , Cryptosporidium , Plasmodium , Criança , Humanos , Criptosporidiose/tratamento farmacológico , Desenvolvimento de MedicamentosRESUMO
Infection with pathogenic free-living amoebae, including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because of catastrophic central nervous system involvement. Efficacious treatment options for these infections are lacking, and the mortality rate due to infection is high. Previously, we evaluated the N. fowleri glucokinase (NfGlck) as a potential target for therapeutic intervention, as glucose metabolism is critical for in vitro viability. Here, we extended these studies to the glucokinases from two other pathogenic free-living amoebae, including Acanthamoeba castellanii (AcGlck) and B. mandrillaris (BmGlck). While these enzymes are similar (49.3% identical at the amino acid level), they have distinct kinetic properties that distinguish them from each other. For ATP, AcGlck and BmGlck have apparent Km values of 472.5 and 41.0 µM, while Homo sapiens Glck (HsGlck) has a value of 310 µM. Both parasite enzymes also have a higher apparent affinity for glucose than the human counterpart, with apparent Km values of 45.9 µM (AcGlck) and 124 µM (BmGlck) compared to ~8 mM for HsGlck. Additionally, AcGlck and BmGlck differ from each other and other Glcks in their sensitivity to small molecule inhibitors, suggesting that inhibitors with pan-amoebic activity could be challenging to generate.
Assuntos
Acanthamoeba , Amebíase , Amoeba , Balamuthia mandrillaris , Naegleria fowleri , Amebíase/tratamento farmacológico , Amebíase/parasitologia , Glucoquinase , HumanosRESUMO
All radical S-adenosylmethionine (radical-SAM) enzymes, including the noncanonical radical-SAM enzyme diphthamide biosynthetic enzyme Dph1-Dph2, require at least one [4Fe-4S](Cys)3 cluster for activity. It is well-known in the radical-SAM enzyme community that the [4Fe-4S](Cys)3 cluster is extremely air-sensitive and requires strict anaerobic conditions to reconstitute activity in vitro. Thus, how such enzymes function in vivo in the presence of oxygen in aerobic organisms is an interesting question. Working on yeast Dph1-Dph2, we found that consistent with the known oxygen sensitivity, the [4Fe-4S] cluster is easily degraded into a [3Fe-4S] cluster. Remarkably, the small iron-containing protein Dph3 donates one Fe atom to convert the [3Fe-4S] cluster in Dph1-Dph2 to a functional [4Fe-4S] cluster during the radical-SAM enzyme catalytic cycle. This mechanism to maintain radical-SAM enzyme activity in aerobic environments is likely general, and Dph3-like proteins may exist to keep other radical-SAM enzymes functional in aerobic environments.
Assuntos
Histidina/análogos & derivados , Proteínas Ferro-Enxofre/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ditionita/metabolismo , Histidina/biossíntese , Ferro/química , Proteínas Ferro-Enxofre/química , Fator 2 de Elongação de Peptídeos/metabolismo , Proteínas Repressoras/química , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Viperin is a radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication by converting cytidine triphosphate (CTP) into 3'-deoxy-3',4'-didehydro-CTP and by additional undefined mechanisms operating through its N- and C-terminal domains. Here, we describe crystal structures of viperin bound to a SAM analogue and CTP or uridine triphosphate (UTP) and report kinetic parameters for viperin-catalyzed reactions with CTP or UTP as substrates. Viperin orients the C4' hydrogen atom of CTP and UTP similarly for abstraction by a 5'-deoxyadenosyl radical, but the uracil moiety introduces unfavorable interactions that prevent tight binding of UTP. Consistently, kcat is similar for CTP and UTP whereas the Km for UTP is much greater. The structures also show that nucleotide binding results in ordering of the C-terminal tail and reveal that this region contains a P-loop that binds the γ-phosphate of the bound nucleotide. Collectively, the results explain the selectivity for CTP and reveal a structural role for the C-terminal tail in binding CTP and UTP.
Assuntos
Citidina Trifosfato/química , Proteínas/química , Proteínas/metabolismo , S-Adenosil-Homocisteína/química , Uridina Trifosfato/química , Animais , Cristalografia por Raios X , Citidina Trifosfato/metabolismo , Cinética , Camundongos , Modelos Moleculares , Estrutura Molecular , Mutação , Proteínas/genética , S-Adenosil-Homocisteína/metabolismo , Especificidade por Substrato , Uridina Trifosfato/metabolismoRESUMO
Archaebacterial and eukaryotic elongation factor 2 (EF-2) and bacterial elongation factor G (EF-G) are five domain GTPases that catalyze the ribosomal translocation of tRNA and mRNA. In the classical mechanism of activation, GTPases are switched on through GDP/GTP exchange, which is accompanied by the ordering of two flexible segments called switch I and II. However, crystal structures of EF-2 and EF-G have thus far not revealed the conformations required by the classical mechanism. Here, we describe crystal structures of Methanoperedens nitroreducens EF-2 (MnEF-2) and MnEF-2-H595N bound to GMPPCP (GppCp) and magnesium displaying previously unreported compact conformations. Domain III forms interfaces with the other four domains and the overall conformations resemble that of SNU114, the eukaryotic spliceosomal GTPase. The gamma phosphate of GMPPCP is detected through interactions with switch I and a P-loop structural element. Switch II is highly ordered whereas switch I shows a variable degree of ordering. The ordered state results in a tight interdomain arrangement of domains I-III and the formation of a portion of a predicted monovalent cation site involving the P-loop and switch I. The side chain of an essential histidine residue in switch II is placed in the inactive conformation observed for the "on" state of elongation factor EF-Tu. The compact conformations of MnEF-2 and MnEF-2-H595N suggest an "on" ribosome-free conformational state.
RESUMO
Elongation factor 2 (EF-2), a five-domain, GTP-dependent ribosomal translocase of archaebacteria and eukaryotes, undergoes post-translational modification to form diphthamide on a specific histidine residue in domain IV prior to binding the ribosome. The first step of diphthamide biosynthesis in archaebacteria is catalyzed by Dph2, a homodimeric radical S-adenosylmethionine (SAM) enzyme having a noncanonical architecture. Here, we describe a 3.5 Å resolution crystal structure of the Methanobrevibacter smithii (Ms) Dph2 homodimer bound to two molecules of MsEF-2, one of which is ordered and the other largely disordered. MsEF-2 is bound to both protomers of MsDph2, with domain IV bound to the active site of one protomer and domain III bound to a surface α-helix of an adjacent protomer. The histidine substrate of domain IV is inserted into the active site, which reveals for the first time the architecture of the Dph2 active site in complex with its target substrate. We also determined a high-resolution crystal structure of isolated MsDph2 bound to 5'-methylthioadenosine that shows a conserved arginine residue preoriented by conserved phenylalanine and aspartate residues for binding the carboxylate group of SAM. Mutagenesis experiments suggest that the arginine plays an important role in the first step of diphthamide biosynthesis.
Assuntos
Proteínas Arqueais/metabolismo , Histidina/análogos & derivados , Oxirredutases/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Arginina/química , Domínio Catalítico , Cristalografia por Raios X , Desoxiadenosinas/metabolismo , Histidina/química , Histidina/metabolismo , Methanobrevibacter/enzimologia , Mutagênese Sítio-Dirigida , Oxirredutases/química , Oxirredutases/genética , Fator 2 de Elongação de Peptídeos/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Tionucleosídeos/metabolismoRESUMO
Approximately 2000 structures of methyltransferases (MTases) are currently available, displaying fifteen different folds for binding a methyl donor and providing molecular level insight into nearly half the human methyltransferome. Several MTases involved in gene expression and regulation are catalytically inefficient when isolated, and their catalytic domains often show inhibitory active site architectures. Recently reported structures of complexes that more closely reflect biological context have begun to reveal the structural basis of activation. DNA and particular histone MTases are allosterically activated by binding histone modifications using reader domains or separate reader proteins, and some MTases operating beyond chromatin are activated by binding an activator protein. In this review, we describe the structural status of the human methyltransferome and then discuss newly revealed structural mechanisms of MTase activation.
Assuntos
Metiltransferases , Sítios de Ligação , Domínio Catalítico , Ativação Enzimática , Ativadores de Enzimas/metabolismo , Humanos , Metiltransferases/química , Metiltransferases/classificação , Metiltransferases/metabolismo , Modelos Moleculares , Nucleossomos/metabolismo , Conformação ProteicaRESUMO
Diphthamide biosynthesis involves a carbon-carbon bond-forming reaction catalyzed by a radical S-adenosylmethionine (SAM) enzyme that cleaves a carbon-sulfur (C-S) bond in SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. Using rapid freezing, we have captured an organometallic intermediate with an iron-carbon (Fe-C) bond between ACP and the enzyme's [4Fe-4S] cluster. In the presence of the substrate protein, elongation factor 2, this intermediate converts to an organic radical, formed by addition of the ACP radical to a histidine side chain. Crystal structures of archaeal diphthamide biosynthetic radical SAM enzymes reveal that the carbon of the SAM C-S bond being cleaved is positioned near the unique cluster Fe, able to react with the cluster. Our results explain how selective C-S bond cleavage is achieved in this radical SAM enzyme.
Assuntos
Proteínas Arqueais/química , Histidina/análogos & derivados , Proteínas Ferro-Enxofre/química , Pyrococcus horikoshii/enzimologia , S-Adenosilmetionina/química , Carbono/química , Cristalografia por Raios X , Histidina/biossíntese , Ferro/química , Compostos Organometálicos/químicaRESUMO
Burkholderia glumae converts the guanine base of guanosine triphosphate into an azapteridine and methylates both the pyrimidine and triazine rings to make toxoflavin. Strains of Burkholderia thailandensis and Burkholderia pseudomallei have a gene cluster encoding seven putative biosynthetic enzymes that resembles the toxoflavin gene cluster. Four of the enzymes are similar in sequence to BgToxBCDE, which have been proposed to make 1,6-didesmethyltoxoflavin (1,6-DDMT). One of the remaining enzymes, BthII1283 in B. thailandensis E264, is a predicted S-adenosylmethionine (SAM)-dependent N-methyltransferase that shows a low level of sequence identity to BgToxA, which sequentially methylates N6 and N1 of 1,6-DDMT to form toxoflavin. Here we show that, unlike BgToxA, BthII1283 catalyzes a single methyl transfer to N1 of 1,6-DDMT in vitro. In addition, we investigated the differences in reactivity and regioselectivity by determining crystal structures of BthII1283 with bound S-adenosylhomocysteine (SAH) or 1,6-DDMT and SAH. BthII1283 contains a class I methyltransferase fold and three unique extensions used for 1,6-DDMT recognition. The active site structure suggests that 1,6-DDMT is bound in a reduced form. The plane of the azapteridine ring system is orthogonal to its orientation in BgToxA. In BthII1283, the modeled SAM methyl group is directed toward the p orbital of N1, whereas in BgToxA, it is first directed toward an sp2 orbital of N6 and then toward an sp2 orbital of N1 after planar rotation of the azapteridine ring system. Furthermore, in BthII1283, N1 is hydrogen bonded to a histidine residue whereas BgToxA does not supply an obvious basic residue for either N6 or N1 methylation.
Assuntos
Proteínas de Bactérias/metabolismo , Burkholderia/enzimologia , Metiltransferases/metabolismo , Modelos Moleculares , Pirimidinonas/metabolismo , S-Adenosilmetionina/metabolismo , Triazinas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Histidina/química , Ligação de Hidrogênio , Metilação , Metiltransferases/química , Metiltransferases/genética , Família Multigênica , Oxirredução , Filogenia , Conformação Proteica , Pirimidinonas/síntese química , Pirimidinonas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , S-Adenosil-Homocisteína/química , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/química , Especificidade da Espécie , Estereoisomerismo , Triazinas/químicaRESUMO
Viperin is an IFN-inducible radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S-adenosylhomocysteine (SAH) or 5'-deoxyadenosine (5'-dAdo) and l-methionine (l-Met). Viperin contains a partial (ßα)6-barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first ß-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5'-dAdo and l-Met (SAM cleavage products) is consistent with the canonical mechanism of 5'-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTP-binding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.
Assuntos
Dobramento de Proteína , Proteínas/química , Animais , Camundongos , Domínios Proteicos , Proteínas/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-AtividadeRESUMO
The quinolinate synthase of prokaryotes and photosynthetic eukaryotes, NadA, contains a [4Fe-4S] cluster with unknown function. We report crystal structures of Pyrococcus horikoshii NadA in complex with dihydroxyacetone phosphate (DHAP), iminoaspartate analogues, and quinolinate. DHAP adopts a nearly planar conformation and chelates the [4Fe-4S] cluster via its keto and hydroxyl groups. The active site architecture suggests that the cluster acts as a Lewis acid in enediolate formation, like zinc in class II aldolases. The DHAP and putative iminoaspartate structures suggest a model for a condensed intermediate. The ensemble of structures suggests a two-state system, which may be exploited in early steps.
Assuntos
Proteínas Arqueais/química , Complexos Multienzimáticos/química , Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Domínio Catalítico , Cristalografia por Raios X , Fosfato de Di-Hidroxiacetona/química , Proteínas Ferro-Enxofre/química , Modelos Moleculares , Conformação Proteica , Pyrococcus horikoshii/enzimologia , Ácido Quinolínico/químicaRESUMO
Toxoflavin is a major virulence factor of the rice pathogen Burkholderia glumae. The tox operon of B. glumae contains five putative toxoflavin biosynthetic genes toxABCDE. ToxA is a predicted S-adenosylmethionine-dependent methyltransferase, and toxA knockouts of B. glumae are less virulent in plant infection models. In this study, we show that ToxA performs two consecutive methylations to convert the putative azapteridine intermediate, 1,6-didemethyltoxoflavin, to toxoflavin. In addition, we report a series of crystal structures of ToxA complexes that reveals the molecular basis of the dual methyltransferase activity. The results suggest sequential methylations with initial methylation at N6 of 1,6-didemethyltoxoflavin followed by methylation at N1. The two azapteridine orientations that position N6 or N1 for methylation are coplanar with a 140° rotation between them. The structure of ToxA contains a class I methyltransferase fold having an N-terminal extension that either closes over the active site or is largely disordered. The ordered conformation places Tyr7 at a position of a structurally conserved tyrosine site of unknown function in various methyltransferases. Crystal structures of ToxA-Y7F consistently show a closed active site, whereas structures of ToxA-Y7A consistently show an open active site, suggesting that the hydroxyl group of Tyr7 plays a role in opening and closing the active site during the multistep reaction.
Assuntos
Proteínas de Bactérias/química , Burkholderia/enzimologia , Metiltransferases/química , Pirimidinonas/química , Triazinas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia/genética , Catálise , Domínio Catalítico , Cristalografia por Raios X , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Estrutura Secundária de Proteína , Pirimidinonas/metabolismo , Triazinas/metabolismoRESUMO
Comparative genomics of the bacterial thiamin pyrimidine synthase (thiC) revealed a paralogue of thiC (bzaF) clustered with anaerobic vitamin B12 biosynthetic genes. Here we demonstrate that BzaF is a radical S-adenosylmethionine enzyme that catalyzes the remarkable conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI). We identify the origin of key product atoms and propose a reaction mechanism. These studies represent the first step in solving a long-standing problem in anaerobic vitamin B12 assembly and reveal an unanticipated intersection of thiamin and vitamin B12 biosynthesis.
Assuntos
Benzimidazóis/metabolismo , Ribonucleotídeos/metabolismo , Tiamina/biossíntese , Vitamina B 12/biossíntese , Anaerobiose , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Modelos Moleculares , Conformação ProteicaRESUMO
Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a ß-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the ß-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.
Assuntos
Proteínas de Bactérias/metabolismo , Cobamidas/metabolismo , Transferases Intramoleculares/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Pirimidinas/biossíntese , S-Adenosilmetionina/metabolismo , Tiamina/biossíntese , Catálise , Domínio Catalítico , Cristalografia por Raios X , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The majority of excitatory neurotransmission in the CNS is mediated by tetrameric AMPA receptors. Channel activation begins with a series of interactions with an agonist that binds to the cleft between the two lobes of the ligand-binding domain of each subunit. Binding leads to a series of conformational transitions, including the closure of the two lobes of the binding domain around the ligand, culminating in ion channel opening. Although a great deal has been learned from crystal structures, determining the molecular details of channel activation, deactivation, and desensitization requires measures of dynamics and stabilities of hydrogen bonds that stabilize cleft closure. The use of hydrogen-deuterium exchange at low pH provides a measure of the variation of stability of specific hydrogen bonds among agonists of different efficacy. Here, we used NMR measurements of hydrogen-deuterium exchange to determine the stability of hydrogen bonds in the GluA2 (AMPA receptor) ligand-binding domain in the presence of several full and partial agonists. The results suggest that the stabilization of hydrogen bonds between the two lobes of the binding domain is weaker for partial than for full agonists, and efficacy is correlated with the stability of these hydrogen bonds. The closure of the lobes around the agonists leads to a destabilization of the hydrogen bonding in another portion of the lobe interface, and removing an electrostatic interaction in Lobe 2 can relieve the strain. These results provide new details of transitions in the binding domain that are associated with channel activation and desensitization.
Assuntos
Simulação de Dinâmica Molecular , Receptores de AMPA/agonistas , Receptores de AMPA/química , Animais , Medição da Troca de Deutério/métodos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Estrutura Terciária de Proteína , Ratos , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Relação Estrutura-AtividadeRESUMO
In Saccharomyces cerevisiae , thiamin pyrimidine is formed from histidine and pyridoxal phosphate (PLP). The origin of all of the pyrimidine atoms has been previously determined using labeling studies and suggests that the pyrimidine is formed using remarkable chemistry that is without chemical or biochemical precedent. Here we report the overexpression of the closely related Candida albicans pyrimidine synthase (THI5p) and the reconstitution and preliminary characterization of the enzymatic activity. A structure of the C. albicans THI5p shows PLP bound at the active site via an imine with Lys62 and His66 in close proximity to the PLP. Our data suggest that His66 of the THI5 protein is the histidine source for pyrimidine formation and that the pyrimidine synthase is a single-turnover enzyme.
Assuntos
Candida albicans/metabolismo , Histidina/metabolismo , Fosfato de Piridoxal/metabolismo , Pirimidinas/biossíntese , Tiamina/biossíntese , Candida albicans/química , Histidina/química , Modelos Moleculares , Estrutura Molecular , Fosfato de Piridoxal/química , Pirimidinas/química , Tiamina/químicaRESUMO
Toxoflavin (an azapteridine) is degraded to a single product by toxoflavin lyase (TflA) in a reaction dependent on reductant, Mn(II), and oxygen. The isolated product was fully characterized by NMR and MS and was identified as a triazine in which the pyrimidine ring was oxidatively degraded. A mechanism for toxoflavin degradation based on the identification of the enzymatic product and the recently determined crystal structure of toxoflavin lyase is proposed.
Assuntos
Liases/metabolismo , Pirimidinonas/química , Triazinas/química , Liases/química , Manganês/química , Manganês/metabolismo , Modelos Moleculares , Estrutura Molecular , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Pirimidinonas/metabolismo , Triazinas/metabolismoRESUMO
High-resolution crystal structures are reported for apo, holo, and substrate-bound forms of a toxoflavin-degrading metalloenzyme (TflA). In addition, the degradation reaction is shown to be dependent on oxygen, Mn(II), and dithiothreitol in vitro. Despite its low sequence identity with proteins of known structure, TflA is structurally homologous to proteins of the vicinal oxygen chelate superfamily. Like other metalloenzymes in this superfamily, the TflA fold contains four modules that associate to form a metal binding site; however, the fold displays a rare rearrangement of the structural modules indicative of domain permutation. Moreover, unlike the 2-His-1-carboxylate facial triad commonly utilized by vicinal oxygen chelate dioxygenases and other dioxygen-activating non-heme Fe(II) enzymes, the metal center in TflA consists of a 1-His-2-carboxylate facial triad. The substrate-bound complex shows square-pyramidal geometry in which one position is occupied by O5 of toxoflavin. The open coordination site is predicted to be the dioxygen binding site. TflA appears to stabilize the reduced form of toxoflavin through second-sphere interactions. This anionic species is predicted to be the electron source responsible for reductive activation of oxygen to produce a peroxytoxoflavin intermediate.