RESUMEN
A recently discovered ornithine-ammonia cycle (OAC) serves as a conduit in the nitrogen storage and remobilization machinery in cyanobacteria. The OAC involves an arginine catabolic reaction catalyzed by the arginine dihydrolase ArgZ whose catalytic mechanism is unknown. Here we determined the crystal structures at 1.2-3.0 Å of unliganded ArgZ from the cyanobacterium Synechocystis sp. PCC6803 and of ArgZ complexed with its substrate arginine, a covalently linked reaction intermediate, or the reaction product ornithine. The structures reveal that a key residue, Asn71, in the ArgZ active center functions as the determinant distinguishing ArgZ from other members of the guanidino group-modifying enzyme superfamily. The structures, along with biochemical evidence from enzymatic assays coupled with electrospray ionization MS techniques, further suggest that ArgZ-catalyzed conversion of arginine to ornithine, ammonia, and carbon dioxide consists of two successive cycles of amine hydrolysis. Finally, we show that arginine dihydrolases are broadly distributed among bacteria and metazoans, suggesting that the OAC may be frequently used for redistribution of nitrogen from arginine catabolism or nitrogen fixation.
Asunto(s)
Catálisis , Hidrolasas/ultraestructura , Conformación Proteica , Synechocystis/ultraestructura , Amoníaco/química , Arginina/química , Dióxido de Carbono/metabolismo , Cristalografía por Rayos X , Hidrolasas/química , Hidrolasas/genética , Nitrógeno/química , Ornitina/química , Synechocystis/enzimologíaRESUMEN
Canonical bacterial transcription activators bind to non-transcribed promoter elements to increase transcription of their target genes. Here we report crystal structures of binary complexes comprising domains of Caulobacter crescentus GcrA, a noncanonical bacterial transcription factor, that support a novel mechanism for transcription activation through the preferential binding of methylated cis-regulatory elements and the promotion of open complex formation through an interaction with region 2 of the principal σ factor, σ70. We present crystal structures of the C-terminal, σ factor-interacting domain (GcrA-SID) in complex with domain 2 of σ70 (σ702), and the N-terminal, DNA-binding domain (GcrA-DBD) in complex with methylated double-stranded DNA (dsDNA). The structures reveal interactions essential for transcription activation and DNA recognition by GcrA. These structures, along with mutational analyses, support a mechanism of transcription activation in which GcrA associates with RNA polymerase (RNAP) prior to promoter binding through GcrA-SID, arming RNAP with a flexible GcrA-DBD. The RNAP-GcrA complex then binds and activates target promoters harboring a methylated GcrA binding site either upstream or downstream of the transcription start site.
Asunto(s)
Proteínas Bacterianas/genética , Caulobacter crescentus/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Activación Transcripcional , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Caulobacter crescentus/metabolismo , Cristalografía por Rayos X , ADN/química , ADN/genética , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Dominios Proteicos , Elementos Reguladores de la Transcripción/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Escherichia coli 6-carboxytetrahydropterin synthase (eCTPS), a homologue of 6-pyruvoyltetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterin in vitro compared with genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin in vivo. Crystal structures of wild-type apo eCTPS and of a Cys27Ala mutant eCTPS complexed with sepiapterin have been determined to 2.3 and 2.5 Å resolution, respectively. The structures are highly conserved at the active site and the Zn(2+) binding site. However, comparison of the eCTPS structures with those of mammalian PTPS homologues revealed that two specific residues, Trp51 and Phe55, that are not found in mammalian PTPS keep the substrate bound by stacking it with their side chains. Replacement of these two residues by site-directed mutagenesis to the residues Met and Leu, which are only found in mammalian PTPS, converted eCTPS to the mammalian PTPS activity. These studies confirm that these two aromatic residues in eCTPS play an essential role in stabilizing the substrate and in the specific enzyme activity that differs from the original PTPS activity. These aromatic residues Trp51 and Phe55 are a key signature of bacterial PTPS enzymes that distinguish them from mammalian PTPS homologues.
Asunto(s)
Liasas de Fósforo-Oxígeno/química , Liasas de Fósforo-Oxígeno/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Mamíferos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fenilalanina/química , Liasas de Fósforo-Oxígeno/genética , Filogenia , Conformación Proteica , Pterinas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Tribulus terrestris fruits are well known for their usage in pharmaceutical preparations and food supplements. The methanol extract of T. terrestris fruits showed potent inhibition against the papain-like protease (PLpro), an essential proteolylic enzyme for protection to pathogenic virus and bacteria. Subsequent bioactivity-guided fractionation of this extract led to six cinnamic amides (1-6) and ferulic acid (7). Compound 6 emerged as new compound possessing the very rare carbinolamide motif. These compounds (1-7) were evaluated for severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro inhibitory activity to identify their potencies and kinetic behavior. Compounds (1-6) displayed significant inhibitory activity with IC50 values in the range 15.8-70.1 µM. The new cinnamic amide 6 was found to be most potent inhibitor with an IC50 of 15.8 µM. In kinetic studies, all inhibitors exhibited mixed type inhibition. Furthermore, the most active PLpro inhibitors (1-6) were proven to be present in the native fruits in high quantities by HPLC chromatogram and liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI/MS).
Asunto(s)
Cinamatos/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Extractos Vegetales/química , Tribulus/química , Proteínas Virales/antagonistas & inhibidores , Amidas , Cinamatos/aislamiento & purificación , Cinamatos/uso terapéutico , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/genética , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Inhibidores de Cisteína Proteinasa/uso terapéutico , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Frutas/química , Humanos , Concentración 50 Inhibidora , Cinética , Estructura Molecular , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Síndrome Respiratorio Agudo Grave/tratamiento farmacológico , Síndrome Respiratorio Agudo Grave/virología , Relación Estructura-Actividad , Proteínas Virales/genéticaRESUMEN
As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.
Asunto(s)
COVID-19 , Glutamina , Humanos , Glutamina/química , SARS-CoV-2 , Cisteína Endopeptidasas/química , Invenciones , Inhibidores de Proteasas/farmacología , Amidas , Antivirales/farmacología , Antivirales/químicaRESUMEN
Mevalonate kinase (MVK), which plays an important role in catalysing the biosynthesis of isoprenoid compounds derived from the mevalonate pathway, transforms mevalonate to 5-phosphomevalonate using ATP as a cofactor. Mevalonate kinase from Methanosarcina mazei (MmMVK) was expressed in Escherichia coli, purified and crystallized for structural analysis. Diffraction-quality crystals of MmMVK were obtained by the vapour-diffusion method using 0.32â M MgCl2, 0.08â M bis-tris pH 5.5, 16%(w/v) PEG 3350. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a=97.11, b=135.92, c=46.03â Å. Diffraction data were collected to 2.08â Å resolution.
Asunto(s)
Methanosarcina/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Adenosina Trifosfato/metabolismo , Catálisis , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Trometamina/análogos & derivados , Difracción de Rayos XRESUMEN
A DJ-1 homologue protein from Arabidopsis thaliana (AtDJ-1D) belongs to the DJ-1/ThiJ/Pfpl superfamily and contains two tandem arrays of DJ-1-like sequences, but no structural information is available to date for this protein. AtDJ-1D was expressed in Escherichia coli, purified and crystallized for structural analysis. A crystal of AtDJ-1D was obtained by the hanging-drop vapour-diffusion method using 0.22 M NaCl, 0.1 M bis-tris pH 6.5, 21% polyethylene glycol 3350. AtDJ-1D crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 56.78, b = 75.21, c = 141.68 Å, ß = 96.87°, and contained a trimer in the asymmetric unit. Diffraction data were collected to 2.05 Å resolution. The structure of AtDJ-1D has been determined using the multiple-wavelength anomalous dispersion (MAD) method.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Cristalización , Cristalografía por Rayos XRESUMEN
Dictyostelium discoideum phenylalanine hydroxylase (DicPAH; residues 1-415) was expressed in Escherichia coli and purified for structural analysis. Apo DicPAH and DicPAH complexed with dihydrobiopterin (BH(2)) and Fe(III) were crystallized using 0.06 M PIPES pH 7.0, 26%(w/v) PEG 2000 by the hanging-drop vapour-diffusion method. Crystals of apo DicPAH and the DicPAH-BH(2)-Fe(III) complex diffracted to 2.6 and 2.07 A resolution, respectively, and belonged to space group P2(1), with unit-cell parameters a = 70.02, b = 85.43, c = 74.86 A, beta = 110.12 degrees and a = 70.97, b = 85.33, c = 74.89 A, beta = 110.23 degrees , respectively. There were two molecules in the asymmetric unit. The structure of DicPAH has been solved by molecular replacement.
Asunto(s)
Biopterinas/análogos & derivados , Dictyostelium/enzimología , Compuestos Férricos/química , Fenilalanina Hidroxilasa/química , Biopterinas/química , Biopterinas/metabolismo , Cristalización , Cristalografía por Rayos X , Compuestos Férricos/metabolismo , Fenilalanina Hidroxilasa/aislamiento & purificación , Fenilalanina Hidroxilasa/metabolismo , Unión ProteicaRESUMEN
Bacterial RNA polymerase employs extra-cytoplasmic function (ECF) σ factors to regulate context-specific gene expression programs. Despite being the most abundant and divergent σ factor class, the structural basis of ECF σ factor-mediated transcription initiation remains unknown. Here, we determine a crystal structure of Mycobacterium tuberculosis (Mtb) RNAP holoenzyme comprising an RNAP core enzyme and the ECF σ factor σH (σH-RNAP) at 2.7 Å, and solve another crystal structure of a transcription initiation complex of Mtb σH-RNAP (σH-RPo) comprising promoter DNA and an RNA primer at 2.8 Å. The two structures together reveal the interactions between σH and RNAP that are essential for σH-RNAP holoenzyme assembly as well as the interactions between σH-RNAP and promoter DNA responsible for stringent promoter recognition and for promoter unwinding. Our study establishes that ECF σ factors and primary σ factors employ distinct mechanisms for promoter recognition and for promoter unwinding.
Asunto(s)
Proteínas Bacterianas/química , ARN Polimerasas Dirigidas por ADN/química , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Factor sigma/química , Iniciación de la Transcripción Genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , ADN Bacteriano/química , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Modelos Moleculares , Regiones Promotoras Genéticas/genética , Factor sigma/metabolismoRESUMEN
Dihydropteridine reductase from Dictyostelium discoideum (dicDHPR) can produce D-threo-BH(4) [6R-(1'R,2'R)-5,6,7,8-tetrahydrobiopterin], a stereoisomer of L-erythro-BH(4), in the last step of tetrahydrobiopterin (BH(4)) recycling. In this reaction, DHPR uses NADH as a cofactor to reduce quinonoid dihydrobiopterin back to BH(4). To date, the enzyme has been purified to homogeneity from many sources. In this report, the dicDHPR-NAD complex has been crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as a precipitant. Rectangular-shaped crystals were obtained. Crystals grew to maximum dimensions of 0.4 x 0.6 x 0.1 mm. The crystal belonged to space group P2(1), with unit-cell parameters a = 49.81, b = 129.90, c = 78.76 A, beta = 100.00 degrees , and contained four molecules in the asymmetric unit, forming two closely interacting dicDHPR-NAD dimers. Diffraction data were collected to 2.16 A resolution using synchrotron radiation. The crystal structure has been determined using the molecular-replacement method.
Asunto(s)
Dictyostelium/enzimología , Dihidropteridina Reductasa/química , Proteínas Protozoarias/química , Animales , Cristalización/métodos , Cristalografía por Rayos X , Dihidropteridina Reductasa/aislamiento & purificación , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Proteínas Protozoarias/aislamiento & purificaciónRESUMEN
Neurofibrillary tangles (NFTs) represent products of insoluble tau protein in the brains of patients with Alzheimer disease (AD). The cerebrospinal fluid (CSF) tau level is a biomarker in AD diagnosis. The soluble portion of tau protein in brain parenchyma is presumably the source for CSF tau but this has not previously been quantified. We measured CSF tau and soluble brain tau at autopsy in temporal and frontal brain tissue samples from 7 cognitive normal, 12 mild cognitively impaired, and 19 AD subjects. Based on the measured brain soluble tau, we calculated the whole brain tau load and estimated tau secretion factor. Our results suggest that the increase in NFT in AD is likely attributable to post-translational processes; the increase in CSF tau in AD patients is due to an accelerated carrier-based secretion. Moreover, cognitive dysfunction assessed by final Mini-Mental State Examination scores correlated with the secretion factor but not with the soluble tau.
Asunto(s)
Enfermedad de Alzheimer/líquido cefalorraquídeo , Encéfalo/metabolismo , Ovillos Neurofibrilares/metabolismo , Proteínas tau/líquido cefalorraquídeo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/psicología , Biomarcadores/líquido cefalorraquídeo , Encéfalo/patología , Escalas de Valoración Psiquiátrica Breve , Femenino , Humanos , Masculino , Ovillos Neurofibrilares/patología , Método Simple CiegoRESUMEN
A UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) enzyme was discovered in the cyanobacterium Synechococcus sp. PCC 7942 which transfers a glucose moiety from UDP-glucose to tetrahydrobiopterin (BH4). BGluT protein was overexpressed with selenomethionine labelling for structure determination by the multi-wavelength anomalous dispersion method. The BGluT protein was purified by nickel-affinity and size-exclusion chromatography. It was then crystallized by the hanging-drop vapour-diffusion method using a well solution consisting of 0.1 M bis-tris pH 5.5, 19%(w/v) polyethylene glycol 3350 with 4%(w/v) D(+)-galactose as an additive. X-ray diffraction data were collected to 1.99 Å resolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 171.35, b = 77.99, c = 53.77 Å, ß = 90.27°.
Asunto(s)
Proteínas Bacterianas/química , Cristalografía por Rayos X/métodos , Glucosiltransferasas/química , Synechococcus/enzimología , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Cristalización , Cartilla de ADN , Glucosiltransferasas/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Conformación ProteicaRESUMEN
Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.
Asunto(s)
Proteínas Bacterianas , Celulasa/genética , Celulasa/metabolismo , Celulosa/metabolismo , Celulasa/aislamiento & purificación , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrólisis , Mutación , Periplasma/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , TemperaturaRESUMEN
We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.
Asunto(s)
Dictyostelium/enzimología , Fenilalanina Hidroxilasa/química , Fenilalanina Hidroxilasa/metabolismo , Fenilalanina/metabolismo , Fenilalanina/farmacología , Biocatálisis , Activación Enzimática/efectos de los fármacos , Estabilidad de Enzimas , Cinética , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
Cinnamyl alcohol dehydrogenase is a zinc- and NADPH-dependent dehydrogenase catalyzing the reversible conversion of p-hydroxycinnamaldehydes to their corresponding hydroxycinnamyl alcohols. A CAD homolog from Helicobacter pylori (HpCAD) possesses broad substrate specificities like the plant CADs and additionally a dismutation activity converting benzaldehyde to benzyl alcohol and benzoic acid. We have determined the crystal structure of HpCAD complexed with NADP(H) at 2.18Å resolution to get a better understanding of this class of CAD outside of plants. The structure of HpCAD is highly homologous to the sinapyl alcohol dehydrogenase and the plant CAD with well-conserved residues involved in catalysis and zinc binding. However, the NADP(H) binding mode of the HpCAD has been found to be significantly different from those of plant CADs.
Asunto(s)
Oxidorreductasas de Alcohol/química , Helicobacter pylori/enzimología , NADP/química , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , ADN Bacteriano/genética , Dimerización , Helicobacter pylori/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , NAD/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformación Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Up to now, d-threo-tetrahydrobiopterin (DH(4), dictyopterin) was detected only in Dictyostelium discoideum, while the isomer L-erythro-tetrahydrobioterin (BH(4)) is common in mammals. To elucidate the mechanism of DH(4) regeneration by D. discoideum dihydropteridine reductase (DicDHPR), we have determined the crystal structure of DicDHPR complexed with NAD(+) at 2.16 Å resolution. Significant structural differences from mammalian DHPRs are found around the coenzyme binding site, resulting in a higher K(m) value for NADH (K(m)=46.51±0.4 µM) than mammals. In addition, we have found that rat DHPR as well as DicDHPR could bind to both substrates quinonoid-BH(2) and quinonoid-DH(2) by docking calculations and have confirmed their catalytic activity by in vitro assay.