RESUMEN
Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN(-) ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO)x(CN)y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (ßα)8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a µ2-sulfide ion to a mononuclear Fe(2+) center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe(CO)x(CN)y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.
Asunto(s)
Proteínas Bacterianas/química , Cristalografía por Rayos X/métodos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Hidrógeno/química , Hidrogenasas/química , Proteínas Hierro-Azufre/química , Dominio Catalítico , Modelos Moleculares , Conformación Proteica , Tirosina/químicaRESUMEN
Lipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) enzyme superfamily. In the present study, we solved crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX4CX5C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site-directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized lipoyl synthase (LipA) complex contains 5'-methylthioadenosine (MTA), a breakdown product of SAM, bound in the likely SAM-binding site. Modelling has identified an 18 Å (1 Å=0.1 nm) deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest that the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enable.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Azufre/metabolismo , Sulfurtransferasas/química , Sulfurtransferasas/metabolismo , Sitios de Unión/fisiología , Cristalización , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
A series of bisubstrate inhibitors for DNA N6 adenine methyltransferase (Dam) have been synthesized by linking an amine analogue of S-adenosylmethionine to an aryl moiety designed to probe the binding pocket of the DNA adenine base. An initial structure-activity relationship study has identified substituents that increase inhibitor potency to the â¼10 µM range and improve selectivity against the human cytosine methyltransferase Dnmt1.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/antagonistas & inhibidores , Sitios de Unión , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Humanos , S-Adenosilmetionina , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
N-6 methylation of adenine destabilises duplex DNA and this can increase the proportion of DNA that dissociates into single strands. We have investigated utilising this property to measure the DNA adenine methyltransferase-catalyzed conversion of hemimethylated to fully methylated DNA through a simple, direct, fluorescence-based assay. The effects of methylation on the kinetics and thermodynamics of hybridisation were measured by comparing a fully methylated oligonucleotide product and a hemimethylated oligonucleotide substrate using a 13-bp duplex labeled on adjacent strands with a fluorophore (fluorescein) and quencher (dabcyl). Enzymatic methylation of the hemimethylated GATC site resulted in destabilisation of the duplex, increasing the proportion of dissociated DNA, and producing an observable increase in fluorescence. The assay provides a direct measurement of methylation rate in real time and is highly reproducible, with a coefficient of variance over 48 independent measurements of 3.6%. DNA methylation rates can be measured as low as 3.55 ± 1.84 fmols(-1) in a 96-well plate format, and the assay has been used to kinetically characterise the Pyrococcus horikoshii DNA adenine methyltransferase.
Asunto(s)
ADN/metabolismo , Fluoresceína/análisis , Pyrococcus horikoshii/metabolismo , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/análisis , Secuencia de Bases , ADN/química , Metilación de ADN , Fluoresceína/química , Cinética , Pyrococcus horikoshii/enzimología , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/química , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Especificidad por Sustrato , Temperatura , Termodinámica , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/análisis , p-Dimetilaminoazobenceno/químicaRESUMEN
The members of the NSD subfamily of lysine methyl transferases are compelling oncology targets due to the recent characterization of gain-of-function mutations and translocations in several hematological cancers. To date, these proteins have proven intractable to small molecule inhibition. Here, we present initial efforts to identify inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal structural methods. On the basis of 2D NMR experiments comparing NSD1 and MMSET structural mobility, we designed an MMSET construct with five point mutations in the N-terminal helix of its SET domain for crystallization experiments and elucidated the structure of the mutant MMSET SET domain at 2.1 Å resolution. Both NSD1 and MMSET crystal systems proved resistant to soaking or cocrystallography with inhibitors. However, use of the close homologue SETD2 as a structural surrogate supported the design and characterization of N-alkyl sinefungin derivatives, which showed low micromolar inhibition against both SETD2 and MMSET.
Asunto(s)
Adenosina/análogos & derivados , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Oncogenes , Proteínas Represoras/antagonistas & inhibidores , Adenosina/química , Adenosina/farmacología , Sitios de Unión , Calorimetría , Cromatografía Liquida , Cristalografía por Rayos X , Diseño de Fármacos , N-Metiltransferasa de Histona-Lisina/genética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Conformación Proteica , Proteínas Represoras/genéticaRESUMEN
Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.