RESUMO
Using myoglobin (Mb) as a model protein, we herein developed a facial approach to modifying the heme active site. A cavity was first generated in the heme distal site by F46â C mutation, and the thiol group of Cys46 was then used for covalently linked to exogenous ligands, 1H-1,2,4-triazole-3-thiol and 1-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione. The engineered proteins, termed F46C-triazole Mb and F46C-phenol Mb, respectively, were characterized by X-ray crystallography, spectroscopic and stopped-flow kinetic studies. The results showed that both the heme coordination state and the protein function such as H2 O2 activation and peroxidase activity could be efficiently regulated, which suggests that this approach might be generally applied to the design of functional heme proteins.
Assuntos
Heme , Mioglobina , Mioglobina/química , Mioglobina/genética , Mioglobina/metabolismo , Domínio Catalítico , Heme/química , Cinética , Conformação Proteica , Compostos de SulfidrilaRESUMO
A series of amides, including α-bromo hydroxamates, N-alkoxyamides, and N-aryloxyamides, were subjected to phosphine-catalyzed ring-opening O-selective addition with cyclopropenones, producing various special α,ß-unsaturated esters containing oxime ether motif, in moderate to excellent yields, with high regioselectivity, and exclusive O-selectivity. The methodology is highly atom-economical, with simple operation procedures, and compatible with a wide substrate scope (more than 44 examples).
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A Cu-Fe bimetallic hydrogel (2-QF-CuFe-G) was constructed through a simple method. The 2-QF-CuFe-G metallohydrogel possesses excellent peroxidase-like activity to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The catalytic mechanism was confirmed by the addition of â¢OH radical scavenger isopropyl alcohol (IPA), tert-butyl alcohol (TBA) and ËOH trapping agent terephthalic acid (TA). Remarkably, the resultant blue ox-TMB system can be used to selectively and sensitively detect ascorbic acid (AA) with an LOD of 0.93 µM in the range of 4-36 µM through the colorimetric method. Moreover, the assay based on the 2-QF-CuFe-G metallohydrogel can be successfully applied to detect AA in fresh fruits.
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The design of functional metalloenzymes is attractive for the biosynthesis of biologically important compounds, such as phenoxazinones and phenazines catalyzed by native phenoxazinone synthase (PHS). To design functional heme enzymes, we used myoglobin (Mb) as a model protein and introduced an artificial CXXC motif into the heme distal pocket by F46C and L49C mutations, which forms a de novo disulfide bond, as confirmed by the X-ray crystal structure. We further introduced a catalytic Tyr43 into the heme distal pocket and found that the F43Y/F46C/L49C Mb triple mutant and the previously designed F43Y/F46S Mb exhibit PHS-like activity (80-98% yields in 5-15 min), with the catalytic efficiency exceeding those of natural metalloenzymes, including o-aminophenol oxidase, laccase, and dye-decolorizing peroxidase. Moreover, we showed that the oxidative coupling product of 1,6-disulfonic-2,7-diaminophenazine is a potential pH indicator, with the orange-magenta color change at pH 4-5 (pKa = 4.40). Therefore, this study indicates that functional heme enzymes can be rationally designed by structural modifications of Mb, exhibiting the functionality of the native PHS for green biosynthesis.
Assuntos
Metaloproteínas , Mioglobina , Mioglobina/química , Heme/química , Oxazinas , Óxido Nítrico SintaseRESUMO
A facile and dual fluorescent chemosensor (named 7-IDF) based on a phenylalanine derivative with an indole group was designed and synthesized. 7-IDF can selectively and sensitively detect Zn2+ via obvious fluorescence enhancement in an aqueous solution. Remarkably, the 7-IDF-Zn complex with blue luminescence has higher selectivity toward cysteine (Cys) and histidine (His) than for other amino acids. Intriguingly, 7-IDF can also be used as an excellent probe to detect Zn2+ in real water samples. Moreover, 7-IDF and 7-IDF-Zn possess excellent biocompatibility and cell permeability, and 7-IDF can consecutively detect Zn2+ and Cys/His in Hela cells through fluorescence imaging experiments. This study suggests that the phenylalanine-based chemosensor possesses great potential applications for the sequential detection of Zn2+ and Cys/His in biosystems.
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Cisteína , Corantes Fluorescentes , Humanos , Corantes Fluorescentes/química , Cisteína/química , Células HeLa , Histidina , Fenilalanina , Espectrometria de Fluorescência , ZincoRESUMO
We report herein a general and effective system achieving cyclization of ß-trifluoromethyl enones with amidines in the presence of 1,3-diiodo-5,5-dimethylhydantoin (DIH), which affords a range of trifluoromethylated 2-imidazolines in synthetically useful yields with good diastereoselectivities (up to 95% yield, up to 98:2 dr) and good functional group tolerance. Furthermore, the one-pot synthesis of trifluoromethylated imidazoles via sequential cyclization and oxidation is demonstrated. More significantly, the reaction mechanism was verified by ESI-MS studies of possible intermediates, and a reasonable reaction mechanism was proposed.
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It is desired to design and construct more efficient enzymes with better performance to catalyze carbene N-H insertions for the synthesis of bioactive molecules. To this end, we exploited and designed a series of human neuroglobin (Ngb) mutants. As shown in this study, a double mutant, A15C/H64G Ngb, with an additional disulfide bond and a modified heme active site, exhibited yields up to >99% and total turnover numbers up to 33000 in catalyzing the carbene N-H insertions for aromatic amine derivatives, including those with a large size such as 1-aminopyrene. Moreover, for o-phenylenediamine derivatives, they underwent two cycles of N-H insertions, followed by cyclization to form quinoxalinones, as confirmed by the X-ray crystal structures. This study suggests that Ngb can be designed into a functional carbene transferase for efficiently catalyzing carbene N-H insertion reactions with a range of substrates. It also represents the first example of the formation of quinoxalinones catalyzed by an engineered heme enzyme.
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Significant efforts have been made in the design of artificial metalloenzymes. Myoglobin (Mb), an O2 carrier, has been engineered to exhibit different functions. Herein, we applied a series of engineered Mb mutants with peroxidase activity for biosynthesis of clofazimine (CFZ), a potential drug with a broad-spectrum antiviral activity, by integration with chemical synthesis. Two of those mutants, F43Y Mb and F43Y/T67R Mb, have been shown to efficiently catalyze the oxidative coupling of 2-N-(4-chlorophenyl) benzene-1,2-diamine (N-4-CPBDA) in the presence of H2O2, with 97% yields. The overall catalytic efficiency (kcat/Km) is 46-fold and 82-fold higher than that of WT Mb, respectively. By further combination of this reaction with chemical synthesis, the production of CFZ was accomplished with an isolated yield of 72%. These results showed that engineered Mbs containing the Tyr-heme cross-link (F43Y Mb and F43Y/T67R Mb) exhibit enhanced activity in the oxidative coupling reaction. This study also indicates that the combination of biocatalysis and chemical synthesis avoids the need for the separation of intermediate products, which offers a convenient approach for the total synthesis of the biological compound CFZ.
Assuntos
Clofazimina , Mioglobina , Mioglobina/genética , Mioglobina/química , Peróxido de Hidrogênio/química , Modelos Moleculares , Heme/químicaRESUMO
The construction of smart materials, especially white light emitting (WLE) hydrogels with multi-stimuli responsive properties, has received widespread attention from researchers. In this study, a WLE hydrogel was obtained by the in situ doping of Eu3+ and Tb3+ into a blue emission low molecular weight gelator (MPF). Remarkably, the prepared WLE hydrogel possessed excellent stimuli responsiveness to pH, temperature and chemicals, and could be used as a soft thermometer and a selective sensor for Cu2+. The correlated color temperature of the WLE hydrogel was calculated to be 5063 K, suggesting a potential application in cool white light. Moreover, a series of metallohydrogels with different colors were obtained by modulating the ratio of MPF, Eu3+ and Tb3+ or changing the excitation wavelength, which was an excellent candidate to construct soft materials of a full-color system. Additionally, the WLE hydrogel could be used for constructing anti-counterfeiting materials. Therefore, this study provides a new approach for preparing smart WLE hydrogels with multiple functions.
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With the increasing demand for blue dyes, it is of vital importance to develop a green and efficient biocatalyst to produce indigo. This study constructed a hydrogen peroxide-dependent catalytic system for the direct conversion of indole to indigo using P450BM3 with the assistance of dual-functional small molecules (DFSM). The arrangements of amino acids at 78, 87, and 268 positions influenced the catalytic activity. F87G/T268V mutant gave the highest catalytic activity with kcat of 1402 min-1 and with a yield of 73%. F87A/T268V mutant was found to produce the indigo product with chemoselectivity as high as 80%. Moreover, F87G/T268A mutant was found to efficiently catalyze indole oxidation with higher activity (kcat/Km = 1388 mM-1 min-1) than other enzymes, such as the NADPH-dependent P450BM3 (2.4-fold), the Ngb (32-fold) and the Mb (117-fold). Computer simulation results indicate that the arrangements of amino acid residues in the active site can significantly affect the catalytic activity of the protein. The DFSM-facilitated P450BM3 peroxygenase system provides an alternative, simple approach for a key step in the bioproduction of indigo.
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Sistema Enzimático do Citocromo P-450 , Índigo Carmim , Sistema Enzimático do Citocromo P-450/metabolismo , Simulação por Computador , Oxirredução , Indóis/metabolismoRESUMO
Human neuroglobin (Ngb) contains a heme group and three Cys residues (Cys46, Cys55, and Cys120) in the polypeptide chain. By introducing an additional Cys at position 15, the X-ray structure of A15C Ngb mutant was solved at a high resolution of 1.35 Å, which reveals the formation of both the native (C46C55) and the engineered (C15C120) disulfide bonds, likely playing a functional and structural role, respectively, according to the geometry analysis. Unexpectedly, 1,4-dioxane from the crystallization reagents was bound not only to the protein surface, but also to the heme distal pocket, providing insights into protein-ligand interactions for the globin and guiding the design of functional heme enzymes.
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Globinas , Proteínas do Tecido Nervoso , Sítios de Ligação , Dissulfetos/química , Globinas/química , Globinas/genética , Globinas/metabolismo , Heme/química , Humanos , Ligantes , Proteínas do Tecido Nervoso/química , Neuroglobina , Raios XRESUMO
Human soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. In this work, fluorescent proteins were employed to study the NO-induced sGC molecular mechanism via mutagenesis at the catalytic domain. The conformational change of sGC by mutant α1C595 was investigated in living cells through fluorescence lifetime imaging microscopy (FLIM). The results indicated that the NO-induced conformational change of the catalytic domain of sGC from "open to "closed" upon GTP-binding was regulated by the hydrogen (H)-bonding network of the catalytic domain. The mutation of C595 caused a big conformational change of catalytic domain with H-bond variation, which not only demonstrates the key role of the C595 site in the process of conformational change of the catalytic domain, but also reveals the regulatory mechanism of sGC at the catalytic domain. This finding would guide the design of small-molecule drugs targeting the catalytic domain to modulate sGC activity.
Assuntos
Guanilato Ciclase , Receptores Citoplasmáticos e Nucleares , Domínio Catalítico , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Humanos , Óxido Nítrico/metabolismo , Guanilil Ciclase Solúvel/genética , Guanilil Ciclase Solúvel/metabolismoRESUMO
Globins are heme proteins such as hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb), playing important roles in biological system. In addition to normal functions, zebrafish Ngb was able to penetrate cell membranes, whereas less was known for other globin members. In this study, to improve the cell-membrane-penetrating activity of globins, we used sperm whale Mb as a model protein and constructed a quadruple mutant of G5K/Q8K/A19K/V21K Mb (termed 4K Mb), by introduction of four positive charges on the protein surface, which was designed according to the amino acid alignment with that of zebrafish Ngb. Spectroscopic and crystallographic studies showed that the four positively charged Lys residues did not affect the protein structure. Cell-membrane-penetrating essay further showed that 4K Mb exhibited enhanced activity compared to that of native Mb. This study provides valuable information for the effect of distribution of charged residues on the protein structure and the cell-membrane-penetrating activity of globins. Therefore, it will guide the design of protein-based biomaterials for biological applications.
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Membrana Celular/metabolismo , Mioglobina/química , Mioglobina/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Cristalografia por Raios X , Fluoresceína-5-Isotiocianato/química , Humanos , Lisina/química , Células MCF-7 , Mutação , Mioglobina/genética , Mioglobina/farmacocinética , Espectrofotometria Ultravioleta , CachaloteRESUMO
A double mutant of human H64M/V71C neuroglobin (Ngb) was engineered, which formed a single thioether bond as that in atypical cytochrome c, whereas the heme distal Met64 was oxidized to both sulfoxide (SO-Met) and sulfone (SO2 -Met). By contrast, no Cys-heme cross-link was formed in V71C Ngb with His64/His96 coordination, as shown by the X-ray crystal structure, which indicates that an open distal site facilitates the activation of heme iron for structural modifications.
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Citocromos c , Sulfetos , Humanos , Citocromos c/genética , Citocromos c/metabolismo , Heme/química , Neuroglobina/química , Neuroglobina/metabolismo , Oxirredução , Engenharia de ProteínasRESUMO
Heme proteins play vital roles in regulating the reactive oxygen/nitrogen species (ROS/RNS) levels in cells. In this study, we overexpressed human wild-type (WT) myoglobin (Mb) and its double mutant, F43H/H64A Mb with enhanced nitrite reductase (NIR) activity, in the typical representative triple-negative breast cancer cell, MDA-MB-231 cells. The results showed that the overexpression of F43H/H64A Mb increased the level of nitric oxide (NO) and the degree of oxidative stress, and then activated Akt/MAPK mediated apoptotic cascade, whereas WT Mb showed the opposite effect. This study indicates that Mb plays an important role in maintaining the balance of the cellular redox system and could thus be a valuable target for cancer therapy.
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Neoplasias da Mama , Mioglobina , Humanos , Feminino , Mioglobina/genética , Mioglobina/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Espécies Reativas de Oxigênio , Neoplasias da Mama/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estresse Oxidativo , Oxigênio/metabolismo , Nitrito Redutases/genética , Nitrito Redutases/metabolismo , NitrogênioRESUMO
The Nid site coordination microenvironment of a truncated acetyl-coenzyme A synthase has been designed systematically for functional conversion to a Ni-SOD-like enzyme. To this end, the first strategy is to introduce an axial histidine ligand, using mutations F598H, S594H and S594H-GP individually. The resulting three mutants obtained Ni-SOD-like activity successfully, although the catalytic activity was about 10-fold lower than in native Ni-SOD. The second strategy is to mimic the H-bond network in the second sphere coordination microenvironment of the native Ni-SOD. Two mutations based on F598H (EFG-F598H and YGP-F598H) were designed. The successful EFG-F598H exhibited ~3-fold Ni-SOD-like activity of F598H. These designed Ni-SOD-like metalloproteins were characterized by UV/Vis, EPR and Cyclic voltammetry while F598H was also characterized by X-ray protein crystallography. The pH titrations were performed to reveal the source of the two protons required for forming H2O2 in the SOD catalytic reaction. Based on all of the results, a proposed catalytic mechanism for the Ni-SOD-like metalloproteins is presented.
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Metaloproteínas , Níquel , Coenzima A , Peróxido de Hidrogênio , Metaloproteínas/química , Níquel/química , Prótons , Superóxido Dismutase/metabolismoRESUMO
Indoleamine 2,3-dioxygenase 1 (IDO1) is an attractive heme enzyme for its significant function in cancer immunotherapy. Potent IDO1 inhibitors have been discovered for decades, whereas no clinical drugs are used for cancer treatment up to now. With the goal of developing medically valuable IDO inhibitors, we performed a systematic study of SAR405838 analogs with a spiro-oxindole skeleton in this study. Based on the expression and purification of human IDO1, the inhibitory activity of spiro-oxindole skeleton compounds to IDO1 was evaluated by IC50 and Ki values. The results demonstrated that inhibitor 3 exhibited the highest IDO1 inhibitory activity with IC50 at 7.9 µM among all inhibitors, which is ~six-fold of the positive control (4-PI). Moreover, inhibitor 3 was found to have the most effective inhibition of IDO1 in MCF-7 cancer cells without toxic effects. Molecular docking analysis revealed that the hydrophobic interaction stabilized the binding of inhibitor 3 to the IDO1 active site and made an explanation for the uncompetitive mode of inhibitors. Therefore, this study provides valuable insights into the screen of more potent IDO1 inhibitors for cancer immunotherapy.
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Inibidores Enzimáticos , Indolamina-Pirrol 2,3,-Dioxigenase , Oxindóis , Compostos de Espiro , Inibidores Enzimáticos/química , Humanos , Imunoterapia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Indóis , Simulação de Acoplamento Molecular , Oxindóis/farmacologia , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
Heme proteins perform a variety of biological functions and also play significant roles in the field of bio-catalysis. The ß-lactamase activity of heme proteins has rarely been reported. Herein, we found, for the first time, that myoglobin (Mb), an O2 carrier, also exhibits novel ß-lactamase activity by catalyzing the hydrolysis of ampicillin. The catalytic proficiency ((kcat/KM)/kuncat) was determined to be 6.25 × 1010, which is much higher than the proficiency reported for designed metalloenzymes, although it is lower than that of natural ß-lactamases. Moreover, we found that this activity could be regulated by an engineered disulfide bond, such as Cys46-Cys61 in F46C/L61C Mb or by the addition of imidazole to directly coordinate to the heme center. These results indicate that the heme active site is responsible for the ß-lactamase activity of Mb. Therefore, the study suggests the potential of heme proteins acting as ß-lactamases, which broadens the diversity of their catalytic functions.
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Heme , Mioglobina , Mioglobina/química , Heme/química , Conformação Proteica , Modelos Moleculares , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Tetracyclines are one class of widely used antibiotics. Meanwhile, due to abuse and improper disposal, they are often detected in wastewater, which causes a series of environmental problems and poses a threat to human health and safety. As an efficient and environmentally friendly method, enzymatic catalysis has attracted much attention. In previous studies, we have designed an efficient peroxidase (F43Y/P88W/F138W Mb, termed YWW Mb) based on the protein scaffold of myoglobin (Mb), an O2 carrier, by modifying the heme active center and introducing two Trp residues. In this study, we further applied it to degrade the tetracycline antibiotics. Both UV-Vis and HPLC studies showed that the triple mutant YWW Mb was able to catalyze the degradation of tetracycline, oxytetracycline, doxycycline, and chlortetracycline effectively, with a degradation rate of ~100%, ~98%, ~94%, and ~90%, respectively, within 5 min by using H2O2 as an oxidant. These activities are much higher than those of wild-type Mb and other heme enzymes such as manganese peroxidase. As further analyzed by UPLC-ESI-MS, we identified multiple degradation products and thus proposed possible degradation mechanisms. In addition, the toxicity of the products was analyzed by using in vitro antibacterial experiments of E. coli. Therefore, this study indicates that the engineered heme enzyme has potential applications for environmental remediation by degradation of tetracycline antibiotics.
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Mioglobina , Tetraciclina , Humanos , Mioglobina/química , Peroxidase , Peróxido de Hidrogênio , Escherichia coli/genética , Escherichia coli/metabolismo , Peroxidases/química , Antibacterianos/farmacologia , Tetraciclinas , Heme/químicaRESUMO
Imbalance in the cellular redox system is thought to be associated with the induction and progression of breast cancers, and heme proteins may regulate the redox balance. Cytochrome b5 (Cyt b5) is a small mitochondrial heme protein. Its function and regulating mechanism in breast cancer remain unknown. In this study, we elucidated the level of endogenous oxidative stress in breast cancer cells, MCF-7 cells (hormone receptor-positive cells) and MDA-MB-231 cells (triple-negative cells), and investigated the difference in Cyt b5 content. Based on the low content of Cyt b5 in MDA-MB-231 cells, the overexpression of Cyt b5 was found to regulate the oxidative stress and apoptosis cascades, including ERK1/2 and Akt signaling pathways. The overexpressed Cyt b5 MDA-MB-231 cells were shown to exhibit decreased oxidative stress, less phosphorylation of ERK1/2 and Akt, and less cleavage of caspases 3 and 9 upon treatment with H2O2, as compared to those of normal MDA-MB-231 cells. Moreover, the overexpressed Cyt b5 most likely functioned by interacting with its protein partner, Cyt c, as suggested by co-immunoprecipitation studies. These results indicated that Cyt b5 has different effects on breast cancer cells of different phenotypes, which provides useful information for understanding the multiple roles of Cyt b5 and provides clues for clinical treatment.