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1.
Nat Methods ; 19(2): 205-215, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35132245

RESUMO

Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.


Assuntos
Metronidazol/farmacologia , Nitrorredutases/metabolismo , Pró-Fármacos/química , Animais , Animais Geneticamente Modificados , Células CHO , Cricetulus , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Metronidazol/farmacocinética , Nitrorredutases/química , Nitrorredutases/genética , Pró-Fármacos/farmacologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Vibrio/enzimologia , Peixe-Zebra/genética
2.
J Am Chem Soc ; 145(34): 18888-18903, 2023 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-37584157

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C-H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in NcAA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity. The presence of Glu or Asp close to the copper lowered the reduction potential and decreased the ratio between the reduction and reoxidation rates by up to 500-fold. All mutants showed increased enzyme inactivation, likely due to changes in the confinement of radical intermediates, and displayed changes in a protective hole-hopping pathway. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies gave virtually identical results for all NcAA9C variants, showing that the mutations do not directly perturb the Cu(II) ligand field. DFT calculations indicated that the higher experimental reoxidation rate observed for the Glu mutant could be reconciled if this residue is protonated. Further, for the glutamic acid form, we identified a Cu(III)-hydroxide species formed in a single step on the H2O2 splitting path. This is in contrast to the Cu(II)-hydroxide and hydroxyl intermediates, which are predicted for the WT and the unprotonated glutamate variant. These results show that this second sphere residue is a crucial determinant of the catalytic functioning of the copper-binding histidine brace and provide insights that may help in understanding LPMOs and LPMO-inspired synthetic catalysts.


Assuntos
Cobre , Oxigenases de Função Mista , Oxigenases de Função Mista/química , Cobre/química , Peróxido de Hidrogênio/metabolismo , Polissacarídeos/metabolismo , Glutamatos
3.
Mol Microbiol ; 105(4): 508-524, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28640457

RESUMO

The first cells probably possessed rudimentary metabolic networks, built using a handful of multifunctional enzymes. The promiscuous activities of modern enzymes are often assumed to be relics of this primordial era; however, by definition these activities are no longer physiological. There are many fewer examples of enzymes using a single active site to catalyze multiple physiologically-relevant reactions. Previously, we characterized the promiscuous alanine racemase (ALR) activity of Escherichia coli cystathionine ß-lyase (CBL). Now we have discovered that several bacteria with reduced genomes lack alr, but contain metC (encoding CBL). We characterized the CBL enzymes from three of these: Pelagibacter ubique, the Wolbachia endosymbiont of Drosophila melanogaster (wMel) and Thermotoga maritima. Each is a multifunctional CBL/ALR. However, we also show that CBL activity is no longer required in these bacteria. Instead, the wMel and T. maritima enzymes are physiologically bi-functional alanine/glutamate racemases. They are not highly active, but they are clearly sufficient. Given the abundance of the microorganisms using them, we suggest that much of the planet's biochemistry is carried out by enzymes that are quite different from the highly-active exemplars usually found in textbooks. Instead, primordial-like enzymes may be an essential part of the adaptive strategy associated with streamlining.


Assuntos
Enzimas/genética , Liases/genética , Alanina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Escherichia coli/genética , Genoma/genética , Genoma Bacteriano/genética , Liases/metabolismo , Redes e Vias Metabólicas , Thermotoga maritima/genética , Wolbachia/genética
4.
Biotechnol Lett ; 40(2): 359-367, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29147875

RESUMO

OBJECTIVES: To characterize the activities of two candidate nitroreductases, Neisseria meningitidis NfsA (NfsA_Nm) and Bartonella henselae (PnbA_Bh), with the nitro-prodrugs, CB1954 and metronidazole, and the environmental pollutants 2,4- and 2,6-dinitrotoluene. RESULTS: NfsA_Nm and PnbA_Bh were evaluated in Escherichia coli over-expression assays and as His6-tagged proteins in vitro. With the anti-cancer prodrug CB1954, both enzymes were more effective than the canonical O2-insensitive nitroreductase E. coli NfsB (NfsB_Ec), NfsA_Nm exhibiting comparable levels of activity to the leading nitroreductase candidate E. coli NfsA (NfsA_Ec). NfsA_Nm is also the first NfsA-family nitroreductase shown to produce a substantial proportion of 4-hydroxylamine end-product. NfsA_Nm and PnbA_Bh were again more efficient than NfsB_Ec at aerobic activation of metronidazole to a cytotoxic form, with NfsA_Nm appearing a promising candidate for improving zebrafish-targeted cell ablation models. NfsA_Nm was also more active than either NfsA_Ec or NfsB_Ec with 2,4- or 2,6-dinitrotoluene substrates, whereas PnbA_Bh was relatively inefficient with either substrate. CONCLUSIONS: NfsA_Nm is a promising new nitroreductase candidate for several diverse biotechnological applications.


Assuntos
Proteínas de Bactérias , Bartonella henselae/enzimologia , Biodegradação Ambiental , Dinitrobenzenos/metabolismo , Neisseria meningitidis/enzimologia , Pró-Fármacos , Antineoplásicos , Aziridinas , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Dinitrobenzenos/análise , Escherichia coli , Terapia Genética , Nitrorredutases , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia
5.
ACS Omega ; 9(21): 23040-23052, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38826537

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, using a single copper cofactor bound in a conserved histidine brace with a more variable second coordination sphere. Cellulose-active LPMOs in the fungal AA9 family and in a subset of bacterial AA10 enzymes contain a His-Gln-Tyr second sphere motif, whereas other cellulose-active AA10s have an Arg-Glu-Phe motif. To shine a light on the impact of this variation, we generated single, double, and triple mutations changing the His216-Gln219-Tyr221 motif in cellulose- and chitin-oxidizing MaAA10B toward Arg-Glu-Phe. These mutations generally reduced enzyme performance due to rapid inactivation under turnover conditions, showing that catalytic fine-tuning of the histidine brace is complex and that the roles of these second sphere residues are strongly interconnected. Studies of copper reactivity showed remarkable effects, such as an increase in oxidase activity following the Q219E mutation and a strong dependence of this effect on the presence of Tyr at position 221. In reductant-driven reactions, differences in oxidase activity, which lead to different levels of in situ generated H2O2, correlated with differences in polysaccharide-degrading ability. The single Q219E mutant displayed a marked increase in activity on chitin in both reductant-driven reactions and reactions fueled by exogenously added H2O2. Thus, it seems that the evolution of substrate specificity in LPMOs involves both the extended substrate-binding surface and the second coordination sphere.

6.
Nat Commun ; 15(1): 3975, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38729930

RESUMO

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein's surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.


Assuntos
Proteínas de Bactérias , Oxigenases de Função Mista , Oxirredução , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Domínio Catalítico , Triptofano/metabolismo , Polissacarídeos/metabolismo , Mutação , Estresse Oxidativo , Tirosina/metabolismo , Modelos Moleculares , Histidina/metabolismo , Histidina/genética
7.
FEBS Lett ; 597(10): 1363-1374, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37081294

RESUMO

Polysaccharide-degrading mono-copper lytic polysaccharide monooxygenases (LPMOs) are efficient peroxygenases that require electron donors (reductants) to remain in the active Cu(I) form and to generate the H2 O2 co-substrate from molecular oxygen. Here, we show how commonly used reductants affect LPMO catalysis in a pH-dependent manner. Between pH 6.0 and 8.0, reactions with ascorbic acid show little pH dependency, whereas reactions with gallic acid become much faster at increased pH. These dependencies correlate with the reductant ionization state, which affects its ability to react with molecular oxygen and generate H2 O2 . The correlation does not apply to l-cysteine because, as shown by stopped-flow kinetics, increased H2 O2 production at higher pH is counteracted by increased binding of l-cysteine to the copper active site. The findings highlight the importance of the choice of reductant and pH in LPMO reactions.


Assuntos
Cisteína , Substâncias Redutoras , Substâncias Redutoras/farmacologia , Oxirredução , Cisteína/metabolismo , Polissacarídeos/metabolismo , Oxigenases de Função Mista/química , Concentração de Íons de Hidrogênio , Oxigênio
8.
Elife ; 102021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34184634

RESUMO

Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in multiple zebrafish and mouse RP models, reasoning drugs effective across species and/or independent of disease mutation may translate better clinically. We first performed a large-scale phenotypic drug screen for compounds promoting rod cell survival in a larval zebrafish model of inducible RP. We tested 2934 compounds, mostly human-approved drugs, across six concentrations, resulting in 113 compounds being identified as hits. Secondary tests of 42 high-priority hits confirmed eleven lead candidates. Leads were then evaluated in a series of mouse RP models in an effort to identify compounds effective across species and RP models, that is, potential pan-disease therapeutics. Nine of 11 leads exhibited neuroprotective effects in mouse primary photoreceptor cultures, and three promoted photoreceptor survival in mouse rd1 retinal explants. Both shared and complementary mechanisms of action were implicated across leads. Shared target tests implicated parp1-dependent cell death in our zebrafish RP model. Complementation tests revealed enhanced and additive/synergistic neuroprotective effects of paired drug combinations in mouse photoreceptor cultures and zebrafish, respectively. These results highlight the value of cross-species/multi-model phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for RP patients.


Photoreceptors are the cells responsible for vision. They are part of the retina: the light-sensing tissue at the back of the eye. They come in two types: rods and cones. Rods specialise in night vision, while cones specialise in daytime colour vision. The death of these cells can cause a disease, called retinitis pigmentosa, that leads to vision loss. Symptoms often start in childhood with a gradual loss of night vision. Later on, loss of cone photoreceptors can lead to total blindness. Unfortunately, there are no treatments available that protect photoreceptor cells from dying. Research has identified drugs that can protect photoreceptors in animal models, but these drugs have failed in humans. The classic way to look for new treatments is to find drugs that target molecules implicated in a disease, and then test them to see if they are effective. Unfortunately, many drugs identified in this way fail in later stages of testing, either because they are ineffective, or because they have unacceptable side effects. One way to reverse this trend is to first test whether a drug is effective at curing a disease in animals, and later determining what it does at a molecular level. This could reveal whether drugs can protect photoreceptors before research to discover their molecular targets begins. Tests like this across different species could maximise the chances of finding a drug that works in humans, because if a drug works in several species, it is more likely to have shared target molecules across species. Applying this reasoning, Zhang et al. tested around 3,000 drug candidates for treating retinitis pigmentosa in a strain of zebrafish that undergoes photoreceptor degeneration similar to the human disease. Most of these drug candidates already have approval for use in humans, meaning that if they were found to be effective for treating retinitis pigmentosa, they could be fast-tracked for use in people. Zhang et al. found three compounds that helped photoreceptors survive both in zebrafish and in retinas grown in the laboratory derived from a mouse strain with degeneration similar to retinitis pigmentosa. Tests to find out how these three compounds worked at the molecular level revealed that they interfered with a protein that can trigger cell death. The tests also found other promising compounds, many of which offered increased protection when combined in pairs. Worldwide there are between 1.5 and 2.5 million people with retinitis pigmentosa. With this disease, loss of vision happens slowly, so identifying drugs that could slow or stop the process could help many people. These results suggest that placing animal testing earlier in the drug discovery process could complement traditional target-based methods. The compounds identified here, and the information about how they work, could expand potential treatment research. The next step in this research is to test whether the drugs identified by Zhang et al. protect mammals other than mice from the degeneration seen in retinitis pigmentosa.


Assuntos
Fármacos Neuroprotetores/farmacologia , Retinose Pigmentar/tratamento farmacológico , Animais , Animais Geneticamente Modificados , Células Cultivadas/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Mutação , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Peixe-Zebra
9.
Elife ; 92020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33185191

RESUMO

Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and newly-encountered substrates to influence the evolutionary trajectory, an aspect that is often overlooked in laboratory directed evolution studies. We selected the Escherichia coli nitro/quinone reductase NfsA for chloramphenicol detoxification by simultaneously randomising eight active-site residues and interrogating ~250,000,000 reconfigured variants. Analysis of every possible intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions. In both cases, improved chloramphenicol detoxification was only observed after an R225 substitution that largely eliminated activity with endogenous quinones. Error-prone PCR mutagenesis reinforced the importance of R225 substitutions, found in 100% of selected variants. This strong activity trade-off demonstrates that endogenous cellular metabolites hold considerable potential to shape evolutionary outcomes. Unselected prodrug-converting activities were mostly unaffected, emphasising the importance of negative selection to effect enzyme specialisation, and offering an application for the evolved genes as dual-purpose selectable/counter-selectable markers.


In the cell, most tasks are performed by big molecules called proteins, which behave like molecular machines. Although proteins are often described as having one job each, this is not always true, and many proteins can perform different roles. Enzymes are a type of protein that facilitate chemical reactions. They are often specialised to one reaction, but they can also accelerate other side-reactions. During evolution, these side-reactions can become more useful and, as a result, the role of the enzyme may change over time. The main role of the enzyme called NfsA in Escherichia coli bacteria is thought to be to convert molecules called quinones into hydroquinones, which can protect the cell from toxic molecules produced in oxidation reactions. As a side-reaction, NfsA has the potential to protect bacteria from an antibiotic called chloramphenicol, but it generally does this with such low efficacy that the effects are negligible. Producing hydroquinones is helpful to the cell in some situations, but if bacteria are regularly exposed to chloramphenicol, NfsA's role aiding antibiotic resistance could become more important. Over time, the enzyme could evolve to become better at neutralising chloramphenicol. Therefore, NfsA provides an opportunity to study the evolution of proteins and how bacteria adapt to antibiotics. To see how evolution might affect the activity of NfsA, Hall et al. generated 250 million E. coli with either random or targeted changes to the gene that codes for the NfsA enzyme. The resulting variants of NfsA that were most effective against chloramphenicol all had a change that eliminated the enzyme's ability to convert quinones. This result demonstrates a key trade-off between roles for NfsA, where one must be lost for the other to improve. These results demonstrate the interplay between a protein's different roles and provide insight into bacterial drug resistance. Additionally, the experiments showed that the bacteria with improved resistance to chloramphenicol also became more sensitive to another antibiotic, metronidazole. These findings could inform the fight against drug-resistant bacterial infections and may also be helpful in guiding the design of proteins with different roles.


Assuntos
Cloranfenicol/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Nitrorredutases/metabolismo , Domínio Catalítico , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Evolução Molecular , Inativação Metabólica , Mutação , Nitrorredutases/química , Nitrorredutases/genética , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
10.
Org Lett ; 22(24): 9427-9432, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33232161

RESUMO

The first total synthesis of (-)-TAN-2483B, a fungal metabolite possessing a densely functionalized furo[3,4-b]pyran-5-one framework, is achieved in 14 steps from d-mannose. Generation of the 2,6-trans-pyran is by cyclopropane ring expansion followed by α-selective alkynylation. Julia-Kocienski olefination introduces the E-propenyl side chain. Alkyne functionalization and carbonylation stereoselectively establish the bicyclic core of (-)-TAN-2483B. Inhibition of kinases Btk and Bmx, bacterial priority pathogens, and cytokine production in splenocytes indicates promising therapeutic potential.


Assuntos
Ciclopropanos/química , Fungos/metabolismo , Lactonas/síntese química , Piranos/síntese química , Fungos/química , Lactonas/química , Estrutura Molecular , Piranos/química , Estereoisomerismo
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