RESUMEN
Alzheimer's disease (AD) was first described by Alois Alzheimer over 100 years ago, but there is still no overarching theory that can explain its cause in detail. There are also no effective therapies to treat either the cause or the associated symptoms of this devastating disease. A potential approach to better understand the pathogenesis of AD could be the development of selective caspase-2 (Casp2) probes, as we have shown that a Casp2-mediated cleavage product of tau (Δtau314) reversibly impairs cognitive and synaptic function in animal models of tauopathies. In this article, we map out the Casp2 binding site through the preparation and assay of a series of 35 pentapeptide inhibitors with the goal of gaining selectivity against caspase-3 (Casp3). We also employed computational docking methods to understand the key interactions in the binding pocket of Casp2 and the differences predicted for binding at Casp3. Moreover, we crystallographically characterized the binding of selected pentapeptides with Casp3. Furthermore, we engineered and expressed a series of recombinant tau mutants and investigated them in an in vitro cleavage assay. These studies resulted in simple peptidic inhibitors with nanomolar affinity, for example, AcVDV(Dab)D-CHO (24) with up to 27.7-fold selectivity against Casp3. Our findings provide a good basis for the future development of selective Casp2 probes and inhibitors that can serve as pharmacological tools in planned in vivo studies and as lead compounds for the design of bioavailable and more drug-like small molecules.
RESUMEN
Hydroxynitrile lyases (HNL's) belonging to the α/ß-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/ß-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 °C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/ß-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol·M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.
Asunto(s)
Aldehído-Liasas/química , Aldehído-Liasas/genética , Dominio Catalítico , Cristalografía por Rayos X/métodos , Esterasas/química , Esterasas/genética , Hevea/genética , Hevea/metabolismo , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida/métodos , Proteínas de Plantas/genética , Pliegue de Proteína , Especificidad por Sustrato , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
RcnR is a transcription factor that regulates the homeostasis of cobalt and nickel in bacterial cells. Escherichia coli RcnR was crystallized with DNA that encompasses the DNA-binding site. X-ray diffraction data were collected to 2.9â Å resolution. The crystal belonged to space group P6122 or P6522, with unit-cell parameters a = b = 73.59, c = 157.66â Å, α = ß = 90, γ = 120°.
Asunto(s)
Cobalto/química , Proteínas de Escherichia coli/química , Níquel/química , Proteínas Represoras/química , Cobalto/metabolismo , Cristalografía por Rayos X , ADN , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Modelos Moleculares , Níquel/metabolismo , Proteínas Represoras/aislamiento & purificación , Difracción de Rayos XRESUMEN
Understanding the biosynthesis of cofactors is fundamental to the life sciences, yet to date a few important pathways remain unresolved. One example is the redox cofactor pyrroloquinoline quinone (PQQ), which is critical for C1 metabolism in many microorganisms, a disproportionate number of which are opportunistic human pathogens. While the initial and final steps of PQQ biosynthesis, involving PqqD/E and PqqC, have been elucidated, the precise nature and order of the remaining transformations in the pathway are unknown. Here we show evidence that the remaining essential biosynthetic enzyme PqqB is an iron-dependent hydroxylase catalyzing oxygen-insertion reactions that are proposed to produce the quinone moiety of the mature PQQ cofactor. The demonstrated reactions of PqqB are unprecedented within the metallo ß-lactamase protein family and expand the catalytic repertoire of nonheme iron hydroxylases. These new findings also generate a nearly complete description of the PQQ biosynthetic pathway.
Asunto(s)
Proteínas Bacterianas/química , Dihidroxifenilalanina/análogos & derivados , Oxigenasas de Función Mixta/química , Catálisis , Dihidroxifenilalanina/química , Hidroxilación , Hierro/química , Methylobacterium extorquens/enzimología , Modelos Químicos , Zinc/químicaRESUMEN
The Radical SAM (RS) enzyme PqqE catalyzes the first step in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, forming a new carbon-carbon bond between two side chains within the ribosomally synthesized peptide substrate PqqA. In addition to the active site RS 4Fe-4S cluster, PqqE is predicted to have two auxiliary Fe-S clusters, like the other members of the SPASM domain family. Here we identify these sites and examine their structure using a combination of X-ray crystallography and Mössbauer and electron paramagnetic resonance (EPR) spectroscopies. X-ray crystallography allows us to identify the ligands to each of the two auxiliary clusters at the C-terminal region of the protein. The auxiliary cluster nearest the RS site (AuxI) is in the form of a 2Fe-2S cluster ligated by four cysteines, an Fe-S center not seen previously in other SPASM domain proteins; this assignment is further supported by Mössbauer and EPR spectroscopies. The second, more remote cluster (AuxII) is a 4Fe-4S center that is ligated by three cysteine residues and one aspartate residue. In addition, we examined the roles these ligands play in catalysis by the RS and AuxII clusters using site-directed mutagenesis coupled with EPR spectroscopy. Lastly, we discuss the possible functional consequences that these unique AuxI and AuxII clusters may have in catalysis for PqqE and how these may extend to additional RS enzymes catalyzing the post-translational modification of ribosomally encoded peptides.
Asunto(s)
Proteínas Bacterianas/química , Endopeptidasas/química , Proteínas Hierro-Azufre/química , Methylobacterium extorquens/química , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Conformación Proteica , TemperaturaRESUMEN
Renewable production of hydrocarbons is being pursued as a petroleum-independent source of commodity chemicals and replacement for biofuels. The bacterial biosynthesis of long-chain olefins represents one such platform. The process is initiated by OleA catalyzing the condensation of two fatty acyl-coenzyme A substrates to form a ß-keto acid. Here, the mechanistic role of the conserved His285 is investigated through mutagenesis, activity assays, and X-ray crystallography. Our data demonstrate that His285 is required for product formation, influences the thiolase nucleophile Cys143 and the acyl-enzyme intermediate before and after transesterification, and orchestrates substrate coordination as a defining component of an oxyanion hole. As a consequence, His285 plays a key role in enabling a mechanistic strategy in OleA that is distinct from other thiolases.
Asunto(s)
Acilcoenzima A/química , Proteínas Bacterianas/química , Xanthomonas campestris/enzimología , Acilcoenzima A/genética , Acilcoenzima A/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Histidina/química , Histidina/genética , Histidina/metabolismo , Xanthomonas campestris/genéticaRESUMEN
In the interest of decreasing dependence on fossil fuels, microbial hydrocarbon biosynthesis pathways are being studied for renewable, tailored production of specialty chemicals and biofuels. One candidate is long-chain olefin biosynthesis, a widespread bacterial pathway that produces waxy hydrocarbons. Found in three- and four-gene clusters, oleABCD encodes the enzymes necessary to produce cis-olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching. The first enzyme in the pathway, OleA, catalyzes the Claisen condensation of two fatty acyl-coenzyme A (CoA) molecules to form a ß-keto acid. In this report, the mechanistic role of Xanthomonas campestris OleA Glu117 is investigated through mutant enzymes. Crystal structures were determined for each mutant as well as their complex with the inhibitor cerulenin. Complemented by substrate modeling, these structures suggest that Glu117 aids in substrate positioning for productive carbon-carbon bond formation. Analysis of acyl-CoA substrate hydrolysis shows diminished activity in all mutants. When the active site lacks an acidic residue in the 117 position, OleA cannot form condensed product, demonstrating that Glu117 has a critical role upstream of the essential condensation reaction. Profiling of pH dependence shows that the apparent pKa for Glu117 is affected by mutagenesis. Taken together, we propose that Glu117 is the general base needed to prime condensation via deprotonation of the second, non-covalently bound substrate during turnover. This is the first example of a member of the thiolase superfamily of condensing enzymes to contain an active site base originating from the second monomer of the dimer.
Asunto(s)
Proteínas Bacterianas/química , Ligasas/química , Xanthomonas campestris/enzimología , Acilcoenzima A/química , Acilcoenzima A/genética , Alquenos/química , Alquenos/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Ácido Glutámico/química , Ácido Glutámico/genética , Ligasas/genética , Mutación Missense , Xanthomonas campestris/genéticaRESUMEN
PqqB is an enzyme involved in the biosynthesis of pyrroloquinoline quinone and a distal member of the metallo-ß-lactamase (MBL) superfamily. PqqB lacks two residues in the conserved signature motif HxHxDH that makes up the key metal-chelating elements that can bind up to two metal ions at the active site of MBLs and other members of its superfamily. Here, we report crystal structures of PqqB bound to Mn2+, Mg2+, Cu2+, and Zn2+. These structures demonstrate that PqqB can still bind metal ions at the canonical MBL active site. The fact that PqqB can adapt its side chains to chelate a wide spectrum of metal ions with different coordination features on a uniform main chain scaffold demonstrates its metal-binding plasticity. This plasticity may provide insights into the structural basis of promiscuous activities found in ensembles of metal complexes within this superfamily. Furthermore, PqqB belongs to a small subclass of MBLs that contain an additional CxCxxC motif that binds a structural Zn2+. Our data support a key role for this motif in dimerization.
Asunto(s)
Proteínas Bacterianas/metabolismo , Metales/metabolismo , Pseudomonas putida/enzimología , beta-Lactamasas/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Metales/química , Modelos Moleculares , Cofactor PQQ/metabolismo , Unión Proteica , Conformación Proteica , Infecciones por Pseudomonas/microbiología , Pseudomonas putida/química , Pseudomonas putida/metabolismo , beta-Lactamasas/químicaRESUMEN
Biosynthesis of the ribosomally synthesized and post-translationally modified peptide (RiPP), pyrroloquinoline quinone (PQQ), is initiated when the precursor peptide, PqqA, is recognized and bound by the RiPP precursor peptide recognition element (RRE), PqqD, for presentation to the first enzyme in the pathway, PqqE. Unlike other RiPP-producing, postribosomal peptide synthesis (PRPS) pathways in which the RRE is a component domain of the first enzyme, PqqD is predominantly a separate scaffolding protein that forms a ternary complex with the precursor peptide and first tailoring enzyme. As PqqD is a stable, independent RRE, this makes the PQQ pathway an ideal PRPS model system for probing RRE interactions using nuclear magnetic resonance (NMR). Herein, we present both the solution NMR structure of Methylobacterium extorquens PqqD and results of 1H-15N HSQC binding experiments that identify the PqqD residues involved in binding the precursor peptide, PqqA, and the enzyme, PqqE. The reported structural model for an independent RRE, along with the mapped binding surfaces, will inform future efforts both to understand and to manipulate PRPS pathways.
Asunto(s)
Proteínas Bacterianas/metabolismo , Methylobacterium extorquens/enzimología , Chaperonas Moleculares/metabolismo , Resonancia Magnética Nuclear Biomolecular , Oxidorreductasas/metabolismo , Cofactor PQQ/biosíntesis , Proteínas Bacterianas/química , Sitios de Unión , Methylobacterium extorquens/metabolismo , Modelos Moleculares , Chaperonas Moleculares/química , Oxidorreductasas/química , Cofactor PQQ/química , Cofactor PQQ/metabolismo , Conformación ProteicaRESUMEN
Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins from Xanthomonas campestris in Escherichia coli and purification over His6 and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of copurified OleBCD assemblies could be reconstituted by adding separately purified OleA. Immunoblots of nondenaturing gels using anti-OleC reacted with X. campestris crude protein lysate indicated the presence of a large protein assembly containing OleC in the native host. Negative-stain electron microscopy of recombinant OleBCD revealed distinct large structures with diameters primarily between 24 and 40 nm. Assembling OleB, OleC, and OleD into a complex may be important to maintain stereochemical integrity of intermediates, facilitate the movement of hydrophobic metabolites between enzyme active sites, and protect the cell against the highly reactive ß-lactone intermediate produced by the OleC-catalyzed reaction.IMPORTANCE Bacteria biosynthesize hydrophobic molecules to maintain a membrane, store carbon, and for antibiotics that help them survive in their niche. The hydrophobic compounds are often synthesized by a multidomain protein or by large multienzyme assemblies. The present study reports on the discovery that long-chain olefinic hydrocarbons made by bacteria from different phyla are produced by multienzyme assemblies in X. campestris The OleBCD multienzyme assemblies are thought to compartmentalize and sequester olefin biosynthesis from the rest of the cell. This system provides additional insights into how bacteria control specific biosynthetic pathways.
Asunto(s)
Alquenos/metabolismo , Vías Biosintéticas , Hidrocarburos/metabolismo , Complejos Multienzimáticos/metabolismo , Xanthomonas campestris/metabolismo , Proteínas Bacterianas/genética , Dominio Catalítico , Escherichia coli/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/aislamiento & purificación , Especificidad por Sustrato , Xanthomonas campestris/químicaRESUMEN
The first ß-lactone synthetase enzyme is reported, creating an unexpected link between the biosynthesis of olefinic hydrocarbons and highly functionalized natural products. The enzyme OleC, involved in the microbial biosynthesis of long-chain olefinic hydrocarbons, reacts with syn- and anti-ß-hydroxy acid substrates to yield cis- and trans-ß-lactones, respectively. Protein sequence comparisons reveal that enzymes homologous to OleC are encoded in natural product gene clusters that generate ß-lactone rings, suggesting a common mechanism of biosynthesis.
Asunto(s)
Proteínas Bacterianas/genética , Coenzima A Ligasas/genética , Regulación Bacteriana de la Expresión Génica , Lactonas/metabolismo , Micrococcus luteus/genética , Stenotrophomonas maltophilia/genética , Streptomyces/genética , Alquenos/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Productos Biológicos/metabolismo , Coenzima A Ligasas/metabolismo , Hidroxiácidos , Micrococcus luteus/enzimología , Familia de Multigenes , Operón , Homología de Secuencia de Aminoácido , Stenotrophomonas maltophilia/enzimología , Streptomyces/enzimologíaRESUMEN
Phylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a ß-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue. Reaction with the first substrate produces a covalent cysteine-thioester tethered acyl group that is transferred to the second substrate through formation of a carbon-carbon bond. Although the basics of thiolase chemistry are precedented, the mechanism by which OleA accommodates two substrates with extended carbon chains and a coenzyme moiety-unusual for a thiolase-are unknown. Gaining insights into this process could enable manipulation of the system for large scale olefin production with hydrocarbon chains lengths equivalent to those of fossil fuels. In this study, mutagenesis of the active site cysteine in Xanthomonas campestris OleA (Cys143) enabled trapping of two catalytically relevant species in crystals. In the resulting structures, long chain alkyl groups (C12 and C14) and phosphopantetheinate define three substrate channels in a T-shaped configuration, explaining how OleA coordinates its two substrates and product. The C143A OleA co-crystal structure possesses a single bound acyl-CoA representing the Michaelis complex with the first substrate, whereas the C143S co-crystal structure contains both acyl-CoA and fatty acid, defining how a second substrate binds to the acyl-enzyme intermediate. An active site glutamate (Gluß117) is positioned to deprotonate bound acyl-CoA and initiate carbon-carbon bond formation.
Asunto(s)
Acetil-CoA C-Acetiltransferasa/metabolismo , Acilcoenzima A/metabolismo , Alquenos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Coenzima A/metabolismo , Xanthomonas campestris/enzimología , Acetil-CoA C-Acetiltransferasa/química , Acetil-CoA C-Acetiltransferasa/genética , Proteínas Bacterianas/genética , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Cisteína/química , Cisteína/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación/genética , Conformación Proteica , Especificidad por Sustrato , Xanthomonas campestris/genéticaRESUMEN
The ribosomally synthesized and post-translationally modified peptide (RiPP), pyrroloquinoline quinone (PQQ), is a dehydrogenase cofactor synthesized by, but not exclusively used by, certain prokaryotes. RiPPs represent a rapidly expanding and diverse class of natural products-many of which have therapeutic potential-and the biosynthetic pathways for these are gaining attention. Five gene products from the pqq operon (PqqA, PqqB, PqqC, PqqD, and PqqE) are essential for PQQ biosynthesis. The substrate is the peptide PqqA, which is presented to the radical SAM enzyme PqqE by the small protein PqqD. PqqA is unstructured in solution, and only binds to PqqE when in complex with PqqD. PqqD is a member of a growing family of RiPP chaperone proteins (or domains in most cases) that present their associated peptide substrates to the initial RiPP biosynthesis enzymes. An X-ray crystal structure exists for dimeric Xanthomonas campestris PqqD (PDB ID: 3G2B), but PqqD is now known to act as a monomer under physiological conditions. In this study, the PqqD truncation from naturally fused Methylobacterium extorquens (Mex) PqqCD was overexpressed in Escherichia coli and MexPqqA was chemically synthesized. Solution NMR (1)H-,(15)N-HSQC chemical shift studies have identified the PqqD residues involved in binding PqqA, and (1)H, (13)C, and (15)N peak assignments for PqqD alone and for PqqD bound to PqqA are reported herein.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Methylobacterium extorquens , Resonancia Magnética Nuclear Biomolecular , Cofactor PQQ/metabolismo , Secuencia de Aminoácidos , Estructura Secundaria de ProteínaRESUMEN
X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.
Asunto(s)
Cristalografía por Rayos X/instrumentación , Enzimas/química , Acústica , Cristalografía por Rayos X/métodos , Modelos Moleculares , Muramidasa/química , Conformación Proteica , Termolisina/químicaRESUMEN
The accumulation of amyloid-ß (Aß) as amyloid fibrils and toxic oligomers is an important step in the development of Alzheimer's disease (AD). However, there are numerous potentially toxic oligomers and little is known about their neurological effects when generated in the living brain. Here we show that Aß oligomers can be assigned to one of at least two classes (type 1 and type 2) based on their temporal, spatial, and structural relationships to amyloid fibrils. The type 2 oligomers are related to amyloid fibrils and represent the majority of oligomers generated in vivo, but they remain confined to the vicinity of amyloid plaques and do not impair cognition at levels relevant to AD. Type 1 oligomers are unrelated to amyloid fibrils and may have greater potential to cause global neural dysfunction in AD because they are dispersed. These results refine our understanding of the pathogenicity of Aß oligomers in vivo.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Placa Amiloide/metabolismo , Agregación Patológica de Proteínas/metabolismo , Estructura Cuaternaria de Proteína , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/clasificación , Animales , Humanos , Ratones , Placa Amiloide/químicaRESUMEN
The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. One heme is low-spin with ligands provided by His205 and Tyr294, and the other is high-spin with a ligand provided by His35. The side chain methyl groups of Thr67 and Leu70 are positioned at a distance of 3.4Å on either side of His35, maintaining a hydrophobic environment in the proximal pocket of the high-spin heme and restricting the movement of this ligand. Mutation of Thr67 to Ala in the proximal pocket of the high-spin heme prevented reduction of the low-spin heme by dithionite, yielding a mixed-valent state. The mutation also enhanced the stabilization of the charge-resonance-transition of the high-valent bis-FeIV state that is generated by addition of H2O2. The rates of electron transfer from TTQ biosynthetic intermediates to the high-valent form of T67A MauG were similar to that of wild-type MauG. These results are compared to those previously reported for mutation of residues in the distal pocket of the high-spin heme that also affected the redox properties and charge resonance transition stabilization of the high-valent state of the hemes. However, given the position of residue 67, the structure of the variant protein and the physical nature of the T67A mutation, the basis for the effects of the T67A mutation must be different from those of the mutations of the residues in the distal heme pocket.
Asunto(s)
Proteínas Bacterianas/química , Compuestos Férricos/química , Compuestos Ferrosos/química , Hemo/química , Hemoproteínas/química , Mutación/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Transporte de Electrón , Compuestos Férricos/metabolismo , Compuestos Ferrosos/metabolismo , Hemo/genética , Hemo/metabolismo , Hemoproteínas/genética , Hemoproteínas/metabolismo , Indolquinonas/metabolismo , Modelos Moleculares , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Paracoccus denitrificans/genética , Paracoccus denitrificans/crecimiento & desarrollo , Paracoccus denitrificans/metabolismo , Procesamiento Proteico-Postraduccional , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/crecimiento & desarrollo , Rhodobacter sphaeroides/metabolismo , Espectrometría Raman , Triptófano/análogos & derivados , Triptófano/metabolismoRESUMEN
The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin biology. Key components are demethylase enzymes, which remove methyl moieties from lysine residues. KDM2A, a member of the Jumonji C domain-containing histone lysine demethylase family, specifically targets lower methylation states of H3K36. Here, structural studies reveal that H3K36 specificity for KDM2A is mediated by the U-shaped threading of the H3K36 peptide through a catalytic groove within KDM2A. The side chain of methylated K36 inserts into the catalytic pocket occupied by Ni(2+) and cofactor, where it is positioned and oriented for demethylation. Key residues contributing to K36me specificity on histone H3 are G33 and G34 (positioned within a narrow channel), P38 (a turn residue), and Y41 (inserts into its own pocket). Given that KDM2A was found to also bind the H3K36me3 peptide, we postulate that steric constraints could prevent α-ketoglutarate from undergoing an "off-line"-to-"in-line" transition necessary for the demethylation reaction. Furthermore, structure-guided substitutions of residues in the KDM2A catalytic pocket abrogate KDM2A-mediated functions important for suppression of cancer cell phenotypes. Together, our results deduce insights into the molecular basis underlying KDM2A regulation of the biologically important methylated H3K36 mark.
Asunto(s)
Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/metabolismo , Modelos Moleculares , Animales , Sitios de Unión , Línea Celular , Inestabilidad Genómica , Histona Demetilasas con Dominio de Jumonji/genética , Metilación , Ratones , Mutación , Unión Proteica , Estructura Cuaternaria de ProteínaRESUMEN
The diheme enzyme MauG catalyzes a six-electron oxidation that is required for the posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived cofactor, tryptophan tryptophylquinone (TTQ). Crystallographic and computational studies have implicated Gln103 in stabilizing the Fe(IV)âO moiety of the bis-Fe(IV) state by hydrogen bonding. The role of Gln103 was probed by site-directed mutagenesis. Q103L and Q103E mutations resulted in no expression and very little expression of the protein, respectively. Q103A MauG exhibited oxidative damage when isolated. Q103N MauG was isolated at levels comparable to that of wild-type MauG and exhibited normal activity in catalyzing the biosynthesis of TTQ from preMADH. The crystal structure of the Q103N MauG-preMADH complex suggests that a water may mediate hydrogen bonding between the shorter Asn103 side chain and the Fe(IV)âO moiety. The Q103N mutation caused the two redox potentials associated with the diferric/diferrous redox couple to become less negative, although the redox cooperativity of the hemes of MauG was retained. Upon addition of H2O2, Q103N MauG exhibits changes in the absorbance spectrum in the Soret and near-IR regions consistent with formation of the bis-Fe(IV) redox state. However, the rate of spontaneous return of the spectrum in the Soret region was 4.5-fold greater for Q103N MauG than for wild-type MauG. In contrast, the rate of spontaneous decay of the absorbance at 950 nm, which is associated with charge-resonance stabilization of the high-valence state, was similar for wild-type MauG and Q103N MauG. This suggests that as a consequence of the mutation a different distribution of resonance structures stabilizes the bis-Fe(IV) state. These results demonstrate that subtle changes in the structure of the side chain of residue 103 can significantly affect the overall protein stability of MauG and alter the redox properties of the hemes.
Asunto(s)
Glutamina , Hemoproteínas/química , Hemoproteínas/metabolismo , Mutagénesis Sitio-Dirigida , Paracoccus denitrificans/enzimología , Cristalografía por Rayos X , Estabilidad de Enzimas , Hemoproteínas/genética , Hierro/metabolismo , Modelos Moleculares , Mutación , Oxidación-Reducción , Conformación ProteicaRESUMEN
The lack of methodological uniformity in enzyme assays has been a long-standing difficulty, a problem for bench researchers, for the interpretation of clinical diagnostic tests, and an issue for investigational drug review. Illustrative of the problem, α-L-iduronidase enzyme catalytic activity is frequently measured with the substrate 4-methylumbelliferyl-α-L-iduronide (4MU-iduronide); however, final substrate concentrations used in different assays vary greatly, ranging from 25 µM to 1425 µM (Km ≈ 180 µM) making it difficult to compare results between laboratories. In this study, α-L-iduronidase was assayed with 15 different substrate concentrations. The resulting activity levels from the same specimens varied greatly with different substrate concentrations but, as a group, obeyed the expectations of Michaelis-Menten kinetics. Therefore, for the sake of improved comparability, it is proposed that α-L-iduronidase enzyme assays should be conducted either (1) under substrate saturating conditions; or (2) when concentrations are significantly below substrate saturation, with results standardized by arithmetic adjustment that considers Michaelis-Menten kinetics. The approach can be generalized to many other enzyme assays.
Asunto(s)
Pruebas de Enzimas/normas , Himecromona/análogos & derivados , Iduronidasa/análisis , Mucopolisacaridosis I/enzimología , Calibración , Humanos , Himecromona/química , Himecromona/normas , Iduronidasa/metabolismo , Cinética , Mucopolisacaridosis I/diagnóstico , Mucopolisacaridosis I/patología , Control de CalidadRESUMEN
The di-heme enzyme MauG catalyzes the oxidative biosynthesis of a tryptophan tryptophylquinone cofactor on a precursor of the enzyme methylamine dehydrogenase (preMADH). Reaction of H2O2 with the diferric form of MauG, or reaction of O2 with diferrous MauG, forms the catalytic intermediate known as bis-Fe(IV), which acts as the key oxidant during turnover. The site of substrate oxidation is more than 40 Å from the high-spin heme iron where H2O2 initially reacts, and catalysis relies on radical hopping through an interfacial residue, Trp199 of MauG. In the absence of preMADH, the bis-Fe(IV) intermediate is remarkably stable, but repeated exposure to H2O2 results in suicide inactivation. Using mass spectrometry, we show that this process involves the oxidation of three Met residues (108, 114, and 116) near the high-spin heme through ancillary electron transfer pathways engaged in the absence of substrate. The mutation of a conserved Pro107 in the distal pocket of the high-spin heme results in a dramatic increase in the level of oxidation of these Met residues. These results illustrate structural mechanisms by which MauG controls reaction with its high-valent heme cofactor and limits uncontrolled oxidation of protein residues and loss of catalytic activity. The conservation of Met residues near the high-spin heme among MauG homologues from different organisms suggests that eventual deactivation of MauG may function in a biological context. That is, methionine oxidation may represent a protective mechanism that prevents the generation of reactive oxygen species by MauG in the absence of preMADH.