RESUMO
In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called "bifid shunt." The three-dimensional structure of PKT from Bifidobacterium longum with co-enzyme thiamine diphosphate (ThDpp) was determined at 2.1 Å resolution by cryo-EM single-particle analysis using 196,147 particles to build up the structural model of a PKT octamer related by D4 symmetry. Although the cryo-EM structure of PKT was almost identical to the X-ray crystal structure previously determined at 2.2 Å resolution, several interesting structural features were observed in the cryo-EM structure. Because this structure was solved at relatively high resolution, it was observed that several amino acid residues adopt multiple conformations. Among them, Q546-D547-H548-N549 (the QN-loop) demonstrate the largest structural change, which seems to be related to the enzymatic function of PKT. The QN-loop is at the entrance to the substrate binding pocket. The minor conformer of the QN-loop is similar to the conformation of the QN-loop in the crystal structure. The major conformer is located further from ThDpp than the minor conformer. Interestingly, the major conformer in the cryo-EM structure of PKT resembles the corresponding loop structure of substrate-bound Escherichia coli transketolase. That is, the minor and major conformers may correspond to "closed" and "open" states for substrate access, respectively. Moreover, because of the high-resolution analysis, many water molecules were observed in the cryo-EM structure of PKT. Structural features of the water molecules in the cryo-EM structure are discussed and compared with water molecules observed in the crystal structure.
Assuntos
Aldeído Liases/química , Bifidobacterium longum/enzimologia , Microscopia Crioeletrônica/métodos , Escherichia coli , Modelos Moleculares , Tiamina Pirofosfato , ÁguaRESUMO
Thermostability is a key factor in the industrial and clinical application of enzymes, and understanding mechanisms of thermostability is valuable for molecular biology and enzyme engineering. In this chapter, we focus on the thermostability of leucine dehydrogenase (LDH, EC 1.4.1.9), an amino acid-metabolizing enzyme that is an NAD+-dependent oxidoreductase which catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH from Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has already been applied to quantify the concentration of BCAAs in biological specimens. However, the molecular mechanism of its thermostability had been unknown because no high-resolution structure was available. Here, we discuss the thermostability of GstLDH on the basis of its structure determined by cryo-electron microscopy. Sequence comparison with other structurally characterized LDHs (from Lysinibacillus sphaericus and Sporosarcina psychrophila) indicated that non-conserved residues in GstLDH, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric stability through intermolecular interactions between protomers. Furthermore, NAD+ binding to GstLDH increased the thermostability of the enzyme as additional intermolecular interactions formed on cofactor binding. This knowledge is important for further applications and development of amino acid metabolizing enzymes in industrial and clinical fields.
Assuntos
Leucina Desidrogenase/química , Leucina Desidrogenase/metabolismo , Bacillaceae/enzimologia , Microscopia Crioeletrônica , Estabilidade Enzimática , Geobacillus stearothermophilus/enzimologia , Leucina Desidrogenase/ultraestrutura , Sporosarcina/enzimologiaRESUMO
Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD+-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has been applied for the quantification or production of BCAAs. Here the cryo-electron microscopy (cryo-EM) structures of apo and NAD+-bound LDH are reported at 3.0 and 3.2â¯Å resolution, respectively. On comparing the structures, the two overall structures are almost identical, but it was observed that the partial conformational change was triggered by the interaction between Ser147 and the nicotinamide moiety of NAD+. NAD+ binding also enhanced the strength of oligomerization interfaces formed by the core domains. Such additional interdomain interaction is in good agreement with our experimental results showing that the residual activity of NAD+-bound form was approximately three times higher than that of the apo form after incubation at 80⯰C. In addition, sequence comparison of three structurally known LDHs indicated a set of candidates for site-directed mutagenesis to improve thermostability. Subsequent mutation analysis actually revealed that non-conserved residues, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric thermostability.
Assuntos
Microscopia Crioeletrônica/métodos , Geobacillus stearothermophilus/enzimologia , Leucina Desidrogenase/química , NAD/química , Sequência de Aminoácidos , Sítios de Ligação , Estrutura Molecular , Mutagênese Sítio-Dirigida , Estabilidade ProteicaRESUMO
The formation of Fc-fusions, in which biologically active molecules and the Fc fragment of antibodies are linked to each other, is one of the most efficient and successful half-life extension technologies to be developed and applied to peptide and protein pharmaceuticals thus far. Fc-fusion compounds are generally produced by recombinant methods. However, these cannot be applied to artificial middle molecules, such as peptides with non-natural amino acids, unnatural cyclic peptides, or pharmaceutical oligonucleotides. Here, we developed a simple, efficient, semisynthetic method for Fc-fusion production involving our previously developed enzymatic N-terminal extension reaction (i.e., NEXT-A reaction) and strain-promoted azide-alkyne cycloaddition, achieving quantitative conversion and high selectivity for the N-terminus of the Fc protein. An Fc-fusion compound prepared by this method showed comparable biological activity to that of the original peptide and a long-circulating plasma half-life. Thus, the proposed method is potentially applicable for the conjugation of a wide range of pharmaceutical components.
Assuntos
Fragmentos Fc das Imunoglobulinas/química , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/farmacocinética , Alcinos/química , Sequência de Aminoácidos , Animais , Azidas/química , Reação de Cicloadição , Meia-Vida , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/químicaRESUMO
l-Histidine analysis is essential in physiological research and clinical applications because l-histidine concentrations in biofluids are associated with various diseases. However, an enzymatic method for l-histidine quantitation has not yet been established. Here, we describe a novel l-histidine quantitation assay using a combination of histidine decarboxylase (HDC) and histamine dehydrogenase (HDH) enzymes. Wild-type HDC is unstable and completely lost its activity within 50 days of storage at 4⯰C in solution. We rationally designed a HDC C57S mutant with markedly improved stability (storage at 4⯰C for over 200 days) without altering the enzyme's substrate specificity. Together with HDH, the HDC C57S mutant was applied to quantify l-histidine concentrations in human plasma. The assay showed high precision (<2.0% inter-assay variation) and high accuracy (<5.8% deviation from the results of LC/MS).
Assuntos
Histidina Descarboxilase/metabolismo , Histidina/sangue , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Cromatografia Líquida de Alta Pressão , Histidina/metabolismo , Histidina Descarboxilase/genética , Humanos , Cinética , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Photobacterium/enzimologia , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por SubstratoRESUMO
G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl-13C1H3-labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of ß2-adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Receptores Adrenérgicos beta 2/química , Coloração e Rotulagem/métodos , Alanina , Animais , Baculoviridae , Deutério , Insetos/citologia , Insetos/virologia , Bicamadas Lipídicas , Micelas , Ligação Proteica , Conformação ProteicaRESUMO
The rhizomes of Acorus tatarinowii Schott and Acorus gramineus Solander are widely used for treating amnesia in traditional Chinese medicine. In contrast, their leaves are usually discarded without their medicinal properties being known. Here, we found that the hot water extract of leaves improved cognition and tau pathology in model mice of frontotemporal dementia, similar to or even better than that of rhizomes. To explore the optimal method of processing, we made three preparations from dried leaves: hot water extract, extraction residue, and non-extracted simple crush powder. Among them, the simple crush powder had the strongest effect on tauopathy in mice. The crush powder also ameliorated Aß and α-synuclein pathologies and restored cognition in mouse models of Alzheimer's disease and dementia with Lewy bodies. These findings suggest the potential of Acorus tatarinowii/gramineus leaves as a dietary source for dementia prevention and reveal that simple crushing is a better way to maximize their efficacy.
Assuntos
Acorus , Demência , Extratos Vegetais , Folhas de Planta , Animais , Folhas de Planta/química , Acorus/química , Camundongos , Extratos Vegetais/farmacologia , Demência/prevenção & controle , Modelos Animais de Doenças , Cognição/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Masculino , Doença de Alzheimer/prevenção & controle , Proteínas tau/metabolismoRESUMO
Phosphoketolase and transketolase are thiamine diphosphate-dependent enzymes and play a central role in the primary metabolism of bifidobacteria: the bifid shunt. The enzymes both catalyze phosphorolytic cleavage of xylulose 5-phosphate or fructose 6-phosphate in the first reaction step, but possess different substrate specificity in the second reaction step, where phosphoketolase and transketolase utilize inorganic phosphate (Pi) and D-ribose 5-phosphate, respectively, as the acceptor substrate. Structures of Bifidobacterium longum phosphoketolase holoenzyme and its complex with a putative inhibitor, phosphoenolpyruvate, were determined at 2.5â Å resolution by serial femtosecond crystallography using an X-ray free-electron laser. In the complex structure, phosphoenolpyruvate was present at the entrance to the active-site pocket and plugged the channel to thiamine diphosphate. The phosphate-group position of phosphoenolpyruvate coincided well with those of xylulose 5-phosphate and fructose 6-phosphate in the structures of their complexes with transketolase. The most striking structural change was observed in a loop consisting of Gln546-Asp547-His548-Asn549 (the QN-loop) at the entrance to the active-site pocket. Contrary to the conformation of the QN-loop that partially covers the entrance to the active-site pocket (`closed form') in the known crystal structures, including the phosphoketolase holoenzyme and its complexes with reaction intermediates, the QN-loop in the current ambient structures showed a more compact conformation with a widened entrance to the active-site pocket (`open form'). In the phosphoketolase reaction, the `open form' QN-loop may play a role in providing the binding site for xylulose 5-phosphate or fructose 6-phosphate in the first step, and the `closed form' QN-loop may help confer specificity for Pi in the second step.
Assuntos
Bifidobacterium longum , Tiamina Pirofosfato , Tiamina Pirofosfato/química , Tiamina Pirofosfato/metabolismo , Bifidobacterium longum/metabolismo , Cristalografia por Raios X , Transcetolase/química , Transcetolase/metabolismo , Fosfoenolpiruvato , Temperatura , Xilulose , Domínio Catalítico , FrutoseRESUMO
Enzymatic amino acid assays are important in physiological research and clinical diagnostics because abnormal amino acid concentrations in biofluids are associated with various diseases. L-histidine decarboxylase from Photobacterium phosphoreum (PpHDC) is a pyridoxal 5'-phosphate-dependent enzyme and a candidate for use in an L-histidine quantitation assay. Previous cysteine substitution experiments demonstrated that the PpHDC C57S mutant displayed improved long-term storage stability and thermostability when compared with those of the wild-type enzyme. In this study, combinational mutation experiments of single cysteine substitution mutants of PpHDC were performed, revealing that the PpHDC C57S/C101V/C282V mutant possessed the highest thermostability. The stabilizing mechanism of these mutations was elucidated by solving the structures of PpHDC C57S and C57S/C101V/C282V mutants by X-ray crystallography. In the crystal structures, two symmetry-related PpHDC molecules form a domain-swapped homodimer. The side chain of S57 is solvent exposed in the structure, indicating that the C57S mutation eliminates chemical oxidation or disulfide bond formation with a free thiol group, thereby providing greater stability. Residues 101 and 282 form hydrophobic interactions with neighboring hydrophobic residues. Mutations C101V and C282V enhanced thermostability of PpHDC by filling a cavity present in the hydrophobic core (C101V) and increasing hydrophobic interactions.
Assuntos
Cisteína , Histidina Descarboxilase , Histidina/genética , PhotobacteriumRESUMO
Olfactory mucus contributes to the specific functions of the olfactory mucosa, but the composition and source of mucus proteins have not been fully elucidated. In this study, we used comprehensive proteome analysis and identified lipocalin 15 (LCN15), a human-specific lipocalin family protein, as an abundant component of the olfactory mucus. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using a newly generated anti-LCN15 antibody showed that LCN15 was concentrated in olfactory mucus samples, but not in respiratory mucus samples. Immunohistochemical staining using anti-LCN15 antibody revealed that LCN15 localized to the cytokeratin 18-positive Bowman's glands of the olfactory cleft mucosa. Quantitative image analysis revealed that the area of LCN15 immunoreactivity along the olfactory cleft mucosa significantly correlated with the area of neuron-specific Protein-Gene Product 9.5 (PGP9.5) immunoreactivity, suggesting that LCN15 is produced in non-degenerated areas of the olfactory neuroepithelium. ELISA demonstrated that the concentration of LCN15 in the mucus was lower in participants with normal olfaction (≥ 50 years) and also tended to be lower in patients with idiopathic olfactory loss (≥ 50 years) than in participants with normal olfaction (< 50 years). Thus, LCN15 may serve as a biomarker for the activity of the Bowman's glands.
Assuntos
Mucosa Olfatória , Olfato , Biomarcadores/metabolismo , Humanos , Lipocalinas/metabolismo , Muco/metabolismo , Mucosa Olfatória/metabolismoRESUMO
Commercially approved conventional antibody-drug conjugates (ADCs) are produced as heterogeneous mixtures containing a stochastic distribution of payloads decorating the antibody molecules resulting in decreased efficacy and thus lowering their therapeutic index. Control of the DAR and conjugation site in the development of next-generation ADCs is believed to assist in increasing the therapeutic index of these targeted biologics leading to overall enhanced clinical efficacy and reduced toxicity. A chemical site-specific conjugation technology termed AJICAP® allows ADC developers to control both the location and quantity of the payload conjugation to an antibody. Furthermore, this simplified ADC composition enables a streamlined chemical analysis. Here we report the chromatographic separation of site-specific ADCs produced by AJICAP® technology using an analytical affinity chromatography HPLC column containing a recombinant FcγIIIa receptor-ligand immobilized on a non-porous polymer resin (NPR). These HPLC analyses provided visually clear chromatogram results reflecting the heterogeneity of each ADC. The affinity strength was also measured by biolayer interferometry (BLI) and predicted by molecular structure analysis. The results indicate that AJICAP® technology is a promising solution to link hydrophobic payloads to antibodies without compromising antibody receptor function. This study also shows that FcγIIIa-NPR column can be used to characterize site-specific conjugated ADCs compared to ADCs synthesized using conventional methods.
Assuntos
Cromatografia de Afinidade/métodos , Imunoconjugados , Receptores de IgG , Proteínas Recombinantes , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Imunoconjugados/análise , Imunoconjugados/química , Imunoconjugados/metabolismo , Modelos Moleculares , Porosidade , Receptores de IgG/análise , Receptores de IgG/química , Receptores de IgG/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The C-terminal region of G-protein-coupled receptors (GPCRs), stimulated by agonist binding, is phosphorylated by GPCR kinases, and the phosphorylated GPCRs bind to arrestin, leading to the cellular responses. To understand the mechanism underlying the formation of the phosphorylated GPCR-arrestin complex, we performed NMR analyses of the phosphorylated ß2-adrenoceptor (ß2AR) and the phosphorylated ß2AR-ß-arrestin 1 complex, in the lipid bilayers of nanodisc. Here we show that the phosphorylated C-terminal region adheres to either the intracellular side of the transmembrane region or lipids, and that the phosphorylation of the C-terminal region allosterically alters the conformation around M2155.54 and M2796.41, located on transemembrane helices 5 and 6, respectively. In addition, we found that the conformation induced by the phosphorylation is similar to that corresponding to the ß-arrestin-bound state. The phosphorylation-induced structures revealed in this study propose a conserved structural motif of GPCRs that enables ß-arrestin to recognize dozens of GPCRs.
Assuntos
Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , beta-Arrestina 1/química , beta-Arrestina 1/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Receptores Adrenérgicos beta 2/genética , beta-Arrestina 1/genéticaRESUMO
A new convergent method for the synthesis of 1alpha,25-dihydroxyvitamin D(3) and its analogues has been developed that involves efficient preparation of the A-ring part 1a, (Z)-(3S,5R)-1-bromomethylene-3,5-bis(tert-butyldimethylsilyloxy)-2-methylenecyclohexane, starting from epichlorohydrin (4) and its Suzuki-Miyaura coupling reaction with the C,D-ring part 12. Thus, (R)-4 was converted to (3S,5R)-5-(tert-butyldimethylsilyloxy)-8-(trimethylsilyl)-oct-1-en-7-yn-3-ol (3a) through a ten-step reaction sequence in 49% overall yield. Compound 3a thus obtained was treated with a Ti(O-i-Pr)(4)/2 i-PrMgCl reagent and then with NBS to afford (Z)-(1S,2S,5R)-2-bromomethyl-3-[bromo(trimethylsilyl)methylene]-5-(tert-butyldimethylsilyloxy)cyclohexanol (10a) in 51% yield, from which 1a was obtained in 87% yield by sequential treatment with TBSCl/imidazole, DBU, and Cs(2)CO(3). The resulting A-ring intermediate 1a was reacted with alkenylboronate 12 in the presence of a PdCl(2)(dppf) catalyst to furnish 1alpha,25-dihydroxyvitamin D(3) in 82% yield after protodesilylation. Similarly, all of the other three possible stereoisomers of A-ring parts 1b, 1c, and 1d were prepared, from which 1-epi-, 3-epi-, and 1,3-di-epi-1alpha,25-dihydroxyvitamin D(3) were synthesized by coupling with 12 in excellent yield, respectively. Starting from 1a and 1c, des-C,D-1alpha,25-dihydroxyvitamin D(3) analogues, retiferol 13 and its 3-epi derivative, were also prepared, respectively.