RESUMO
Non-CpG methylation is associated with several cellular processes, especially neuronal development and cancer, while its effect on DNA structure remains unclear. We have determined the crystal structures of DNA duplexes containing -CGCCG- regions as CCG repeat motifs that comprise a non-CpG site with or without cytosine methylation. Crystal structure analyses have revealed that the mC:G base-pair can simultaneously form two alternative conformations arising from non-CpG methylation, including a unique water-mediated cis Watson-Crick/Hoogsteen, (w)cWH, and Watson-Crick (WC) geometries, with partial occupancies of 0.1 and 0.9, respectively. NMR studies showed that an alternative conformation of methylated mC:G base-pair at non-CpG step exhibits characteristics of cWH with a syn-guanosine conformation in solution. DNA duplexes complexed with the DNA binding drug echinomycin result in increased occupancy of the (w)cWH geometry in the methylated base-pair (from 0.1 to 0.3). Our structural results demonstrated that cytosine methylation at a non-CpG step leads to an antiâsyntransition of its complementary guanosine residue toward the (w)cWH geometry as a partial population of WC, in both drug-bound and naked mC:G base pairs. This particular geometry is specific to non-CpG methylated dinucleotide sites in B-form DNA. Overall, the current study provides new insights into DNA conformation during epigenetic regulation.
Assuntos
Pareamento de Bases , Citosina , Metilação de DNA , DNA , Conformação de Ácido Nucleico , Água , DNA/química , Citosina/química , Água/química , Cristalografia por Raios X , Modelos MolecularesRESUMO
Arsenite-induced stress granule (SG) formation can be cleared by the ubiquitin-proteasome system aided by the ATP-dependent unfoldase p97. ZFAND1 participates in this pathway by recruiting p97 to trigger SG clearance. ZFAND1 contains two An1-type zinc finger domains (ZF1 and ZF2), followed by a ubiquitin-like domain (UBL); but their structures are not experimentally determined. To shed light on the structural basis of the ZFAND1-p97 interaction, we determined the atomic structures of the individual domains of ZFAND1 by solution-state NMR spectroscopy and X-ray crystallography. We further characterized the interaction between ZFAND1 and p97 by methyl NMR spectroscopy and cryo-EM. 15N spin relaxation dynamics analysis indicated independent domain motions for ZF1, ZF2, and UBL. The crystal structure and NMR structure of UBL showed a conserved ß-grasp fold homologous to ubiquitin and other UBLs. Nevertheless, the UBL of ZFAND1 contains an additional N-terminal helix that adopts different conformations in the crystalline and solution states. ZFAND1 uses the C-terminal UBL to bind to p97, evidenced by the pronounced line-broadening of the UBL domain during the p97 titration monitored by methyl NMR spectroscopy. ZFAND1 binding induces pronounced conformational heterogeneity in the N-terminal domain of p97, leading to a partial loss of the cryo-EM density of the N-terminal domain of p97. In conclusion, this work paved the way for a better understanding of the interplay between p97 and ZFAND1 in the context of SG clearance.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Grânulos de Estresse , Proteína com Valosina , Humanos , Arsenitos/metabolismo , Arsenitos/química , Cristalografia por Raios X , Ligação Proteica , Domínios Proteicos , Grânulos de Estresse/metabolismo , Ubiquitina/metabolismo , Proteína com Valosina/metabolismo , Proteína com Valosina/química , Proteína com Valosina/genética , Dedos de Zinco , Dobramento de Proteína , Imageamento por Ressonância Magnética , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Some mutations which occur in the α/ß-discordant region (resides 15 to 23) of ß-amyloid peptide (Aß) lead to familial Alzheimer's disease (FAD). In vitro studies have shown that these genetic mutations could accelerate Aß aggregation. We recently showed that mutations in this region could alter the structural propensity, resulting in a different aggregative propensity of Aß. Whether these genetic mutations display similar effects remains largely unknown. Here, we characterized the structural propensity and aggregation kinetics of Dutch-type Aß40 (Aß40(E22Q)) and its L17A/F19A-substituted mutant (Aß40(L17A/F19A/E22Q)) using circular dichroism spectroscopy, nuclear magnetic spectroscopy, and thioflavin T fluorescence assay. In comparison with wild-type Aß40, we found that Dutch-type mutation, unlike Artic-type mutation (E22G), does not reduce the α-helical propensity of the α/ß-discordant region in sodium dodecyl sulfate micellar solution. Moreover, we found that Aß40(L17A/F19A/E22Q) displays a higher α-helical propensity of the α/ß-discordant region and a slower aggregation rate than Aß40(E22Q), suggesting that the inhibition of aggregation might be via increasing the α-helical propensity of the α/ß-discordant region, similar to that observed in wild-type and Artic-type Aß40. Taken together, Dutch-type and Artic-type mutations adopt different mechanisms to promote Aß aggregation, however, the L17A/F19A mutation could increase the α-helical propensities of both Dutch-type and Artic-type Aß40 and inhibit their aggregation.
Assuntos
Substituição de Aminoácidos/genética , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Fragmentos de Peptídeos/genética , Doença de Alzheimer/genética , Humanos , Mutação/genética , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína/genética , Dodecilsulfato de Sódio/químicaRESUMO
Transketolase (TK) catalyzes a reversible transfer of a two-carbon (C2 ) unit between phosphoketose donors and phosphoaldose acceptors, for which the group-transfer reaction that follows a one- or two-electron mechanism and the force that breaks the C2"-C3" bond of the ketose donors remain unresolved. Herein, we report ultrahigh-resolution crystal structures of a TK (TKps) from Pichia stipitis in previously undiscovered intermediate states and support a diradical mechanism for a reversible group-transfer reaction. In conjunction with MS, NMR spectroscopy, EPR and computational analyses, it is concluded that the enzyme-catalyzed non-Kekulé diradical cofactor brings about the C2"-C3" bond cleavage/formation for the C2 -unit transfer reaction, for which suppression of activation energy and activation and destabilization of enzymatic intermediates are facilitated.
Assuntos
Pichia/enzimologia , Transcetolase/química , Biocatálise , Cristalografia por Raios X , Escherichia coli/genética , Cinética , Modelos Moleculares , OxirreduçãoRESUMO
It is theoretically plausible that thiazolium mesomerizes to congeners other than carbene in a low effective dielectric binding site; especially given the energetics and uneven electronegativity of carbene groups. However, such a phenomenon has never been reported. Nine crystal structures of transketolase obtained from Pichia stipitis (TKps) are reported with subatomic resolution, where thiazolium displays an extraordinary ring-bending effect. The bent thiazolium congeners correlate with non-Kekulé diradicals because there is no gain or loss of electrons. In conjunction with biophysical and biochemical analyses, it is concluded that ring bending is a result of tautomerization of thiazolium with its non- Kekulé diradicals, exclusively in the binding site of TKps. The chemophysical properties of these thiazolium mesomers may account for the great variety of reactivities carried out by thiamine-diphosphate-containing (ThDP) enzymes. The stability of ThDP in living systems can be regulated by the levels of substrates, and hydration and dehydration, as well as diradical-mediated oxidative degradation.
Assuntos
Proteínas Fúngicas/metabolismo , Pichia/enzimologia , Tiazóis/metabolismo , Transcetolase/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Isomerismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Tiamina Pirofosfato/metabolismo , Tiazóis/química , Transcetolase/química , Transcetolase/genéticaRESUMO
Field-dependent NMR studies of bio-molecular systems using a sample shuttling hardware operating on a high-field NMR apparatus have provided valuable structural and dynamic information. We have recently published a design of a compact sample transportation device, called "field-cycler", which was installed in a commercial spectrometer and which provided highly precise positioning and stability during high speed shuttling. In this communication, we demonstrate the first use of a sample shuttling device on a commercial high field standard bore NMR spectrometer, equipped with a commercial triple resonance cryogenically cooled NMR probe. The performance and robustness of the hardware operating in 1D and 2D field cycling experiments, as well as the impact of the sample shuttling time on the signal intensity are discussed.
Assuntos
Espectroscopia de Ressonância Magnética/instrumentação , Espectroscopia de Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/normas , Ressonância Magnética Nuclear Biomolecular/instrumentação , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Reprodutibilidade dos TestesRESUMO
The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane sensor, RstB and its cognate response regulator (RR) RstA. The RstA of Klebsiella pneumoniae (kpRstA) consists of an N-terminal receiver domain (RD, residues 1-119) and a C-terminal DNA-binding domain (DBD, residues 130-236). Phosphorylation of kpRstA induces dimerization, which allows two kpRstA DBDs to bind to a tandem repeat, called the RstA box, and regulate the expression of downstream genes. Here we report the solution and crystal structures of the free kpRstA RD, DBD and DBD/RstA box DNA complex. The structure of the kpRstA DBD/RstA box complex suggests that the two protomers interact with the RstA box in an asymmetric fashion. Equilibrium binding studies further reveal that the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner. Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the first kpRstA DBD protomer to anchor protein-DNA interaction, whereas the second protomer plays a key role in ensuring correct recognition of the RstA box.
Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Klebsiella pneumoniae/genética , Regiões Promotoras Genéticas , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Cytotoxic ribonucleases found in the oocytes and early embryos of frogs with antitumor activity are well-documented. RC-RNase 2, a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana, consists of 105 residues linked with 4 disulfide bridges and belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Among the RC-RNases, the base preference for RNase 2 is UpG but CpG for RC-RNase 4; while RC-RNase possesses the base specificity of both UpG and CpG. Interestingly, RC-RNase 2 or 4 has much lower catalytic activity but only three-fold less cytotoxicity than RC-RNase. Here, we report the NMR solution structure of rRC-RNase 2, comprising three alpha-helices and two sets of antiparallel beta-sheets. The differences of side-chain conformations of subsite residues among RNase A, RC-RNase, RC-RNase 4 and rRNase 2 are related to their distinct catalytic activities and base preferences. Furthermore, the substrate-related residues in the base specificity among native RC-RNases are derived using the chemical shift perturbation on ligand binding.
Assuntos
Proteínas de Anfíbios/química , Endorribonucleases/química , Animais , Bovinos , Ressonância Magnética Nuclear Biomolecular , Oócitos/enzimologia , Estrutura Secundária de Proteína , Rana catesbeianaRESUMO
The E3 ubiquitin ligase RNF4 (RING finger protein 4) contains four tandem SIM [SUMO (small ubiquitin-like modifier)-interaction motif] repeats for selective interaction with poly-SUMO-modified proteins, which it targets for degradation. We employed a multi-faceted approach to characterize the structure of the RNF4-SIMs domain and the tetra-SUMO2 chain to elucidate the interaction between them. In solution, the SIM domain was intrinsically disordered and the linkers of the tetra-SUMO2 were highly flexible. Individual SIMs of the RNF4-SIMs domains bind to SUMO2 in the groove between the ß2-strand and the α1-helix parallel to the ß2-strand. SIM2 and SIM3 bound to SUMO with a high affinity and together constituted the recognition module necessary for SUMO binding. SIM4 alone bound to SUMO with low affinity; however, its contribution to tetra-SUMO2 binding avidity is comparable with that of SIM3 when in the RNF4-SIMs domain. The SAXS data of the tetra-SUMO2-RNF4-SIMs domain complex indicate that it exists as an ordered structure. The HADDOCK model showed that the tandem RNF4-SIMs domain bound antiparallel to the tetra-SUMO2 chain orientation and wrapped around the SUMO protamers in a superhelical turn without imposing steric hindrance on either molecule.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos/fisiologia , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Ubiquitina-Proteína Ligases/metabolismo , Difração de Raios XRESUMO
Lon belongs to a unique group of AAA+ proteases that bind DNA. However, the DNA-mediated regulation of Lon remains elusive. Here, the crystal structure of the α subdomain of the Lon protease from Brevibacillus thermoruber (Bt-Lon) is presented, together with biochemical data, and the DNA-binding mode is delineated, showing that Arg518, Arg557 and Arg566 play a crucial role in DNA binding. Electrostatic interactions contributed by arginine residues in the AAA+ module are suggested to be important to DNA binding and allosteric regulation of enzymatic activities. Intriguingly, Arg557, which directly binds DNA in the α subdomain, has a dual role in the negative regulation of ATPase stimulation by DNA and in the domain-domain communication in allosteric regulation of Bt-Lon by substrate. In conclusion, structural and biochemical evidence is provided to show that electrostatic interaction in the AAA+ module is important for DNA binding by Lon and allosteric regulation of its enzymatic activities by DNA and substrate.
Assuntos
Arginina/química , Proteínas de Bactérias/química , Brevibacillus/química , DNA Bacteriano/química , Protease La/química , Regulação Alostérica , Arginina/metabolismo , Proteínas de Bactérias/genética , Brevibacillus/enzimologia , Domínio Catalítico , Cristalografia por Raios X , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Mutagênese , Protease La/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Eletricidade Estática , TermodinâmicaRESUMO
Toxoplasmosis, caused by Toxoplasma gondii, poses risks to vulnerable populations. TgPDCD5, a secreted protein of T. gondii, induces apoptosis through heparan sulfate-mediated endocytosis. The entry mechanism of TgPDCD5 has remained elusive. Here, we present the solution structure of TgPDCD5 as a helical bundle with an extended N-terminal helix, exhibiting molten globule characteristics. NMR perturbation studies reveal heparin/heparan sulfate binding involving the heparan sulfate/heparin proteoglycans-binding motif and the core region, influenced by proline isomerization of P107 residue. The heterogeneous proline recruits a cyclophilin TgCyp18, accelerating interconversion between conformers and regulating heparan/heparin binding. These atomic-level insights elucidate the binary switch's functionality, expose novel heparan sulfate-binding surfaces, and illuminate the unconventional cellular entry of pathogenic TgPDCD5.
RESUMO
Alzheimer's disease (AD) is a devastating, progressive neurodegenerative disease affecting the elderly in the world. The pathological hallmark senile plaques are mainly composed of amyloid-ß (Aß), in which the main isoforms are Aß40 and Aß42. Aß is prone to aggregate and ultimately forms amyloid fibrils in the brains of AD patients. Factors that alter the Aß aggregation process have been considered to be potential targets for treatments of AD. Modifier of aggregation 4 (MOAG-4)/small EDRK-rich factor (SERF) was previously selected from a chemical mutagenesis screen and identified as an amyloid modifier that promotes amyloid aggregation for α-synuclein, huntingtin, and Aß40. The interaction and effect of yeast ScSERF on Aß40 were previously described. Here, we examined the human SERF1a effect on Aß40 and Aß42 fibrillization by the Thioflavin T assay and found that SERF1a accelerated Aß fibrillization in a dose-dependent manner without changing the fibril amount and without incorporation. By Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM), we found that SERF1a altered the secondary structures and the morphology of Aß fibrils. The electrospray ionization mass spectrometry (ESI-MS) and analytical ultracentrifugation (AUC) results showed that SERF1a binds to Aß in a 1:1 stoichiometry. Moreover, the NMR study showed that SERF1a interacts with Aß via its N-terminal region. Cytotoxicity assay demonstrated that SERF1a enhanced toxicity of Aß intermediates, and the effect can be rescued by SERF1a antibody. Overall, our study provides the underlying molecular mechanism for the SERF1a effect on Aß fibrillization and facilitates the therapeutic development of AD.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Proteínas do Tecido Nervoso , Idoso , Humanos , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/química , Proteínas do Tecido Nervoso/metabolismoRESUMO
Antimicrobial peptides (AMPs) are natural molecules that function within the innate immune system to counteract pathogenic invasion and minimize the detrimental consequences of infection. However, utilizing these molecules for medical applications has been challenging. In this study, we selected a model AMP with poor stability, Tilapia Piscidin 4 (TP4), and modified its sequence and chirality (TP4-γ) to improve its potential for clinical application. The strategy of chirality inversion was inspired by the cereulide peptide, which has a DDLL enantiomer pattern and exhibits exceptional stability. Sequential substitution of key residues and selective chirality inversion yielded a less toxic peptide with enhanced stability and notable antimicrobial activity. In addition to its superior stability profile and antimicrobial activity, TP4-γ treatment reduced the level of LPS-induced nitric oxide (NO) release in a macrophage cell line. This reduction in NO release may reflect anti-inflammatory properties, as NO is widely known to promote inflammatory processes. Hence, our heterochiral peptide construct shows a more suitable pharmacokinetic profile than its parental compound, and further studies are warranted to develop the molecule for potential clinical application.
Assuntos
Anti-Infecciosos , Tilápia , Animais , Peptídeos Antimicrobianos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Linhagem Celular , Anti-Infecciosos/farmacologiaRESUMO
Because antimicrobial peptides (AMPs) often exhibit broad-spectrum bactericidal potency, we sought to develop peptide-based antimicrobials for potential clinical use against drug-resistant pathogens. To accomplish this goal, we first optimized the amino acid sequence of a broad-spectrum AMP known as Tilapia Piscidin 4 (TP4). Then, we used the optimized sequence to create a pair of heterochiral variants (TP4-α and TP4-ß) with different percentages of D-enantiomers, as poly-L peptides often exhibit poor pharmacokinetic profiles. The conformations of the peptide pair exhibited inverted chirality according to CD and NMR spectroscopic analyses. Both heterochiral peptides displayed enhanced stability and low hemolysis activities. Irrespective of their different d-enantiomer contents, both heterochiral peptides exhibited bactericidal activities in the presence of human serum or physiological enzymes. However, the peptide with higher d-amino acid content (TP4-ß) caused better bacterial clearance when tested in mice infected with NDM-1 K. pneumoniae. In addition, we observed a relatively higher hydrogen bonding affinity in a simulation of the interaction between TP4-ß and a model bacterial membrane. In sum, our results demonstrate that the current design strategy may be applicable for development of new molecules with enhanced stability and in vivo antimicrobial activity.
Assuntos
Anti-Infecciosos , Tilápia , Humanos , Animais , Camundongos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Sequência de Aminoácidos , Testes de Sensibilidade MicrobianaRESUMO
The aberrant fibrillization of huntingtin exon 1 (Httex1) characterized by an expanded polyglutamine (polyQ) tract is a defining feature of Huntington's disease, a neurodegenerative disorder. Recent investigations underscore the involvement of a small EDRK-rich factor 1a (SERF1a) in promoting Httex1 fibrillization through interactions with its N terminus. By establishing an integrated approach with size-exclusion-column-based small- and wide-angle X-ray scattering (SEC-SWAXS), NMR, and molecular simulations using Rosetta, the analysis here reveals a tight binding of two NT17 fragments of Httex1 (comprising the initial 17 amino acids at the N terminus) to the N-terminal region of SERF1a. In contrast, examination of the complex structure of SERF1a with a coiled NT17-polyQ peptide (33 amino acids in total) indicates sparse contacts of the NT17 and polyQ segments with the N-terminal side of SERF1a. Furthermore, the integrated SEC-SWAXS and molecular-simulation analysis suggests that the coiled NT17 segment can transform into a helical conformation when associated with a polyQ segment exhibiting high helical content. Intriguingly, NT17-polyQ peptides with enhanced secondary structures display diminished interactions with SERF1a. This insight into the conformation-dependent binding of NT17 provides clues to a catalytic association mechanism underlying SERF1a's facilitation of Httext1 fibrillization.
RESUMO
Iron-inducible transcription of the ap65-1 gene in Trichomonas vaginalis involves at least three Myb-like transcriptional factors (tvMyb1, tvMyb2 and tvMyb3) that differentially bind to two closely spaced promoter sites, MRE-1/MRE-2r and MRE-2f. Here, we defined a fragment of tvMyb2 comprising residues 40-156 (tvMyb240â156) as the minimum structural unit that retains near full binding affinity with the promoter DNAs. Like c-Myb in vertebrates, the DNA-free tvMyb240â156 has a flexible and open conformation. Upon binding to the promoter DNA elements, tvMyb240â156 undergoes significant conformational re-arrangement and structure stabilization. Crystal structures of tvMyb240â156 in complex with promoter element-containing DNA oligomers showed that 5'-a/gACGAT-3' is the specific base sequence recognized by tvMyb240â156, which does not fully conform to that of the Myb binding site sequence. Furthermore, Lys49, which is upstream of the R2 motif (amino acids 52-102) also participates in specific DNA sequence recognition. Intriguingly, tvMyb240â156 binds to the promoter elements in an orientation opposite to that proposed in the HADDOCK model of the tvMyb135â141/MRE-1-MRE-2r complex. These results shed new light on understanding the molecular mechanism of Myb-DNA recognition and provide a framework to study the molecular basis of transcriptional regulation of myriad Mybs in T. vaginalis.
Assuntos
Moléculas de Adesão Celular/genética , Regiões Promotoras Genéticas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Proteínas de Protozoários/metabolismo , Termodinâmica , Trichomonas vaginalis/genéticaRESUMO
Flavonoids are associated with health benefits, but most of them have poor oral bioavailability due to their extremely low aqueous solubility. Flavonoid O-phosphorylation suggests a potent modification to solve the problems. Here, we isolated, identified and characterized an unprecedented phosphotransferase, flavonoid phosphate synthetase (BsFPS), from B. subtilis. The enzyme catalyzes the ATP-dependent phosphorylation of flavonoid to generate flavonoid monophosphates, AMP and orthophosphate. BsFPS is a promiscuous phosphotransferase that efficiently catalyzes structurally-diverse flavonoids, including isoflavones, flavones, flavonols, flavanones and flavonolignans. Based on MS and NMR analysis, the phosphorylation mainly occurs on the hydroxyl group at C-7 of A-ring or C-4' of B-ring in flavonoid skeleton. Notably, BsFPS is regioselective for the ortho-3',4'-dihydroxy moiety of catechol-containing structures, such as luteolin and quercetin, to produce phosphate conjugates at C-4' or C-3' of B-ring. Our findings highlight the potential for developing biosynthetic platform to obtain new phosphorylated flavonoids for pharmaceutical and nutraceutical applications.
Assuntos
Flavanonas , Flavonas , Flavonolignanos , Isoflavonas , Monofosfato de Adenosina , Trifosfato de Adenosina , Bacillus subtilis , Catecóis , Flavonoides/química , Ligases , Luteolina , Fosfatos , Fosfotransferases , QuercetinaRESUMO
Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of which the allosteric redox switch formed by the lysine-cysteine NOS bridge is an example. Here, we report a noncanonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin to form the antibiotic BD-12. X-ray crystallography was used to investigate this complex enzymatic process, which showed Orf1 has two substrate-binding sites that sit 13.5 Å apart unlike canonical FAD-dependent oxidoreductases. One site could accommodate glycine and the other glycinothricin or glycylthricin. Moreover, an intermediate-enzyme adduct with a NOS-covalent linkage was observed in the later site, where it acts as a two-scissile-bond linkage facilitating nucleophilic addition and cofactor-free decarboxylation. The chain length of nucleophilic acceptors vies with bond cleavage sites at either N-O or O-S accounting for N-formimidoylation or N-iminoacetylation. The resultant product is no longer sensitive to aminoglycoside-modifying enzymes, a strategy that antibiotic-producing species employ to counter drug resistance in competing species.
Assuntos
Aminoglicosídeos , Cisteína , Cisteína/química , Ligantes , Sítios de Ligação , Antibacterianos , Cristalografia por Raios X , GlicinaRESUMO
Abnormal polyglutamine (polyQ) expansion and fibrillization occur in Huntington's disease (HD). Amyloid modifier SERF enhances amyloid formation, but the underlying mechanism is not revealed. Here, the fibrillization and toxicity effect of SERF1a on Htt-exon1 are examined. SERF1a enhances the fibrillization of and interacts with mutant thioredoxin (Trx)-fused Httex1. NMR studies with Htt peptides show that TrxHttex1-39Q interacts with the helical regions in SERF1a and SERF1a preferentially interacts with the N-terminal 17 residues of Htt. Time-course analysis shows that SERF1a induces mutant TrxHttex1 to a single conformation enriched of ß-sheet. Co-expression of SERF1a and Httex1-polyQ in neuroblastoma and lentiviral infection of SERF1a in HD-induced polypotent stem cell (iPSC)-derived neurons demonstrates the detrimental effect of SERF1a in HD. Higher level of SERF1a transcript or protein is detected in HD iPSC, transgenic mice, and HD plasma. Overall, this study provides molecular mechanism for SERF1a and mutant Httex1 to facilitate therapeutic development for HD.
Assuntos
Proteínas Amiloidogênicas , Doença de Huntington , Animais , Camundongos , Peptídeos/genética , Fatores de Transcrição , Éxons , Doença de Huntington/genética , Camundongos TransgênicosRESUMO
Luteolin (LUT), a plant-derived flavone, exhibits various bioactivities; however, the poor aqueous solubility hampers its applications. Here, we revealed bioconversion of LUT by Bacillus subtilis BCRC 80517, yielding three water-soluble phosphate conjugates. These derivatives were identified as luteolin 4'-O-phosphate (L4'P), luteolin 3'-O-phosphate (L3'P), and luteolin 7-O-phosphate (L7P) by LC-ESI-MS/MS and NMR. Besides, we found that Bacillus subtilis BCRC 80517 was able to convert different levels of LUT but showed a limited conversion rate. By observing bacterial morphology with transmission electron microscopy and confocal fluorescence microscopy, we found that LUT disrupted the bacterial membrane integrity, which explained the incomplete conversion. Additionally, we revealed a spontaneous intramolecular transesterification of L4'P to L3'P, the thermodynamically more stable form, under acidic conditions and proposed the possible mechanism involving a cyclic phosphate as the intermediate. This study provides insight into development of a potent structural modification strategy to enhance the solubility of LUT through biophosphorylation.