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1.
Curr Biol ; 31(24): 5622-5632.e7, 2021 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-34727522

RESUMEN

Stomata are the pores in the epidermal surface of plant leaves that regulate the exchange of water and CO2 with the environment thus controlling leaf gas exchange.1 In the model dicot plant Arabidopsis thaliana, the transcription factors SPEECHLESS (SPCH) and MUTE sequentially control formative divisions in the stomatal lineage by forming heterodimers with ICE1.2 SPCH regulates entry into the stomatal lineage and its stability or activity is regulated by a mitogen-activated protein kinase (MAPK) signaling cascade, mediated by its interaction with ICE1.3-6 This MAPK pathway is regulated by extracellular epidermal patterning factor (EPFs) peptides, which bind a transmembrane receptor complex to inhibit (EPF1 and EPF2) or promote (STOMAGEN/EPFL9) stomatal development.7-9 MUTE controls the transition to guard mother cell identity and is regulated by the HD-ZIP transcription factor HDG2, which is expressed exclusively in stomatal lineage cells.10,11 Light signals acting through phytochrome and cryptochrome photoreceptors positively regulate stomatal development in response to increased irradiance.12,13 Here we report that stomatal development is also regulated by the redox state of the photosynthetic electron transport chain (PETC). Oxidation of the plastoquinone (PQ) pool inhibits stomatal development by negatively regulating SPCH and MUTE expression. This mechanism is dependent on MPK6 and forms part of the response to lowering irradiance, which is distinct to the photoreceptor dependent response to increasing irradiance. Our results show that environmental signals can act through the PETC, demonstrating that photosynthetic signals regulate the development of the pores through which CO2 enters the leaf.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Oxidación-Reducción , Estomas de Plantas/fisiología , Plastoquinona/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
2.
Sci Rep ; 11(1): 6447, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33742033

RESUMEN

Tripartite members of the ClyA family of α-PFTs have recently been identified in a number of pathogenic Gram-negative bacteria, including the human pathogen Serratia marcescens. Structures of a Gram-negative A component and a tripartite α-PFT complete pore are unknown and a mechanism for pore formation is still uncertain. Here we characterise the tripartite SmhABC toxin from S. marcescens and propose a mechanism of pore assembly. We present the structure of soluble SmhA, as well as the soluble and pore forms of SmhB. We show that the ß-tongue soluble structure is well conserved in the family and propose two conserved latches between the head and tail domains that are broken on the soluble to pore conformational change. Using the structures of individual components, sequence analysis and docking predictions we illustrate how the A, B and C protomers would assemble on the membrane to produce a complete tripartite α-PFT pore.

3.
Commun Biol ; 4(1): 376, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33742128

RESUMEN

In infections by apicomplexan parasites including Plasmodium, Toxoplasma gondii, and Eimeria, host interactions are mediated by proteins including families of membrane-anchored cysteine-rich surface antigens (SAGs) and SAG-related sequences (SRS). Eimeria tenella causes caecal coccidiosis in chickens and has a SAG family with over 80 members making up 1% of the proteome. We have solved the structure of a representative E. tenella SAG, EtSAG19, revealing that, despite a low level of sequence similarity, the entire Eimeria SAG family is unified by its three-layer αßα fold which is related to that of the CAP superfamily. Furthermore, sequence comparisons show that the Eimeria SAG fold is conserved in surface antigens of the human coccidial parasite Cyclospora cayetanensis but this fold is unrelated to that of the SAGs/SRS proteins expressed in other apicomplexans including Plasmodium species and the cyst-forming coccidia Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. However, despite having very different structures, Consurf analysis showed that Eimeria SAG and Toxoplasma SRS families each exhibit marked hotspots of sequence hypervariability that map to their surfaces distal to the membrane anchor. This suggests that the primary and convergent purpose of the different structures is to provide a platform onto which sequence variability can be imposed.


Asunto(s)
Antígenos de Protozoos/metabolismo , Eimeria tenella/metabolismo , Proteínas Protozoarias/metabolismo , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Cristalografía por Rayos X , Eimeria tenella/genética , Evolución Molecular , Variación Genética , Modelos Moleculares , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Relación Estructura-Actividad
4.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 12): 577-582, 2020 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-33263568

RESUMEN

Tripartite α-pore-forming toxins are constructed of three proteins (A, B and C) and are found in many bacterial pathogens. While structures of the B and C components from Gram-negative bacteria have been described, the structure of the A component of a Gram-negative α-pore-forming toxin has so far proved elusive. SmhA, the A component from the opportunistic human pathogen Serratia marcescens, has been cloned, overexpressed and purified. Crystals were grown of selenomethionine-derivatized protein and anomalous data were collected. Phases were calculated and an initial electron-density map was produced.


Asunto(s)
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Serratia marcescens/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/aislamiento & purificación , Clonación Molecular , Cristalografía por Rayos X
6.
J Biol Chem ; 294(48): 18077-18091, 2019 11 29.
Artículo en Inglés | MEDLINE | ID: mdl-31624143

RESUMEN

Alginate is a polymer containing two uronic acid epimers, ß-d-mannuronate (M) and α-l-guluronate (G), and is a major component of brown seaweed that is depolymerized by alginate lyases. These enzymes have diverse specificity, cleaving the chain with endo- or exotype activity and with differential selectivity for the sequence of M or G at the cleavage site. Dp0100 is a 201-kDa multimodular, broad-specificity endotype alginate lyase from the marine thermophile Defluviitalea phaphyphila, which uses brown algae as a carbon source, converting it to ethanol, and bioinformatics analysis suggested that its catalytic domain represents a new polysaccharide lyase family, PL39. The structure of the Dp0100 catalytic domain, determined at 2.07 Å resolution, revealed that it comprises three regions strongly resembling those of the exotype lyase families PL15 and PL17. The conservation of key catalytic histidine and tyrosine residues belonging to the latter suggests these enzymes share mechanistic similarities. A complex of Dp0100 with a pentasaccharide, M5, showed that the oligosaccharide is located in subsites -2, -1, +1, +2, and +3 in a long, deep canyon open at both ends, explaining the endotype activity of this lyase. This contrasted with the hindered binding sites of the exotype enzymes, which are blocked such that only one sugar moiety can be accommodated at the -1 position in the catalytic site. The biochemical and structural analyses of Dp0100, the first for this new class of endotype alginate lyases, have furthered our understanding of the structure-function and evolutionary relationships within this important class of enzymes.


Asunto(s)
Proteínas Bacterianas/química , Clostridiales/enzimología , Polisacárido Liasas/química , Proteínas Bacterianas/genética , Clostridiales/genética , Cristalografía por Rayos X , Polisacárido Liasas/genética , Dominios Proteicos
7.
Commun Biol ; 2: 345, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31552298

RESUMEN

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 µM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.

8.
Nat Commun ; 10(1): 2900, 2019 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-31263098

RESUMEN

The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.


Asunto(s)
Aeromonas hydrophila/metabolismo , Toxinas Bacterianas/química , Proteínas Hemolisinas/química , Proteínas Citotóxicas Formadoras de Poros/química , Aeromonas hydrophila/química , Aeromonas hydrophila/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Cristalografía por Rayos X , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo
9.
Biomolecules ; 9(6)2019 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-31159273

RESUMEN

The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.


Asunto(s)
Glucosa-6-Fosfato Isomerasa/genética , Glucosa-6-Fosfato Isomerasa/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Pyrococcus furiosus/enzimología , Temperatura , Inhibidores Enzimáticos/farmacología , Glucosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Glucosa-6-Fosfato Isomerasa/química , Manganeso/metabolismo , Simulación de Dinámica Molecular , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/química , Conformación Proteica , Ingeniería de Proteínas , Agua/metabolismo
10.
Nat Commun ; 9(1): 4765, 2018 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-30420757

RESUMEN

The type VI secretion system (T6SS) is a multi-protein complex that injects bacterial effector proteins into target cells. It is composed of a cell membrane complex anchored to a contractile bacteriophage tail-like apparatus consisting of a sharpened tube that is ejected by the contraction of a sheath against a baseplate. We present structural and biochemical studies on TssA subunits from two different T6SSs that reveal radically different quaternary structures in comparison to the dodecameric E. coli TssA that arise from differences in their C-terminal sequences. Despite this, the different TssAs retain equivalent interactions with other components of the complex and position their highly conserved N-terminal ImpA_N domain at the same radius from the centre of the sheath as a result of their distinct domain architectures, which includes additional spacer domains and highly mobile interdomain linkers. Together, these variations allow these distinct TssAs to perform a similar function in the complex.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/ultraestructura , Biología Computacional , Filogenia , Dominios Proteicos , Proteolisis , Relación Estructura-Actividad
11.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 9): 536-542, 2018 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-30198885

RESUMEN

TssA is a core component of the type VI secretion system, and phylogenetic analysis of TssA subunits from different species has suggested that these proteins fall into three distinct clades. Whilst representatives of two clades, TssA1 and TssA2, have been the subjects of investigation, no members of the third clade (TssA3) have been studied. Constructs of TssA from Burkholderia cenocepacia, a representative of clade 3, were expressed, purified and subjected to crystallization trials. Data were collected from crystals of constructs of the N-terminal and C-terminal domains. Analysis of the data from the crystals of these constructs and preliminary structure determination indicates that the C-terminal domain forms an assembly of 32 subunits in D16 symmetry, whereas the N-terminal domain is not involved in subunit assocation.


Asunto(s)
Proteínas Bacterianas/química , Burkholderia cenocepacia/química , Electrones , Proteínas de la Membrana/química , Subunidades de Proteína/química , Sistemas de Secreción Tipo VI/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia cenocepacia/clasificación , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Filogenia , Conformación Proteica en Hélice alfa , Dominios Proteicos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 9): 578-582, 2018 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-30198891

RESUMEN

TssA is a core subunit of the type VI secretion system, which is a major player in interspecies competition in Gram-negative bacteria. Previous studies on enteroaggregative Escherichia coli TssA suggested that it is comprised of three putative domains: a conserved N-terminal domain, a middle domain and a ring-forming C-terminal domain. X-ray studies of the latter two domains have identified their respective structures. Here, the results of the expression and purification of full-length and domain constructs of TssA from Aeromonas hydrophila are reported, resulting in diffraction-quality crystals for the middle domain (Nt2) and a construct including the middle and C-terminal domains (Nt2-CTD).


Asunto(s)
Aeromonas hydrophila/química , Proteínas Bacterianas/química , Proteínas de la Membrana/química , Sistemas de Secreción Tipo VI/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido
13.
PLoS One ; 13(7): e0198827, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29969448

RESUMEN

Acanthamoeba is normally free-living, but sometimes facultative and occasionally opportunistic parasites. Current therapies are, by necessity, arduous and yet poorly effective due to their inabilities to kill cyst stages or in some cases to actually induce encystation. Acanthamoeba can therefore survive as cysts and cause disease recurrence. Herein, in pursuit of better therapies and to understand the biochemistry of this understudied organism, we characterize its histidine biosynthesis pathway and explore the potential of targeting this with antimicrobials. We demonstrate that Acanthamoeba is a histidine autotroph, but with the ability to scavenge preformed histidine. It is able to grow in defined media lacking this amino acid, but is inhibited by 3-amino-1,2,4-triazole (3AT) that targets Imidazoleglycerol-Phosphate Dehydratase (IGPD) the rate limiting step of histidine biosynthesis. The structure of Acanthamoeba IGPD has also been determined in complex with 2-hydroxy-3-(1,2,4-triazol-1-yl) propylphosphonate [(R)-C348], a recently described novel inhibitor of Arabidopsis thaliana IGPD. This compound inhibited the growth of four Acanthamoeba species, having a 50% inhibitory concentration (IC50) ranging from 250-526 nM. This effect could be ablated by the addition of 1 mM exogenous free histidine, but importantly not by physiological concentrations found in mammalian tissues. The ability of 3AT and (R)-C348 to restrict the growth of four strains of Acanthamoeba spp. including a recently isolated clinical strain, while not inducing encystment, demonstrates the potential therapeutic utility of targeting the histidine biosynthesis pathway in Acanthamoeba.


Asunto(s)
Acanthamoeba/enzimología , Amitrol (Herbicida)/química , Antiprotozoarios/química , Histidina/antagonistas & inhibidores , Hidroliasas/química , Acanthamoeba/efectos de los fármacos , Acanthamoeba/genética , Acanthamoeba/crecimiento & desarrollo , Amitrol (Herbicida)/farmacología , Antiprotozoarios/farmacología , Procesos Autotróficos/efectos de los fármacos , Procesos Autotróficos/genética , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Histidina/biosíntesis , Hidroliasas/antagonistas & inhibidores , Hidroliasas/genética , Hidroliasas/metabolismo , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinámica
14.
Proc Natl Acad Sci U S A ; 115(8): 1795-1800, 2018 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-29434040

RESUMEN

Histidine biosynthesis is an essential process in plants and microorganisms, making it an attractive target for the development of herbicides and antibacterial agents. Imidazoleglycerol-phosphate dehydratase (IGPD), a key enzyme within this pathway, has been biochemically characterized in both Saccharomyces cerevisiae (Sc_IGPD) and Arabidopsis thaliana (At_IGPD). The plant enzyme, having been the focus of in-depth structural analysis as part of an inhibitor development program, has revealed details about the reaction mechanism of IGPD, whereas the yeast enzyme has proven intractable to crystallography studies. The structure-activity relationship of potent triazole-phosphonate inhibitors of IGPD has been determined in both homologs, revealing that the lead inhibitor (C348) is an order of magnitude more potent against Sc_IGPD than At_IGPD; however, the molecular basis of this difference has not been established. Here we have used single-particle electron microscopy (EM) to study structural differences between the At and Sc_IGPD homologs, which could influence the difference in inhibitor potency. The resulting EM maps at ∼3 Šare sufficient to de novo build the protein structure and identify the inhibitor binding site, which has been validated against the crystal structure of the At_IGPD/C348 complex. The structure of Sc_IGPD reveals that a 24-amino acid insertion forms an extended loop region on the enzyme surface that lies adjacent to the active site, forming interactions with the substrate/inhibitor binding loop that may influence inhibitor potency. Overall, this study provides insights into the IGPD family and demonstrates the power of using an EM approach to study inhibitor binding.


Asunto(s)
Proteínas de Arabidopsis/antagonistas & inhibidores , Arabidopsis/enzimología , Inhibidores Enzimáticos/química , Hidroliasas/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/enzimología , Arabidopsis/química , Arabidopsis/efectos de los fármacos , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/ultraestructura , Sitios de Unión , Microscopía por Crioelectrón , Cristalografía por Rayos X , Herbicidas/química , Hidroliasas/química , Hidroliasas/ultraestructura , Modelos Moleculares , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura
15.
Sci Rep ; 6: 38879, 2016 12 09.
Artículo en Inglés | MEDLINE | ID: mdl-27934966

RESUMEN

Most organisms are exposed to the genotoxic chemical formaldehyde, either from endogenous or environmental sources. Therefore, biology has evolved systems to perceive and detoxify formaldehyde. The frmRA(B) operon that is present in many bacteria represents one such system. The FrmR protein is a transcriptional repressor that is specifically inactivated in the presence of formaldehyde, permitting expression of the formaldehyde detoxification machinery (FrmA and FrmB, when the latter is present). The X-ray structure of the formaldehyde-treated Escherichia coli FrmR (EcFrmR) protein reveals the formation of methylene bridges that link adjacent Pro2 and Cys35 residues in the EcFrmR tetramer. Methylene bridge formation has profound effects on the pattern of surface charge of EcFrmR and combined with biochemical/biophysical data suggests a mechanistic model for formaldehyde-sensing and derepression of frmRA(B) expression in numerous bacterial species.


Asunto(s)
Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/fisiología , Formaldehído/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Cristalografía por Rayos X , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Formaldehído/farmacología , Inactivación Metabólica , Interferometría , Modelos Moleculares , Operón , Regiones Promotoras Genéticas/genética , Conformación Proteica , Proteínas Represoras/química , Proteínas Represoras/genética , Selenometionina/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética
16.
Angew Chem Int Ed Engl ; 55(43): 13485-13489, 2016 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-27717128

RESUMEN

Programs of drug discovery generally exploit one enantiomer of a chiral compound for lead development following the principle that enantiomer recognition is central to biological specificity. However, chiral promiscuity has been identified for a number of enzyme families, which have shown that mirror-image packing can enable opposite enantiomers to be accommodated in an enzyme's active site. Reported here is a series of crystallographic studies of complexes between an enzyme and a potent experimental herbicide whose chiral center forms an essential part of the inhibitor pharmacophore. Initial studies with a racemate at 1.85 Šresolution failed to identify the chirality of the bound inhibitor, however, by extending the resolution to 1.1 Šand by analyzing high-resolution complexes with the enantiopure compounds, we determined that both enantiomers make equivalent pseudosymmetric interactions in the active site, thus mimicking an achiral reaction intermediate.

17.
Nat Struct Mol Biol ; 23(7): 640-6, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27273516

RESUMEN

Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.9-2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate's single-stranded branch approaches, threads through and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual 'fly-casting, thread, bend and barb' mechanism.


Asunto(s)
ADN de Cadena Simple/química , ADN Viral/química , Exodesoxirribonucleasas/química , Oligonucleótidos/química , Siphoviridae/química , Proteínas Virales/química , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Expresión Génica , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oligonucleótidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Siphoviridae/enzimología , Relación Estructura-Actividad , Especificidad por Sustrato , Proteínas Virales/genética , Proteínas Virales/metabolismo
18.
Structure ; 23(7): 1236-45, 2015 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-26095028

RESUMEN

Imidazoleglycerol-phosphate dehydratase (IGPD) catalyzes the Mn(II)-dependent dehydration of imidazoleglycerol phosphate (IGP) to 3-(1H-imidazol-4-yl)-2-oxopropyl dihydrogen phosphate during biosynthesis of histidine. As part of a program of herbicide design, we have determined a series of high-resolution crystal structures of an inactive mutant of IGPD2 from Arabidopsis thaliana in complex with IGP. The structures represent snapshots of the enzyme trapped at different stages of the catalytic cycle and show how substrate binding triggers a switch in the coordination state of an active site Mn(II) between six- and five-coordinate species. This switch is critical to prime the active site for catalysis, by facilitating the formation of a high-energy imidazolate intermediate. This work not only provides evidence for the molecular processes that dominate catalysis in IGPD, but also describes how the manipulation of metal coordination can be linked to discrete steps in catalysis, demonstrating one way that metalloenzymes exploit the unique properties of metal ions to diversify their chemistry.


Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Hidroliasas/química , Dominio Catalítico , Complejos de Coordinación/química , Cristalografía por Rayos X , Herbicidas/química , Imidazoles/química , Manganeso/química , Modelos Moleculares , Fosfatos/química , Unión Proteica
19.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 2): 189-93, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25664794

RESUMEN

Msmeg_0515, a gene from Mycobacterium smegmatis strain 155 encoding the ligand-binding domain, AgaE, of a putative ABC sugar transporter system, has been cloned into a pET-28a vector system, overexpressed in Escherichia coli and purified. The truncated protein lacking the first 27 residues, which correspond to a N-terminal signal sequence, was crystallized using the sitting-drop vapour-diffusion technique. The crystals of this protein diffracted to 1.48 Å resolution and belonged to space group P212121, with unit-cell parameters a = 64.06, b = 69.26, c = 100.74 Å, α = ß = γ = 90° and with one molecule in the asymmetric unit.


Asunto(s)
Proteínas Bacterianas/química , Mycobacterium smegmatis/química , Receptores de Superficie Celular/química , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Ligandos , Datos de Secuencia Molecular
20.
J Biol Chem ; 289(43): 29471-82, 2014 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-25193663

RESUMEN

CO and NO are small toxic gaseous molecules that play pivotal roles in biology as gasotransmitters. During bacterial infection, NO, produced by the host via the inducible NO synthase, exerts critical antibacterial effects while CO, generated by heme oxygenases, enhances phagocytosis of macrophages. In Escherichia coli, other bacteria and fungi, the flavohemoglobin Hmp is the most important detoxification mechanism converting NO and O2 to the ion nitrate (NO3(-)). The protoheme of Hmp binds not only O2 and NO, but also CO so that this ligand is expected to be an inhibitor of NO detoxification in vivo and in vitro. CORM-3 (Ru(CO)(3)Cl(glycinate)) is a metal carbonyl compound extensively used and recently shown to have potent antibacterial properties. In this study, attenuation of the NO resistance of E. coli by CORM-3 is demonstrated in vivo. However, polarographic measurements showed that CO gas, but not CORM-3, produced inhibition of the NO detoxification activity of Hmp in vitro. Nevertheless, CO release from CORM-3 in the presence of soluble cellular compounds is demonstrated by formation of carboxy-Hmp. We show that the inability of CORM-3 to inhibit the activity of purified Hmp is due to slow release of CO in protein solutions alone i.e. when sodium dithionite, widely used in previous studies of CO release from CORM-3, is excluded. Finally, we measure intracellular CO released from CORM-3 by following the formation of carboxy-Hmp in respiring cells. CORM-3 is a tool to explore the concerted effects of CO and NO in vivo.


Asunto(s)
Monóxido de Carbono/metabolismo , Dihidropteridina Reductasa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hemoproteínas/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Óxido Nítrico/metabolismo , Compuestos Organometálicos/metabolismo , Anaerobiosis/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Espacio Intracelular/metabolismo , Hierro/metabolismo , Solubilidad , Sulfatos/farmacología , Suspensiones
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