RESUMEN
Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as Pseudomonas putida CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N-demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial N-demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial N1- and N3-specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper N-demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during N-demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications.
Asunto(s)
Cafeína/metabolismo , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/metabolismo , Pseudomonas putida/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Cafeína/química , Cristalografía por Rayos X , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Dominios Proteicos , Especificidad por SustratoRESUMEN
NdmB genes from Pseudomonas putida CBB5 and GO genes from spinach, which encode N-demethylase B (NdmB) and Glycolate oxidase (GO) respectively, were separately ligated into expression vectors of pACYCDuet-1 and pET32a to construct recombinant plasmids of pACYCDuet-1-ndmBHis (pBH) and pET32a-GOHis (pGOH). Then the two plasmids were both transformed in Escherichia coli (E. coli) strain BL21 (DE3) and screening the recombinants (pBHGOH) using ampicillin and chloramphonicol as two antibiotics in Luria-Bertani medium. After induction with IPTG, both recombinant ndmB and GO genes were coexpressed in E. coli. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the estimated molecular weight of NdmB and GO was 35 kDa and 40 kDa, respectively. By two-step purification of Ni affinity chromatography and Q-Sepharose chromatography, the coexpressed NdmB and GO were separated and resulted in a 15.8-fold purification with 8.7% yield and 12.8-fold purification with 7.2% yield, respectively.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas de Alcohol/aislamiento & purificación , Oxidorreductasas de Alcohol/metabolismo , Cromatografía de Afinidad/métodos , Clonación Molecular , Electroforesis en Gel de Poliacrilamida/métodos , Escherichia coli/genética , Expresión Génica/efectos de los fármacos , Vectores Genéticos , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Oxidorreductasas N-Desmetilantes/metabolismo , Plásmidos , Pseudomonas putida/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes/genéticaRESUMEN
Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8alpha-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8alpha-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.
Asunto(s)
Eschscholzia/enzimología , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/metabolismo , Cristalografía por Rayos X , Expresión Génica , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Oxidación-Reducción , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Unión Proteica , Conformación ProteicaRESUMEN
The X-ray structure of monomeric N-methyltryptophan oxidase from Escherichia coli (MTOX) has been solved at 3.2 A resolution by molecular replacement methods using Bacillus sp. sarcosine oxidase structure (MSOX, 43% sequence identity) as search model. The analysis of the substrate binding site highlights the structural determinants that favour the accommodation of the bulky N-methyltryptophan residue in MTOX. In fact, although the nature and geometry of the catalytic residues within the first contact shell of the FAD moiety appear to be virtually superposable in MTOX and MSOX, the presence of a Thr residue in position 239 in MTOX (Met245 in MSOX) located at the entrance of the active site appears to play a key role for the recognition of the amino acid substrate side chain. Accordingly, a 15 fold increase in k(cat) and 100 fold decrease in K(m) for sarcosine as substrate has been achieved in MTOX upon T239M mutation, with a concomitant three-fold decrease in activity towards N-methyltryptophan. These data provide clear evidence for the presence of a catalytic core, common to the members of the methylaminoacid oxidase subfamily, and of a side chain recognition pocket, located at the entrance of the active site, that can be adjusted to host diverse aminoacids in the different enzyme species. The site involved in the covalent attachment of flavin has also been addressed by screening degenerate mutants in the relevant positions around Cys308-FAD linkage. Lys341 appears to be the key residue involved in flavin incorporation and covalent linkage.
Asunto(s)
Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Oxidorreductasas N-Desmetilantes/análisis , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Flavina-Adenina Dinucleótido/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Mutación , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Espectrofotometría Ultravioleta , Espectrometría Raman , Especificidad por Sustrato , Treonina/químicaAsunto(s)
Histonas/metabolismo , Oxidorreductasas N-Desmetilantes , Procesamiento Proteico-Postraduccional , Epigénesis Genética , Proteínas F-Box , Histona Demetilasas , Humanos , Histona Demetilasas con Dominio de Jumonji , Metilación , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Estructura Terciaria de ProteínaRESUMEN
Histone methylation plays important roles in the regulation of chromatin dynamics and transcription. Steady-state levels of histone lysine methylation are regulated by a balance between enzymes that catalyze either the addition or removal of methyl groups. Using an activity-based biochemical approach, we recently uncovered the JmjC domain as an evolutionarily conserved signature motif for histone demethylases. Furthermore, we demonstrated that Jhd1, a JmjC domain-containing protein in Saccharomyces cerevisiae, is an H3K36-specific demethylase. Here we report further characterization of Jhd1. Similar to its mammalian homolog, Jhd1-catalyzed histone demethylation requires iron and alpha-ketoglutarate as cofactors. Mutation and deletion studies indicate that the JmjC domain and adjacent sequences are critical for Jhd1 enzymatic activity, while the N-terminal PHD domain is dispensable. Overexpression of JHD1 results in a global reduction of H3K36 methylation in vivo. Finally, chromatin immunoprecipitation-coupled microarray studies reveal subtle changes in the distribution of H3K36me2 upon overexpression or deletion of JHD1. Our studies establish Jhd1 as a histone demethylase in budding yeast and suggest that Jhd1 functions to maintain the fidelity of histone methylation patterns along transcription units.
Asunto(s)
Histonas/metabolismo , Lisina/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Eliminación de Gen , Células HeLa , Humanos , Hierro/farmacología , Histona Demetilasas con Dominio de Jumonji , Ácidos Cetoglutáricos/farmacología , Metilación/efectos de los fármacos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Fenotipo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
Lysine-specific demethylase 1 (LSD1) is a transcriptional repressor and a flavin-dependent amine oxidase that is responsible for the removal of methyl from lysine 4 of histone H3. In this study, we characterize the mechanism and scope of LSD1 inhibition by a propargylamine-derivatized histone H3 substrate (1). Unlike aziridinyl and cyclopropylamine-derivatized histone H3 peptide substrate analogues, compound 1 appears to covalently modify and irreversibly inactivate LSD1 with high potency. Accompanying this inactivation is a spectroscopic change, which shifts the absorbance maximum to 392 nm. Spectral changes associated with the 1-LSD1 complex and reactivity to decreased pH and sodium borohydride treatment were suggestive of a structure involving a flavin-linked inhibitor conjugate between N5 of the flavin and the terminal carbon of the inhibitor. Using a 13C-labeled inhibitor, NMR analysis of the 1-flavin conjugate was consistent with this structural assignment. Kinetic analysis of the spectroscopic shift induced by 1 showed that the flavin adduct formed in a reaction with kinetic constants similar to those of the LSD1 inactivation process. Taken together, these data support a mechanism of LSD1 inactivation by 1 involving amine oxidation followed by Michael addition to the propargylic imine. We further examined the potential for a biotinylated analogue of 1 (1-Btn) to be used as a tool in affinity pulldown experiments. Using 1-Btn, it was feasible to selectively pull down spiked and endogenous LSD1 from HeLa cell nuclear extracts, setting the stage for activity-based demethylase proteomics.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Oxidorreductasas N-Desmetilantes/química , Secuencia de Aminoácidos , Biotinilación , Inhibidores Enzimáticos/química , Flavinas/farmacología , Glutatión/metabolismo , Células HeLa , Histona Demetilasas , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Oxidación-Reducción , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , EspectrofotometríaRESUMEN
The catalytic domain of the flavin-dependent human histone demethylase lysine-specific demethylase 1 (LSD1) belongs to the family of amine oxidases including polyamine oxidase and monoamine oxidase (MAO). We previously assessed monoamine oxidase inhibitors (MAOIs) for their ability to inhibit the reaction catalyzed by LSD1 [Lee, M. G., et al. (2006) Chem. Biol. 13, 563-567], demonstrating that trans-2-phenylcyclopropylamine (2-PCPA, tranylcypromine, Parnate) was the most potent with respect to LSD1. Here we show that 2-PCPA is a time-dependent, mechanism-based irreversible inhibitor of LSD1 with a KI of 242 microM and a kinact of 0.0106 s-1. 2-PCPA shows limited selectivity for human MAOs versus LSD1, with kinact/KI values only 16-fold and 2.4-fold higher for MAO B and MAO A, respectively. Profiles of LSD1 activity and inactivation by 2-PCPA as a function of pH are consistent with a mechanism of inactivation dependent upon enzyme catalysis. Mass spectrometry supports a role for FAD as the site of covalent modification by 2-PCPA. These results will provide a foundation for the design of cyclopropylamine-based inhibitors that are selective for LSD1 to probe its role in vivo.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Flavinas/química , Inhibidores de la Monoaminooxidasa/química , Oxidorreductasas N-Desmetilantes/química , Tranilcipromina/farmacología , Secuencia de Aminoácidos , Dominio Catalítico , Inhibidores Enzimáticos/química , Escherichia coli/genética , Flavinas/metabolismo , Histona Demetilasas , Humanos , Concentración de Iones de Hidrógeno , Cinética , Lisina/metabolismo , Espectrometría de Masas , Metilación , Modelos Biológicos , Datos de Secuencia Molecular , Estructura Molecular , Inhibidores de la Monoaminooxidasa/metabolismo , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Oxidorreductasas N-Desmetilantes/metabolismo , Estructura Terciaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Temperatura , Tranilcipromina/químicaRESUMEN
Posttranslational histone modifications play an important role in regulating chromatin dynamics and function. One of the modifications, methylation, occurs on both lysine and arginine residues and participates in diverse range of biological processes including heterochromatin formation, X-chromosome inactivation, and transcriptional regulation. While acetylation, phosphorylation, and ubiquitylation are dynamically regulated by enzymes that catalyze the addition and removal of a particular modification, enzymes that are capable of removing methyl groups were not known until recently. Thus far, two families of histone demethylases with distinct cofactor requirements and reaction mechanisms have been identified. One is the FAD (flavin adenine dinucleotide)-dependent amine oxidase family LSD1 (lysine-specific demethylase), the other is the Fe(II) and alpha-KG (alpha-ketoglutarate)-dependent dioxygenase family JHDM (JmjC domain-containing histone demethylase). Identification and characterization of these histone demethylases is an important step towards understanding both the function and regulation of histone methylation. Here, we describe assays currently used for measuring histone demethylase activity and chromatography strategies used in purifying histone demethylases from HeLa cells.
Asunto(s)
Histonas/metabolismo , Complejos Multiproteicos/aislamiento & purificación , Oxidorreductasas N-Desmetilantes/análisis , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Cromatografía/métodos , Proteínas F-Box , Células HeLa , Histona Demetilasas , Humanos , Histona Demetilasas con Dominio de Jumonji , MetilaciónRESUMEN
Histone methylation is involved in the regulation of many cellular processes. In the past 2 years, several histone demethylases including BHC110/LSD1 have been characterized. BHC110, the first known histone lysine demethylase, removes methyl groups from methylated histone H3 lysine 4 and has been found in many multi-protein complexes. Using one-step affinity purification, we have isolated enzymatically active BHC110-containing complexes. Here, we detail the methods used for the isolation and characterization of these histone demethylase complexes from a human stable cell line.
Asunto(s)
Cromatografía de Afinidad/métodos , Histonas/metabolismo , Metiltransferasas/aislamiento & purificación , Complejos Multiproteicos/aislamiento & purificación , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Línea Celular , Humanos , Lisina/metabolismo , Metilación , Metiltransferasas/metabolismo , Complejos Multiproteicos/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Transfección/métodosRESUMEN
The reversible methylation of specific lysine residues in histone tails is crucial in epigenetic gene regulation. LSD1, the first known lysine-specific demethylase, selectively removes monomethyl and dimethyl, but not trimethyl modifications of Lys4 or Lys9 of histone-3. Here, we present the crystal structure of LSD1 at 2.9-A resolution. LSD1 forms a highly asymmetric, closely packed domain structure from which a long helical 'tower' domain protrudes. The active site cavity is spacious enough to accommodate several residues of the histone tail substrate, but does not appear capable of recognizing the different methylation states of the substrate lysine. This supports the hypothesis that trimethylated lysine is chemically rather than sterically discriminated. We present a biochemical analysis of LSD1 mutants that identifies crucial residues in the active site cavity and shows the importance of the SWIRM and tower domains for catalysis.
Asunto(s)
Oxidorreductasas N-Desmetilantes/química , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Histona Demetilasas , Histonas/metabolismo , Humanos , Lisina/metabolismo , Modelos Moleculares , Mutagénesis , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
Covalent modification of histones plays an important role in regulating chromatin dynamics and transcription. Histone methylation was thought to be an irreversible modification until recently. Using a biochemical assay coupled with chromatography, we have purified a JmjC domain-containing protein, JHDM2A, which specifically demethylates mono- and dimethyl-H3K9. Similar to JHDM1, JHDM2A-mediated histone demethylation requires cofactors Fe(II) and alpha-ketoglutarate. Mutational studies indicate that a JmjC domain and a zinc finger present in JHDM2A are required for its enzymatic activity. Overexpression of JHDM2A greatly reduced the H3K9 methylation level in vivo. Knockdown of JHDM2A results in an increase in the dimethyl-K9 levels at the promoter region of a subset of genes concomitant with decrease in their expression. Finally, JHDM2A exhibits hormone-dependent recruitment to androgen-receptor target genes, resulting in H3K9 demethylation and transcriptional activation. Thus, our work identifies a histone demethylase and links its function to hormone-dependent transcriptional activation.
Asunto(s)
Histonas/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Receptores Androgénicos/metabolismo , Transactivadores/metabolismo , Activación Transcripcional/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Dominio Catalítico/genética , Chlorocebus aethiops , Regulación hacia Abajo/fisiología , Hormonas Esteroides Gonadales/metabolismo , Células HeLa , Histonas/genética , Humanos , Hierro/metabolismo , Histona Demetilasas con Dominio de Jumonji , Ácidos Cetoglutáricos/metabolismo , Ratones , Datos de Secuencia Molecular , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Regiones Promotoras Genéticas/fisiología , Estructura Terciaria de Proteína/genética , Transactivadores/genética , Transactivadores/aislamiento & purificaciónRESUMEN
Improvement of CYP2B6 expression was examined by co-expression with molecular chaperones GroES/EL. Although a CO-reduced difference spectrum was not detected in Escherichia coli transformed only by the CYP2B6-expressing vector, co-expression of GroES/EL resulted in high-level expression which reached over 2000 nmol P450/L. CYP2B6 was purified from the E. coli membrane with a high yield. Purified CYP2B6 showed 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity in a reconstitution system. This expression system would be useful for the production of large amounts of active CYP2B6 and for the detailed analysis of the enzyme.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/biosíntesis , Proteínas Bacterianas/biosíntesis , Chaperonina 10/biosíntesis , Escherichia coli , Expresión Génica , Proteínas de Choque Térmico/biosíntesis , Oxidorreductasas N-Desmetilantes/biosíntesis , Proteínas Recombinantes/biosíntesis , Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/aislamiento & purificación , Proteínas Bacterianas/genética , Chaperonina 10/genética , Chaperoninas , Cumarinas/química , Citocromo P-450 CYP2B6 , Proteínas de Escherichia coli , Proteínas de Choque Térmico/genética , Humanos , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The covalently bound FAD in native monomeric sarcosine oxidase (MSOX) is attached to the protein by a thioether bond between the 8alpha-methyl group of the flavin and Cys315. Large amounts of soluble apoenzyme are produced by controlled expression in a riboflavin-dependent Escherichia coli strain. A time-dependent increase in catalytic activity is observed upon incubation of apoMSOX with FAD, accompanied by the covalent incorporation of FAD to approximately 80% of the level observed with the native enzyme. The spectral and catalytic properties of the reconstituted enzyme are otherwise indistinguishable from those of native MSOX. The reconstitution reaction exhibits apparent second-order kinetics (k = 139 M(-)(1) min(-)(1) at 23 degrees C) and is accompanied by the formation of a stoichiometric amount of hydrogen peroxide. A time-dependent reduction of FAD is observed when the reconstitution reaction is conducted under anaerobic conditions. The results provide definitive evidence for autoflavinylation in a reaction that proceeds via a reduced flavin intermediate and requires only apoMSOX and FAD. Flavinylation of apoMSOX is not observed with 5-deazaFAD or 1-deazaFAD, an outcome attributed to a decrease in the acidity of the 8alpha-methyl group protons. Covalent flavin attachment is observed with 8-nor-8-chloroFAD in an aromatic nucleophilic displacement reaction that proceeds via a quininoid intermediate but not a reduced flavin intermediate. The reconstituted enzyme contains a modified cysteine-flavin linkage (8-nor-8-S-cysteinyl) as compared with native MSOX (8alpha-S-cysteinyl), a difference that may account for its approximately 10-fold lower catalytic activity.
Asunto(s)
Flavina-Adenina Dinucleótido/análogos & derivados , Flavina-Adenina Dinucleótido/metabolismo , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Oxidorreductasas N-Desmetilantes/metabolismo , Apoenzimas/biosíntesis , Apoenzimas/genética , Apoenzimas/aislamiento & purificación , Apoenzimas/metabolismo , Bacillus/enzimología , Bacillus/genética , Sitios de Unión , Cisteína/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Flavina-Adenina Dinucleótido/síntesis química , Flavina-Adenina Dinucleótido/aislamiento & purificación , Cinética , Mutagénesis , Oxidorreductasas N-Desmetilantes/biosíntesis , Oxidorreductasas N-Desmetilantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sarcosina-Oxidasa , EspectrofotometríaRESUMEN
Posttranslational modifications of histone N-terminal tails impact chromatin structure and gene transcription. While the extent of histone acetylation is determined by both acetyltransferases and deacetylases, it has been unclear whether histone methylation is also regulated by enzymes with opposing activities. Here, we provide evidence that LSD1 (KIAA0601), a nuclear homolog of amine oxidases, functions as a histone demethylase and transcriptional corepressor. LSD1 specifically demethylates histone H3 lysine 4, which is linked to active transcription. Lysine demethylation occurs via an oxidation reaction that generates formaldehyde. Importantly, RNAi inhibition of LSD1 causes an increase in H3 lysine 4 methylation and concomitant derepression of target genes, suggesting that LSD1 represses transcription via histone demethylation. The results thus identify a histone demethylase conserved from S. pombe to human and reveal dynamic regulation of histone methylation by both histone methylases and demethylases.
Asunto(s)
Histonas/metabolismo , Lisina/metabolismo , Metilación , Proteínas Nucleares/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Secuencia Conservada/genética , Formaldehído/metabolismo , Regulación de la Expresión Génica , Células HeLa , Histona Demetilasas , Humanos , Espectrometría de Masas , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Interferencia de ARN , Proteínas Recombinantes , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/aislamiento & purificación , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
Histamine dehydrogenase from Nocardioides simplex is a homodimeric enzyme and catalyzes oxidative deamination of histamine. The gene encoding this enzyme has been sequenced and cloned by polymerase chain reactions and overexpressed in Escherichia coli. The sequence of the complete open reading frame, 2073 bp coding for a protein of 690 amino acids, was determined on both strands. The amino acid sequence of histamine dehydrogenase is closely related to those of trimethylamine dehydrogenase and dimethylamine dehydrogenase containing an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-flavin mononucleotide, and one 4Fe-4S cluster as redox active cofactors in each subunit of the homodimer. The presence of the identical redox cofactors in histamine dehydrogenase has been confirmed by sequence alignment analysis, mass spectral analysis, UV-vis and EPR spectroscopy, and chemical analysis of iron and acid-labile sulfur. These results suggest that the structure of histamine dehydrogenase in the vicinity of the two redox centers is almost identical to that of trimethylamine dehydrogenase as a whole. The structure modeling study, however, demonstrated that a putative substrate-binding cavity in histamine dehydrogenase is quite distinct from that of trimethylamine dehydrogenase.
Asunto(s)
Bacterias Grampositivas/enzimología , Oxidorreductasas N-Desmetilantes/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión , Dominio Catalítico , Clonación Molecular , Mononucleótido de Flavina/química , Proteínas Hierro-Azufre/química , Modelos Moleculares , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/aislamiento & purificación , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Alineación de Secuencia , Análisis de Secuencia de Proteína , Análisis EspectralRESUMEN
Based on information of the nucleotide sequence obtained from rat genome clones, a new CYP3A (CYP3A62) cDNA was isolated from the cDNA library of a rat liver. The CYP3A62 cDNA was 1746 base pairs (bp) in length, which included 1491 bp of an open reading frame and 93 bp and 209 bp of the respective 5'- and 3'-noncoding regions. Amino acid sequence deduced from CYP3A62 cDNA shared the highest similarity with rat CYP3A9 (79.9%) among human and rat CYP3A forms previously reported. CYP3A62 mRNA and protein were consistently detected in small intestines as well as livers. CYP3A62 was a major form in small intestines of both sexes but was a female-predominant form in livers of adult rats. CYP3A62 in both tissues of male and female rats were clearly enhanced by the treatment with dexamethasone. These expression profiles resembled those of CYP3A9. Despite clear detection of CYP3A62, no detectable levels of CYP3A1 and CYP3A2 proteins, as well as those of mRNAs, were found in the intestinal tract. Therefore, CYP3A62 may play major roles together with CYP3A9 and CYP3A18 in endogenous or exogenous detoxification at the absorption site.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Intestinos/enzimología , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Secuencia de Bases , Catálisis , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , Femenino , Hígado/enzimología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Oxidorreductasas N-Desmetilantes/genética , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
As many prokaryotic molybdoenzymes, the trimethylamine oxide reductase (TorA) of Escherichia coli requires the insertion of a bis(molybdopterin guanine dinucleotide)molybdenum cofactor in its catalytic site to be active and translocated to the periplasm. We show in vitro that the purified apo form of TorA was activated weakly when an appropriate bis(molybdopterin guanine dinucleotide)molybdenum source was provided, whereas addition of the TorD chaperone increased apoTorA activation up to 4-fold, allowing maturation of most of the apoprotein. We demonstrate that TorD alone is sufficient for the efficient activation of apoTorA by performing a minimal in vitro assay containing only the components for the cofactor synthesis, apoTorA and TorD. Interestingly, incubation of apoTorA with TorD before cofactor addition led to a significant increase of apoTorA activation, suggesting that TorD acts on apoTorA before cofactor insertion. This result is consistent with the fact that TorD binds to apoTorA and probably modifies its conformation in the absence of cofactor. Therefore, we propose that TorD is involved in the first step of TorA maturation to make it competent to receive the cofactor.
Asunto(s)
Proteínas de Escherichia coli/farmacología , Escherichia coli/enzimología , Chaperonas Moleculares/farmacología , Oxidorreductasas N-Desmetilantes/metabolismo , Apoproteínas/química , Apoproteínas/aislamiento & purificación , Apoproteínas/metabolismo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Proteínas de Escherichia coli/aislamiento & purificación , Nucleótidos de Guanina/farmacología , Cinética , Chaperonas Moleculares/aislamiento & purificación , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Conformación Proteica/efectos de los fármacos , Pterinas/farmacologíaRESUMEN
Nikkomycin antibiotics are potent inhibitors of chitin synthase, effective as therapeutic antifungal agents in humans and easily degradable insecticides in agriculture. NikD is a novel flavoprotein that catalyzes the oxidation of Delta(1)- or Delta(2)-piperideine-2-carboxylate, a key step in the biosynthesis of nikkomycin antibiotics. The resulting dihydropicolinate product may be further oxidized by nikD or converted to picolinate in a nonenzymic reaction. Saturated nitrogen heterocycles (L-pipecolate, L-proline) and 3,4-dehydro-L-proline act as alternate substrates. The ability of nikD to oxidize 3,4-dehydro-L-proline, but not 1-cyclohexenoate, suggests that the enzyme is specific for the oxidation of a carbon-nitrogen bond. An equivalent reaction is possible with the enamine (Delta(2)), but not the imine (Delta(1)), form of the natural piperideine-2-carboxylate substrate. Apparent steady-state kinetic parameters for the reaction of nikD with Delta(1)- or Delta(2)-piperideine-2-carboxylate (k(cat) = 64 min(-1); K(m) = 5.2 microM) or 3,4-dehydro-L-proline (k(cat) = 18 min(-1); K(m) = 13 mM) were determined in air-saturated buffer by measuring hydrogen peroxide formation in a coupled assay. NikD appears to be a new member of the monomeric sarcosine oxidase (MSOX) family of amine oxidizing enzymes. The enzyme contains 1 mol of flavin adenine dinucleotide (FAD) covalently linked to Cys321. The covalent flavin attachment site and two residues that bind substrate carboxylate in MSOX are conserved in nikD. NikD, however, exhibits an unusual long-wavelength absorption band, attributed to charge-transfer interaction between FAD and an ionizable (pK(a) = 7.3) active-site residue. Similar long-wavelength absorption bands have been observed for flavoproteins containing an active site cysteine or cysteine sulfenic acid. Interestingly, Cys273 in nikD aligns with an active-site histidine in MSOX (His269) that is, otherwise, a highly conserved residue within the MSOX family.
Asunto(s)
Aminoglicósidos , Antibacterianos/biosíntesis , Flavoproteínas/química , Oxidorreductasas N-Desmetilantes/química , Prolina/análogos & derivados , Riboflavina/análogos & derivados , Secuencia de Aminoácidos , Antifúngicos/biosíntesis , Ácido Edético/química , Flavoproteínas/antagonistas & inhibidores , Flavoproteínas/genética , Flavoproteínas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Luz , Datos de Secuencia Molecular , Oxidación-Reducción , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/aislamiento & purificación , Paraquat/química , Ácidos Pipecólicos/química , Prolina/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Riboflavina/química , Sarcosina-Oxidasa , Espectrofotometría , Streptomyces/enzimología , Streptomyces/genética , Especificidad por SustratoRESUMEN
Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme that was reported to contain 1 mol of covalently bound FAD and 1 mol of non-covalently-bound FAD. This work describes the result of reinvestigation of the cofactors in this enzyme. The enzyme was found to contain 1 mol of non-covalently-bound NAD+, 1 mol of non-covalently-bound FAD, and 1 mol of covalent FMN. The covalent FMN was identified by the mass and amino acid sequence analyses of the flavin peptide.