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1.
Chem Sci ; 14(11): 2826-2838, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36937599

RESUMO

[FeFe] hydrogenases are highly efficient metalloenyzmes for hydrogen conversion. Their active site cofactor (the H-cluster) is composed of a canonical [4Fe-4S] cluster ([4Fe-4S]H) linked to a unique organometallic di-iron subcluster ([2Fe]H). In [2Fe]H the two Fe ions are coordinated by a bridging 2-azapropane-1,3-dithiolate (ADT) ligand, three CO and two CN- ligands, leaving an open coordination site on one Fe where substrates (H2 and H+) as well as inhibitors (e.g. O2, CO, H2S) may bind. Here, we investigate two new active site states that accumulate in [FeFe] hydrogenase variants where the cysteine (Cys) in the proton transfer pathway is mutated to alanine (Ala). Our experimental data, including atomic resolution crystal structures and supported by calculations, suggest that in these two states a third CN- ligand is bound to the apical position of [2Fe]H. These states can be generated both by "cannibalization" of CN- from damaged [2Fe]H subclusters as well as by addition of exogenous CN-. This is the first detailed spectroscopic and computational characterisation of the interaction of exogenous CN- with [FeFe] hydrogenases. Similar CN--bound states can also be generated in wild-type hydrogenases, but do not form as readily as with the Cys to Ala variants. These results highlight how the interaction between the first amino acid in the proton transfer pathway and the active site tunes ligand binding to the open coordination site and affects the electronic structure of the H-cluster.

2.
Nat Commun ; 14(1): 458, 2023 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-36709327

RESUMO

Eukaryotic arginylation is an essential post-translational modification that modulates protein stability and regulates protein half-life. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite their conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we show that ATE1s bind a previously undiscovered [Fe-S] cluster that is conserved across evolution. We characterize the nature of this [Fe-S] cluster and find that the presence of the [Fe-S] cluster in ATE1 is linked to its arginylation activity, both in vitro and in vivo, and the initiation of the yeast stress response. Importantly, the ATE1 [Fe-S] cluster is oxygen-sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Taken together, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.


Assuntos
Aminoaciltransferases , Proteínas Ferro-Enxofre , Aminoaciltransferases/genética , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Ferro-Enxofre/genética
3.
J Biol Inorg Chem ; 28(2): 187-204, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36527507

RESUMO

Iron-sulfur clusters are ubiquitous cofactors required for fundamental biological processes. Structural and spectroscopic analysis of Fe-S proteins is often limited by low cluster occupancy in recombinantly produced proteins. In this work, we report a systematic comparison of different maturation strategies for three well-established [4Fe-4S] proteins. Aconitase B, HMBPP reductase (IspH), and quinolinate synthase (NadA) were used as model proteins as they have previously been characterized. The protein production strategies include expression of the gene of interest in BL21(DE3) cells, maturation of the apo protein using chemical or semi-enzymatic reconstitution, co-expression with two different plasmids containing the iron-sulfur cluster (isc) or sulfur formation (suf) operon, a cell strain lacking IscR, the transcriptional regulator of the ISC machinery, and an engineered "SufFeScient" derivative of BL21(DE3). Our results show that co-expression of a Fe-S biogenesis pathway influences the protein yield and the cluster content of the proteins. The presence of the Fe-S cluster is contributing to correct folding and structural stability of the proteins. In vivo maturation reduces the formation of Fe-S aggregates, which occur frequently when performing chemical reconstitution. Furthermore, we show that the in vivo strategies can be extended to the radical SAM protein ThnB, which was previously only maturated by chemical reconstitution. Our results shed light on the differences of in vitro and in vivo Fe-S cluster maturation and points out the pitfalls of chemical reconstitution.


Assuntos
Proteínas de Escherichia coli , Proteínas Ferro-Enxofre , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Escherichia coli/metabolismo , Oxirredutases/metabolismo , Ferro/metabolismo , Enxofre/metabolismo
4.
Methods Mol Biol ; 2439: 79-89, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35226316

RESUMO

DNAzymes are biocatalysts that have been selected in vitro and their function inside cells (in vivo) is extremely low. Thus, almost all studies have been carried out in diluted solutions (in vitro). The cellular presence of molecules such as amino acids, polypeptides, alcohols, and sugars introduces forces that modify the kinetics and thermodynamics of DNAzyme-mediated catalysis. The crowded intracellular environment referred to as molecular crowding can be mimicked by adding high concentrations of natural or synthetic macromolecules to the reaction conditions. Here, we investigate the activity of the 10-23 DNAzyme and the stability of the DNAzyme:RNA complex under molecular crowding conditions. Therefore, we use a Förster resonance energy transfer (FRET)-based activity assay in combination with denaturing urea polyacrylamide gel electrophoresis and circular dichroism (CD) spectroscopy.


Assuntos
DNA Catalítico , DNA Catalítico/química , Eletroforese em Gel de Poliacrilamida , Transferência Ressonante de Energia de Fluorescência , Cinética , Termodinâmica
5.
Methods Mol Biol ; 2439: 105-115, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35226318

RESUMO

X-ray crystallography is one of the most prominent techniques for determining high-resolution structures of nucleic acids. The major challenges are to obtain well-diffracting single crystals and to solve the phase problem. The absence of structural information impedes the elucidation of the molecular details of biological processes. A particularly intriguing example is the RNA-cleavage catalyzed by the 10-23 deoxyribozyme (DNAzyme). This DNAzyme consists of a catalytic core that is flanked by two substrate binding arms, which can be designed to bind any RNA of interest. Structure elucidation of the 10-23 DNAzyme in a biologically relevant conformation faces three major challenges: (1) stabilization of the RNA substrate to capture the DNA:RNA complex in the pre-catalytic conformation, (2) prevention of the formation of an artificial duplex conformation due to a self-complementary sequence in the catalytic core of the DNAzyme, and (3) the crystallization of nucleic acids with their uniform surfaces. Here, we provide a protocol for an innovative strategy facilitating the crystallization of protein:nucleic acid complexes using a soaking approach and discuss on how to apply this protocol for the structure elucidation of the 10-23 DNAzyme. For this purpose, we describe the purification procedure of an optimized variant of the RNA-binding protein U1A, the crystallization of this specific U1A variant, the soaking process with its specific RNA hairpin loop, and finally suggest a strategy for applying this procedure on the 10-23 DNAzyme in complex with its specific RNA target.


Assuntos
Ácidos Nucleicos , Ribonucleoproteína Nuclear Pequena U1 , Cristalografia por Raios X , Conformação de Ácido Nucleico , RNA/química , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo
6.
Nature ; 601(7891): 144-149, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34949858

RESUMO

The 10-23 DNAzyme is one of the most prominent catalytically active DNA sequences1,2. Its ability to cleave a wide range of RNA targets with high selectivity entails a substantial therapeutic and biotechnological potential2. However, the high expectations have not yet been met, a fact that coincides with the lack of high-resolution and time-resolved information about its mode of action3. Here we provide high-resolution NMR characterization of all apparent states of the prototypic 10-23 DNAzyme and present a comprehensive survey of the kinetics and dynamics of its catalytic function. The determined structure and identified metal-ion-binding sites of the precatalytic DNAzyme-RNA complex reveal that the basis of the DNA-mediated catalysis is an interplay among three factors: an unexpected, yet exciting molecular architecture; distinct conformational plasticity; and dynamic modulation by metal ions. We further identify previously hidden rate-limiting transient intermediate states in the DNA-mediated catalytic process via real-time NMR measurements. Using a rationally selected single-atom replacement, we could considerably enhance the performance of the DNAzyme, demonstrating that the acquired knowledge of the molecular structure, its plasticity and the occurrence of long-lived intermediate states constitutes a valuable starting point for the rational design of next-generation DNAzymes.


Assuntos
Biocatálise , DNA Catalítico/química , DNA Catalítico/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , RNA/metabolismo , Cinética , Metais/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fatores de Tempo
7.
J Biol Inorg Chem ; 26(1): 93-108, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33544225

RESUMO

The Schizosaccharomyces pombe Asp1 protein is a bifunctional kinase/pyrophosphatase that belongs to the highly conserved eukaryotic diphosphoinositol pentakisphosphate kinase PPIP5K/Vip1 family. The N-terminal Asp1 kinase domain generates specific high-energy inositol pyrophosphate (IPP) molecules, which are hydrolyzed by the C-terminal Asp1 pyrophosphatase domain (Asp1365-920). Thus, Asp1 activities regulate the intracellular level of a specific class of IPP molecules, which control a wide number of biological processes ranging from cell morphogenesis to chromosome transmission. Recently, it was shown that chemical reconstitution of Asp1371-920 leads to the formation of a [2Fe-2S] cluster; however, the biological relevance of the cofactor remained under debate. In this study, we provide evidence for the presence of the Fe-S cluster in Asp1365-920 inside the cell. However, we show that the Fe-S cluster does not influence Asp1 pyrophosphatase activity in vitro or in vivo. Characterization of the as-isolated protein by electronic absorption spectroscopy, mass spectrometry, and X-ray absorption spectroscopy is consistent with the presence of a [2Fe-2S]2+ cluster in the enzyme. Furthermore, we have identified the cysteine ligands of the cluster. Overall, our work reveals that Asp1 contains an Fe-S cluster in vivo that is not involved in its pyrophosphatase activity.


Assuntos
Proteínas do Citoesqueleto/química , Proteínas Ferro-Enxofre/química , Pirofosfatases/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/enzimologia , Biocatálise , Cisteína/química , Proteínas do Citoesqueleto/genética , Proteínas Ferro-Enxofre/genética , Enzimas Multifuncionais/química , Enzimas Multifuncionais/genética , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Pirofosfatases/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética
8.
Molecules ; 25(13)2020 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-32646019

RESUMO

Deoxyribozymes (DNAzymes) with RNA hydrolysis activity have a tremendous potential as gene suppression agents for therapeutic applications. The most extensively studied representative is the 10-23 DNAzyme consisting of a catalytic loop and two substrate binding arms that can be designed to bind and cleave the RNA sequence of interest. The RNA substrate is cleaved between central purine and pyrimidine nucleotides. The activity of this DNAzyme in vitro is considerably higher than in vivo, which was suggested to be related to its divalent cation dependency. Understanding the mechanism of DNAzyme catalysis is hindered by the absence of structural information. Numerous biological studies, however, provide comprehensive insights into the role of particular deoxynucleotides and functional groups in DNAzymes. Here we provide an overview of the thermodynamic properties, the impact of nucleobase modifications within the catalytic loop, and the role of different metal ions in catalysis. We point out features that will be helpful in developing novel strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. Consideration of these features will enable to develop improved strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. These insights provide the basis for improving activity in cells and pave the way for developing DNAzyme applications.


Assuntos
DNA Catalítico/química , DNA de Cadeia Simples/química , Metais/química , Conformação de Ácido Nucleico , Cátions Bivalentes
9.
Angew Chem Int Ed Engl ; 59(38): 16786-16794, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32488975

RESUMO

[FeFe] hydrogenases are the most active H2 converting catalysts in nature, but their extreme oxygen sensitivity limits their use in technological applications. The [FeFe] hydrogenases from sulfate reducing bacteria can be purified in an O2 -stable state called Hinact . To date, the structure and mechanism of formation of Hinact remain unknown. Our 1.65 Šcrystal structure of this state reveals a sulfur ligand bound to the open coordination site. Furthermore, in-depth spectroscopic characterization by X-ray absorption spectroscopy (XAS), nuclear resonance vibrational spectroscopy (NRVS), resonance Raman (RR) spectroscopy and infrared (IR) spectroscopy, together with hybrid quantum mechanical and molecular mechanical (QM/MM) calculations, provide detailed chemical insight into the Hinact state and its mechanism of formation. This may facilitate the design of O2 -stable hydrogenases and molecular catalysts.


Assuntos
Clostridium beijerinckii/enzimologia , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Oxigênio/metabolismo , Enxofre/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Hidrogênio/química , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxigênio/química , Espectrofotometria Infravermelho , Análise Espectral Raman , Enxofre/química , Espectroscopia por Absorção de Raios X
10.
J Struct Biol ; 210(2): 107480, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32070773

RESUMO

The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing. Co-crystallization with proteins is a straight forward approach to overcome these challenges. The human RNA-binding protein U1A has previously been established as crystallization module, however, the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U1A system. Here, we report three crystal structures of tryptophan-containing U1A variants, which expand the crystallization toolbox for nucleic acids. Analysis of the structures complemented by SAXS, NMR spectroscopy, and optical spectroscopy allow for insights into the potential of the U1A variants to serve as crystallization modules for nucleic acids. In addition, we report a fast and efficient protocol for crystallization of RNA by soaking and present a fluorescence-based approach for detecting RNA-binding in crystallo. Our results provide a new tool set for the crystallization of RNA and RNA:DNA complexes.


Assuntos
Ácidos Nucleicos/química , Ribonucleoproteína Nuclear Pequena U1/química , Cristalização , Espectroscopia de Ressonância Magnética , Espalhamento a Baixo Ângulo , Difração de Raios X
11.
Biol Chem ; 402(1): 99-111, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33544488

RESUMO

Deoxyribozymes (DNAzymes) are single-stranded DNA molecules that catalyze a broad range of chemical reactions. The 10-23 DNAzyme catalyzes the cleavage of RNA strands and can be designed to cleave essentially any target RNA, which makes it particularly interesting for therapeutic and biosensing applications. The activity of this DNAzyme in vitro is considerably higher than in cells, which was suggested to be a result of the low intracellular concentration of bioavailable divalent cations. While the interaction of the 10-23 DNAzyme with divalent metal ions was studied extensively, the influence of monovalent metal ions on its activity remains poorly understood. Here, we characterize the influence of monovalent and divalent cations on the 10-23 DNAzyme utilizing functional and biophysical techniques. Our results show that Na+ and K+ affect the binding of divalent metal ions to the DNAzyme:RNA complex and considerably modulate the reaction rates of RNA cleavage. We observe an opposite effect of high levels of Na+ and K+ concentrations on Mg2+- and Mn2+-induced reactions, revealing a different interplay of these metals in catalysis. Based on these findings, we propose a model for the interaction of metal ions with the DNAzyme:RNA complex.


Assuntos
DNA Catalítico/metabolismo , DNA de Cadeia Simples/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Sítios de Ligação , Biocatálise , DNA Catalítico/química , DNA de Cadeia Simples/química , Íons/química , Íons/metabolismo , Potássio/química , Sódio/química
12.
Biol Chem ; 399(7): 787-798, 2018 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-29894292

RESUMO

Pseudomonas putida rubredoxin-2 (Rxn2) is an essential member of the alkane hydroxylation pathway and transfers electrons from a reductase to the membrane-bound hydroxylase. The regioselective hydroxylation of linear alkanes is a challenging chemical transformation of great interest for the chemical industry. Herein, we report the preparation and spectroscopic characterization of cobalt-substituted P. putida Rxn2 and a truncated version of the protein consisting of the C-terminal domain of the protein. Our spectroscopic data on the Co-substituted C-terminal domain supports a high-spin Co(II) with a distorted tetrahedral coordination environment. Investigation of the two-domain protein Rxn2 provides insights into the metal-binding properties of the N-terminal domain, the role of which is not well understood so far. Circular dichroism, electron paramagnetic resonance and X-ray absorption spectroscopies support an alternative Co-binding site within the N-terminal domain, which appears to not be relevant in nature. We have shown that chemical reconstitution in the presence of Co leads to incorporation of Co(II) into the active site of the C-terminal domain, but not the N-terminal domain of Rxn2 indicating distinct roles for the two rubredoxin domains.


Assuntos
Cobalto/química , Rubredoxinas/química , Domínio Catalítico , Dicroísmo Circular , Cobalto/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Pseudomonas putida/química , Rubredoxinas/metabolismo , Espectrometria por Raios X , Espectrofotometria Ultravioleta
13.
Mol Cell Biol ; 38(9)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29440310

RESUMO

The generation of two daughter cells with the same genetic information requires error-free chromosome segregation during mitosis. Chromosome transmission fidelity is dependent on spindle structure/function, which requires Asp1 in the fission yeast Schizosaccharomyces pombe Asp1 belongs to the diphosphoinositol pentakisphosphate kinase (PPIP5K)/Vip1 family which generates high-energy inositol pyrophosphate (IPP) molecules. Here, we show that Asp1 is a bifunctional enzyme in vivo: Asp1 kinase generates specific IPPs which are the substrates of the Asp1 pyrophosphatase. Intracellular levels of these IPPs directly correlate with microtubule stability: pyrophosphatase loss-of-function mutants raised Asp1-made IPP levels 2-fold, thus increasing microtubule stability, while overexpression of the pyrophosphatase decreased microtubule stability. Absence of Asp1-generated IPPs resulted in an aberrant, increased spindle association of the S. pombe kinesin-5 family member Cut7, which led to spindle collapse. Thus, chromosome transmission is controlled via intracellular IPP levels. Intriguingly, identification of the mitochondrion-associated Met10 protein as the first pyrophosphatase inhibitor revealed that IPPs also regulate mitochondrial distribution.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Fuso Acromático/metabolismo , Segregação de Cromossomos/fisiologia , Inositol , Fosfatos de Inositol/metabolismo , Fosfatos de Inositol/fisiologia , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos , Microtúbulos , Mitose/fisiologia , Enzimas Multifuncionais , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Pirofosfatases , Schizosaccharomyces , Fuso Acromático/fisiologia , Sulfito Redutase (NADPH)/metabolismo
14.
J Am Chem Soc ; 138(38): 12615-28, 2016 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-27646786

RESUMO

We report rapid photoinitiated intracomplex electron transfer (ET) within a "charge-disproportionated" myoglobin (Mb) dimer with greatly enhanced affinity. Two mutually supportive Brownian Dynamics (BD) interface redesign strategies, one a new "heme-filtering" approach, were employed to "break the symmetry" of a Mb homodimer by pairing Mb constructs with complementary highly positive and highly negative net surface charges, introduced through D/E → K and K → E mutations, respectively. BD simulations using a previously developed positive mutant, Mb(+6) = Mb(D44K/D60K/E85K), led to construction of the complementary negative mutant Mb(-6) = Mb(K45E, K63E, K95E). Simulations predict the pair will form a well-defined complex comprising a tight ensemble of conformations with nearly parallel hemes, at a metal-metal distance ∼18-19 Å. Upon expression and X-ray characterization of the partners, BD predictions were verified through ET photocycle measurements enabled by Zn-deuteroporphyrin substitution, forming the [ZnMb(-6), Fe(3+)Mb(+6)] complex. Triplet ET quenching shows charge disproportionation increases the binding constant by no less than ∼5 orders of magnitude relative to wild-type Mb values. All progress curves for charge separation (CS) and charge recombination (CR) are reproduced by a generalized kinetic model for the interprotein ET photocycle. The intracomplex ET rate constants for both CS and CR are increased by over 5 orders of magnitude, and their viscosity independence is indicative of true interprotein ET, rather than dynamic gating as seen in previous studies. The complex displays an unprecedented timecourse for CR of the CS intermediate I. After a laser flash, I forms through photoinduced CS, accumulates to a maximum concentration, then dies away through CR. However, before completely disappearing, I reappears without another flash and reaches a second maximum before disappearing completely.


Assuntos
Eletroquímica , Simulação de Dinâmica Molecular , Mioglobina/química , Citocromos b5 , Transporte de Elétrons , Modelos Moleculares , Mutação , Mioglobina/metabolismo , Processos Fotoquímicos , Ligação Proteica , Conformação Proteica
15.
J Am Chem Soc ; 137(39): 12442-5, 2015 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-26371805

RESUMO

E3 ligases are genetically implicated in many human diseases, yet E3 enzyme mechanisms are not fully understood, and there is a strong need for pharmacological probes of E3s. We report the discovery that the HECT E3 Nedd4-1 is a processive enzyme and that disruption of its processivity by biochemical mutations or small molecules switches Nedd4-1 from a processive to a distributive mechanism of polyubiquitin chain synthesis. Furthermore, we discovered and structurally characterized the first covalent inhibitor of Nedd4-1, which switches Nedd4-1 from a processive to a distributive mechanism. To visualize the binding mode of the Nedd4-1 inhibitor, we used X-ray crystallography and solved the first structure of a Nedd4-1 family ligase bound to an inhibitor. Importantly, our study shows that processive Nedd4-1, but not the distributive Nedd4-1:inhibitor complex, is able to synthesize polyubiquitin chains on the substrate in the presence of the deubiquitinating enzyme USP8. Therefore, inhibition of E3 ligase processivity is a viable strategy to design E3 inhibitors. Our study provides fundamental insights into the HECT E3 mechanism and uncovers a novel class of HECT E3 inhibitors.


Assuntos
Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Ubiquitina-Proteína Ligases Nedd4 , Proteínas/química , Ubiquitina-Proteína Ligases/química
16.
J Am Chem Soc ; 136(22): 7926-32, 2014 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-24813236

RESUMO

(E)-1-Hydroxy-2-methylbut-2-enyl 4-diphosphate reductase (IspH) is a [Fe4S4] cluster-containing enzyme involved in isoprenoid biosynthesis in many bacteria as well as in malaria parasites and is an important drug target. Several inhibitors including amino and thiol substrate analogues, as well as acetylene and pyridine diphosphates, have been reported. Here, we investigate the mode of binding of four pyridine diphosphates to Escherichia coli IspH by using X-ray crystallography. In three cases, one of the iron atoms in the cluster is absent, but in the structure with (pyridin-3-yl)methyl diphosphate, the most potent pyridine-analogue inhibitor reported previously, the fourth iron of the [Fe4S4] cluster is present and interacts with the pyridine ring of the ligand. Based on the results of quantum chemical calculations together with the crystallographic results we propose a side-on η(2) coordination of the nitrogen and the carbon in the 2-position of the pyridine ring to the unique fourth iron in the cluster, which is in the reduced state. The X-ray structure enables excellent predictions using density functional theory of the (14)N hyperfine coupling and quadrupole coupling constants reported previously using HYSCORE spectroscopy, as well as providing a further example of the ability of such [Fe4S4]-containing proteins to form organometallic complexes.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Oxirredutases/metabolismo , Piridinas/metabolismo , Enxofre/metabolismo , Cristalografia por Raios X , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas Ferro-Enxofre/química , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Oxirredutases/química , Ligação Proteica , Conformação Proteica , Piridinas/química , Teoria Quântica
18.
Nat Commun ; 3: 1042, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22948824

RESUMO

The final step of the methylerythritol phosphate isoprenoid biosynthesis pathway is catalysed by the iron-sulphur enzyme IspH, producing the universal precursors of terpenes: isopentenyl diphosphate and dimethylallyl diphosphate. Here we report an unforeseen reaction discovered during the investigation of the interaction of IspH with acetylene inhibitors by X-ray crystallography, Mößbauer, and nuclear magnetic resonance spectroscopy. In addition to its role as a 2H(+)/2e(-) reductase, IspH can hydrate acetylenes to aldehydes and ketones via anti-Markovnikov/Markovnikov addition. The reactions only occur with the oxidised protein and proceed via η(1)-O-enolate intermediates. One of these is characterized crystallographically and contains a C4 ligand oxygen bound to the unique, fourth iron in the 4Fe-4S cluster: this intermediate subsequently hydrolyzes to produce an aldehyde product. This unexpected side to IspH reactivity is of interest in the context of the mechanism of action of other acetylene hydratases, as well as in the design of antiinfectives targeting IspH.


Assuntos
Acetileno/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Oxirredutases/metabolismo , Acetileno/química , Aldeídos/química , Aldeídos/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ferro/metabolismo , Modelos Moleculares , Estrutura Molecular , Oxirredução , Oxirredutases/química , Oxirredutases/genética
19.
J Am Chem Soc ; 134(27): 11225-34, 2012 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-22687151

RESUMO

The [4Fe-4S] protein IspH in the methylerythritol phosphate isoprenoid biosynthesis pathway is an important anti-infective drug target, but its mechanism of action is still the subject of debate. Here, by using electron paramagnetic resonance (EPR) spectroscopy and (2)H, (17)O, and (57)Fe isotopic labeling, we have characterized and assigned two key reaction intermediates in IspH catalysis. The results are consistent with the bioorganometallic mechanism proposed earlier, and the mechanism is proposed to have similarities to that of ferredoxin, thioredoxin reductase, in that one electron is transferred to the [4Fe-4S](2+) cluster, which then performs a formal two-electron reduction of its substrate, generating an oxidized high potential iron-sulfur protein (HiPIP)-like intermediate. The two paramagnetic reaction intermediates observed correspond to the two intermediates proposed in the bioorganometallic mechanism: the early π-complex in which the substrate's 3-CH(2)OH group has rotated away from the reduced iron-sulfur cluster, and the next, η(3)-allyl complex formed after dehydroxylation. No free radical intermediates are observed, and the two paramagnetic intermediates observed do not fit in a Birch reduction-like or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy and X-ray crystallography that two substrate analogues (4 and 5) follow the same reaction mechanism.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Radicais Livres/metabolismo , Oxirredutases/metabolismo , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutação , Oxirredutases/química , Oxirredutases/genética
20.
J Mol Biol ; 416(1): 1-9, 2012 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-22137895

RESUMO

Isoprenoids derive from two universal precursors, isopentenyl diphosphate and dimethylallyl diphosphate, which in most human pathogens are synthesized in the deoxyxylulose phosphate pathway. The last step of this pathway is the conversion of (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate into a mixture of isopentenyl diphosphate and dimethylallyl diphosphate catalyzed by the iron-sulfur protein IspH. The crystal structures reported here of the IspH mutant proteins T167C, E126D and E126Q reveal an alternative substrate conformation compared to the wild-type structure. Thus, the previously observed alkoxide complex decomposes, and the substrate's hydroxymethyl group rotates to interact with Glu126. The carboxyl group of Glu126 then donates a proton to the hydroxyl group to enable water elimination. The structural and functional studies provide further knowledge of the IspH reaction mechanism, which opens up new routes to inhibitor design against malaria and tuberculosis.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Hemiterpenos/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Organofosfatos/metabolismo , Compostos Organofosforados/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Terpenos/química , Água
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