Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.907
Filtrar
1.
J Agric Food Chem ; 69(39): 11616-11625, 2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34553918

RESUMO

Salicylic acid (SA) decarboxylase from Trichosporon moniliiforme (TmSdc), which reversibly catalyses the decarboxylation of SA to yield phenol, is of significant interest because of its potential for the production of benzoic acid derivatives under environmentally friendly reaction conditions. TmSdc showed a preference for C2 hydroxybenzoate derivatives, with kcat/Km of SA being 3.2 × 103 M-1 s-1. Here, we presented the first crystal structures of TmSdc, including a complex with SA. The three conserved residues of Glu8, His169, and Asp298 are the catalytic residues within the TIM-barrel domain of TmSdc. Trp239 forms a unique hydrophobic recognition site by interacting with the phenyl ring of SA, while Arg235 is responsible for recognizing the hydroxyl group at the C2 of SA via hydrogen bond interactions. Using a semi-rational combinatorial active-site saturation test, we obtained the TmSdc mutant MT3 (Y64T/P191G/F195V/E302D), which exhibited a 26.4-fold increase in kcat/Km with SA, reaching 8.4 × 104 M-1 s-1. Steered molecular dynamics simulations suggested that the structural changes in MT3 relieved the steric hindrance within the substrate access channel and enlarged the substrate-binding pocket, leading to the increased activity by improving substrate access. Our data elucidate the unique substrate recognition mode and the substrate entrance tunnel of SA decarboxylase.


Assuntos
Basidiomycota/enzimologia , Carboxiliases , Ácido Salicílico , Carboxiliases/genética , Carboxiliases/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Especificidade por Substrato
2.
Nat Commun ; 12(1): 5300, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489427

RESUMO

Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group.


Assuntos
Alcenos/metabolismo , Carboxiliases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Mononucleotídeo de Flavina/química , Glucose/metabolismo , Alcenos/química , Biocatálise , Carboxiliases/genética , Crotonatos/metabolismo , Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentação , Mononucleotídeo de Flavina/metabolismo , Glucose/química , Hypocreales/enzimologia , Hypocreales/genética , Ácido Mevalônico/metabolismo , Prenilação
3.
Nat Commun ; 12(1): 5065, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417452

RESUMO

The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics.


Assuntos
Carboxiliases/química , Carboxiliases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Reação de Cicloadição , Descarboxilação , Mononucleotídeo de Flavina/metabolismo , Cinética , Modelos Moleculares , Oxigênio/farmacologia , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Solventes , Relação Estrutura-Atividade , Especificidade por Substrato , Viscosidade
4.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361722

RESUMO

Off-flavors produced by undesirable microbial spoilage are a major concern in wineries, as they affect wine quality. This situation is worse in warm areas affected by global warming because of the resulting higher pHs in wines. Natural biotechnologies can aid in effectively controlling these processes, while reducing the use of chemical preservatives such as SO2. Bioacidification reduces the development of spoilage yeasts and bacteria, but also increases the amount of molecular SO2, which allows for lower total levels. The use of non-Saccharomyces yeasts, such as Lachancea thermotolerans, results in effective acidification through the production of lactic acid from sugars. Furthermore, high lactic acid contents (>4 g/L) inhibit lactic acid bacteria and have some effect on Brettanomyces. Additionally, the use of yeasts with hydroxycinnamate decarboxylase (HCDC) activity can be useful to promote the fermentative formation of stable vinylphenolic pyranoanthocyanins, reducing the amount of ethylphenol precursors. This biotechnology increases the amount of stable pigments and simultaneously prevents the formation of high contents of ethylphenols, even when the wine is contaminated by Brettanomyces.


Assuntos
Brettanomyces/metabolismo , Aromatizantes/metabolismo , Tecnologia de Alimentos/métodos , Odorantes/análise , Saccharomycetales/metabolismo , Vinho/análise , Antocianinas/metabolismo , Carboxiliases/metabolismo , Fermentação , Proteínas Fúngicas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Dióxido de Enxofre/farmacologia , Vitis/metabolismo , Vitis/microbiologia , Vinho/microbiologia
5.
Inorg Chem ; 60(17): 13539-13549, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34382397

RESUMO

The peroxide-dependent coproheme decarboxylase ChdC from Geobacillus stearothermophilus catalyzes two key steps in the synthesis of heme b, i.e., two sequential oxidative decarboxylations of coproporphyrinogen III (coproheme III) at propionate groups P2 and P4. In the binding site of coproheme III, P2 and P4 are anchored by different residues (Tyr144, Arg217, and Ser222 for P2 and Tyr113, Lys148, and Trp156 for P4); however, strong experimental evidence supports that the generated Tyr144 radical acts as an unique intermediary for hydrogen atom transfer (HAT) from both reactive propionates. So far, the reaction details are still unclear. Herein, we carried out quantum mechanics/molecular mechanics calculations to explore the decarboxylation mechanism of coproheme III. In our calculations, the coproheme Cpd I, Fe(IV) = O coupled to a porphyrin radical cation (por•+) with four propionate groups, was used as a reactant model. Our calculations reveal that Tyr144 is directly involved in the decarboxylation of propionate group P2. First, the proton-coupled electron transfer (PCET) occurs from Tyr144 to P2, generating a Tyr144 radical, which then abstracts a hydrogen atom from the Cß of P2. The ß-H extraction was calculated to be the rate-limiting step of decarboxylation. It is the porphyrin radical cation (por•+) that makes the PCET from Tyr144 to P2 to be quite easy to initiate the decarboxylation. Finally, the electron transfers from the Cß• through the porphyrin to the iron center, leading to the decarboxylation of P2. Importantly, the decarboxylation of P4 mediated by Lys148 was calculated to be very difficult, which suggests that after the P2 decarboxylation, the generated harderoheme III intermediate should rebind or rotate in the active site so that the propionate P4 occupies the binding site of P2, and Tyr144 again mediates the decarboxylation of P4. Thus, our calculations support the fact that Tyr144 is responsible for the decarboxylation of both P2 and P4.


Assuntos
Proteínas de Bactérias/química , Carboxiliases/química , Coproporfirinogênios/química , Proteínas de Bactérias/metabolismo , Carboxiliases/metabolismo , Domínio Catalítico , Coproporfirinogênios/metabolismo , Descarboxilação , Elétrons , Geobacillus stearothermophilus/enzimologia , Listeria monocytogenes/enzimologia , Modelos Químicos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Oxirredução , Ligação Proteica , Prótons , Teoria Quântica , Tirosina/química
6.
Toxicol Lett ; 349: 115-123, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34089817

RESUMO

Cisplatin, the most widely used platinum-based anticancer drug, often causes progressive and irreversible sensorineural hearing loss in cancer patients. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. Nicotinamide adenine dinucleotide (NAD+), a co-substrate for the sirtuin family and PARPs, has emerged as a potent therapeutic molecular target in various diseases. In our investigates, we observed that NAD+ level was changed in the cochlear explants of mice treated with cisplatin. Supplementation of a specific inhibitor (TES-1025) of α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), a rate-limiting enzyme of NAD+de novo synthesis pathway, promoted SIRT1 activity, increased mtDNA contents and enhanced AMPK expression, thus significantly reducing hair cells loss and deformation. The protection was blocked by EX527, a specific SIRT1 inhibitor. Meanwhile, the use of NMN, a precursor of NAD+ salvage synthesis pathway, had shown beneficial effect on hair cell under cisplatin administration, effectively suppressing PARP1. In vivo experiments confirmed the hair cell protection of NAD+ modulators in cisplatin treated mice and zebrafish. In conclusion, we demonstrated that modulation of NAD+ biosynthesis via the de novo synthesis pathway and the salvage synthesis pathway could both prevent ototoxicity of cisplatin. These results suggested that direct modulation of cellular NAD+ levels could be a promising therapeutic approach for protection of hearing from cisplatin-induced ototoxicity.


Assuntos
Inibidores Enzimáticos/farmacologia , Células Ciliadas Auditivas/efeitos dos fármacos , Perda Auditiva/prevenção & controle , Audição/efeitos dos fármacos , NAD/biossíntese , Ototoxicidade/prevenção & controle , Sirtuína 1/metabolismo , Animais , Animais Geneticamente Modificados , Carboxiliases/antagonistas & inibidores , Carboxiliases/metabolismo , Cisplatino , Modelos Animais de Doenças , Ativação Enzimática , Células Ciliadas Auditivas/enzimologia , Células Ciliadas Auditivas/patologia , Perda Auditiva/induzido quimicamente , Perda Auditiva/enzimologia , Perda Auditiva/fisiopatologia , Sistema da Linha Lateral/efeitos dos fármacos , Sistema da Linha Lateral/enzimologia , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Ototoxicidade/enzimologia , Ototoxicidade/etiologia , Ototoxicidade/fisiopatologia , Peixe-Zebra
7.
Nat Commun ; 12(1): 3983, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172745

RESUMO

Despite the well-established chemical processes for C-D bond formation, the toolbox of enzymatic methodologies for deuterium incorporation has remained underdeveloped. Here we describe a photodecarboxylase from Chlorella variabilis NC64A (CvFAP)-catalyzed approach for the decarboxylative deuteration of various carboxylic acids by employing D2O as a cheap and readily available deuterium source. Divergent protein engineering of WT-CvFAP is implemented using Focused Rational Iterative Site-specific Mutagenesis (FRISM) as a strategy for expanding the substrate scope. Using specific mutants, several series of substrates including different chain length acids, racemic substrates as well as bulky cyclic acids are successfully converted into the deuterated products (>40 examples). In many cases WT-CvFAP fails completely. This approach also enables the enantiocomplementary kinetic resolution of racemic acids to afford chiral deuterated products, which can hardly be accomplished by existing methods. MD simulations explain the results of improved catalytic activity and stereoselectivity of WT CvFAP and mutants.


Assuntos
Carboxiliases/química , Carboxiliases/metabolismo , Chlorella/enzimologia , Deutério/química , Carboxiliases/genética , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Catálise , Descarboxilação , Óxido de Deutério/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
8.
ACS Chem Biol ; 16(6): 1030-1039, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33984234

RESUMO

A common strategy employed in antibacterial drug discovery is the targeting of biosynthetic processes that are essential and specific for the pathogen. Specificity in particular avoids undesirable interactions with potential enzymatic counterparts in the human host, and it ensures on-target toxicity. Synthesis of pantothenate (Vitamine B5), which is a precursor of the acyl carrier coenzyme A, is an example of such a pathway. In Mycobacterium tuberculosis (Mtb), which is the causative agent of tuberculosis (TB), pantothenate is formed by pantothenate synthase, utilizing D-pantoate and ß-Ala as substrates. ß-Ala is mainly formed by the decarboxylation of l-aspartate, generated by the decarboxylase PanD, which is a homo-oliogomer in solution. Pyrazinoic acid (POA), which is the bioactive form of the TB prodrug pyrazinamide, binds and inhibits PanD activity weakly. Here, we generated a library of recombinant Mtb PanD mutants based on structural information and PZA/POA resistance mutants. Alterations in oligomer formation, enzyme activity, and/or POA binding were observed in respective mutants, providing insights into essential amino acids for Mtb PanD's proper structural assembly, decarboxylation activity and drug interaction. This information provided the platform for the design of novel POA analogues with modifications at position 3 of the pyrazine ring. Analogue 2, which incorporates a bulky naphthamido group at this position, displayed a 1000-fold increase in enzyme inhibition, compared to POA, along with moderately improved antimycobacterial activity. The data demonstrate that an improved understanding of mechanistic and enzymatic features of key metabolic enzymes can stimulate design of more-potent PanD inhibitors.


Assuntos
Antituberculosos/farmacologia , Carboxiliases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Pirazinamida/análogos & derivados , Antituberculosos/química , Carboxiliases/metabolismo , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/química , Pirazinamida/farmacologia , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
9.
APMIS ; 129(8): 503-511, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34046926

RESUMO

The key regulator of the polyamine biosynthetic pathway is ornithine decarboxylase (ODC). ODC is activated by antizyme inhibitor 1 (AZIN1) and 2 (AZIN2). AZIN1 and recently AZIN2 have been related to cancer; however, their functions in adenoid cystic carcinoma (ACC) have not been studied. We performed immunohistochemical study on minor salivary and mucous gland ACC tissue samples of patients treated at the Helsinki University Hospital (Helsinki, Finland) during 1974-2012. We scored AZIN1 and 2 immunoexpression in 42 and 45 tumor tissue samples, respectively, and correlated them with clinicopathological factors and survival. Enhanced AZIN2 expression was associated with better survival. In addition, both AZINs were seen more commonly in cribriform and tubular than in solid growth patterns. AZIN1 expression did not correlate with the studied clinicopathological factors. It seems that AZIN2 expression is higher in cancer tissue with secretory functions. In ACC tissue, high AZIN2 expression could be related to well-differentiated histological type which still has a functioning vesicle transportation system. Thus, AZIN2 could be a prognostic factor for better survival of ACC patients.


Assuntos
Carboxiliases/metabolismo , Carcinoma Adenoide Cístico/diagnóstico , Neoplasias das Glândulas Salivares/diagnóstico , Adulto , Idoso , Carboxiliases/genética , Carcinoma Adenoide Cístico/genética , Carcinoma Adenoide Cístico/metabolismo , Carcinoma Adenoide Cístico/mortalidade , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Feminino , Finlândia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/metabolismo , Neoplasias das Glândulas Salivares/mortalidade , Glândulas Salivares Menores/metabolismo , Sobrevida
10.
Int J Mol Sci ; 22(6)2021 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-33806862

RESUMO

Considering the widespread occurrence of oxalate in nature and its broad impact on a host of organisms, it is surprising that so little is known about the turnover of this important acid. In plants, oxalate oxidase is the most well-studied enzyme capable of degrading oxalate, but not all plants possess this activity. Recently, acyl-activating enzyme 3 (AAE3), encoding an oxalyl-CoA synthetase, was identified in Arabidopsis. This enzyme has been proposed to catalyze the first step in an alternative pathway of oxalate degradation. Since this initial discovery, this enzyme and proposed pathway have been found to be important to other plants and yeast as well. In this study, we identify, in Arabidopsis, an oxalyl-CoA decarboxylase (AtOXC) that is capable of catalyzing the second step in this proposed pathway of oxalate catabolism. This enzyme breaks down oxalyl-CoA, the product of AtAAE3, into formyl-CoA and CO2. AtOXC:GFP localization suggested that this enzyme functions within the cytosol of the cell. An Atoxc knock-down mutant showed a reduction in the ability to degrade oxalate into CO2. This reduction in AtOXC activity resulted in an increase in the accumulation of oxalate and the enzyme substrate, oxalyl-CoA. Size exclusion studies suggest that the enzyme functions as a dimer. Computer modeling of the AtOXC enzyme structure identified amino acids of predicted importance in co-factor binding and catalysis. Overall, these results suggest that AtOXC catalyzes the second step in this alternative pathway of oxalate catabolism.


Assuntos
Arabidopsis/fisiologia , Carboxiliases/metabolismo , Oxalatos/metabolismo , Fenômenos Fisiológicos Vegetais , Sequência de Aminoácidos , Carboxiliases/química , Carboxiliases/genética , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Ativação Enzimática , Regulação da Expressão Gênica de Plantas , Redes e Vias Metabólicas , Modelos Moleculares , Oxirredução , Desenvolvimento Vegetal/genética , Conformação Proteica , Transporte Proteico
11.
Nat Commun ; 12(1): 2195, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850144

RESUMO

The C4 unsaturated compound 1,3-butadiene is an important monomer in synthetic rubber and engineering plastic production. However, microorganisms cannot directly produce 1,3-butadiene when glucose is used as a renewable carbon source via biological processes. In this study, we construct an artificial metabolic pathway for 1,3-butadiene production from glucose in Escherichia coli by combining the cis,cis-muconic acid (ccMA)-producing pathway together with tailored ferulic acid decarboxylase mutations. The rational design of the substrate-binding site of the enzyme by computational simulations improves ccMA decarboxylation and thus 1,3-butadiene production. We find that changing dissolved oxygen (DO) levels and controlling the pH are important factors for 1,3-butadiene production. Using DO-stat fed-batch fermentation, we produce 2.13 ± 0.17 g L-1 1,3-butadiene. The results indicate that we can produce unnatural/nonbiological compounds from glucose as a renewable carbon source via a rational enzyme design strategy.


Assuntos
Butadienos/metabolismo , Carboxiliases/genética , Carboxiliases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas de Cultura Celular por Lotes , Butadienos/química , Carboxiliases/química , Fermentação , Glucose/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Simulação de Acoplamento Molecular
12.
Science ; 372(6538)2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33833098

RESUMO

Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.


Assuntos
Carboxiliases/química , Carboxiliases/metabolismo , Chlorella/enzimologia , Ácidos Graxos/metabolismo , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Alcanos/metabolismo , Substituição de Aminoácidos , Aminoácidos/metabolismo , Bicarbonatos/metabolismo , Biocatálise , Dióxido de Carbono/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Descarboxilação , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/química , Ligação de Hidrogênio , Luz , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxirredução , Fótons , Conformação Proteica , Temperatura
13.
J Biosci Bioeng ; 132(1): 18-24, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33846091

RESUMO

2,5-Furandicarboxylic acid (FDCA) is a valuable compound that can be synthesized from biomass-derived hydroxymethylfurfural (HMF), and holds great potential as a promising replacement for petroleum-based terephthalic acid in the production of polyamides, polyesters, and polyurethanes used universally. However, an economical large-scale production strategy for HMF from lignocellulosic biomass is yet to be established. This study aimed to design a synthetic pathway that can yield FDCA from furfural, whose industrial production from lignocellulosic biomass has already been established. This artificial pathway consists of an oxidase and a prenylated flavin mononucleotide (prFMN)-dependent reversible decarboxylase, catalyzing furfural oxidation and carboxylation of 2-furoic acid, respectively. The prFMN-dependent reversible decarboxylase was identified in an isolated strain, Paraburkholderia fungorum KK1, whereas an HMF oxidase from Methylovorus sp. MP688 exhibited furfural oxidation activity and was used as a furfural oxidase. Using Escherichia coli cells coexpressing these proteins, as well as a flavin prenyltransferase, FDCA could be produced from furfural via 2-furoic acid in one pot.


Assuntos
Biocatálise , Ácidos Dicarboxílicos/metabolismo , Furaldeído/metabolismo , Furanos/metabolismo , Biomassa , Burkholderiaceae/enzimologia , Burkholderiaceae/metabolismo , Carboxiliases/metabolismo , Oxirredução , Oxirredutases/metabolismo
14.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672882

RESUMO

The carbon-carbon bond formation has always been one of the most important reactions in C1 resource utilization. Compared to traditional organic synthesis methods, biocatalytic C-C bond formation offers a green and potent alternative for C1 transformation. In recent years, with the development of synthetic biology, more and more carboxylases and C-C ligases have been mined and designed for the C1 transformation in vitro and C1 assimilation in vivo. This article presents an overview of C-C bond formation in biocatalytic C1 resource utilization is first provided. Sets of newly mined and designed carboxylases and ligases capable of catalyzing C-C bond formation for the transformation of CO2, formaldehyde, CO, and formate are then reviewed, and their catalytic mechanisms are discussed. Finally, the current advances and the future perspectives for the development of catalysts for C1 resource utilization are provided.


Assuntos
Dióxido de Carbono/química , Monóxido de Carbono/química , Carbono/química , Técnicas de Química Sintética/métodos , Formaldeído/química , Formiatos/química , Biocatálise , Carboxiliases/metabolismo , Ligases/metabolismo , Biologia Sintética/métodos
15.
Methods Mol Biol ; 2280: 219-227, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33751438

RESUMO

Prenylated flavin (prFMN) is a modified FMN cofactor, the isoalloxazine is extended by an additional six membered nonaromatic ring. The modification confers azomethine ylide characteristics on the oxidised prFMN, allowing it to support the reversible nonoxidative decarboxylation of unsaturated acids by the UbiD family of decarboxylases. In absence of a chemical synthesis route for prFMN, enzymatic production by the flavin prenyltransferase, UbiX, is required for in vitro reconstitution of prFMN-dependent enzymes. Here we provide an overview of the methods for producing prFMN in vitro using the flavin prenyltransferase UbiX, and the subsequent reconstitution and activation of UbiD enzymes.


Assuntos
Carboxiliases/metabolismo , Flavinas/metabolismo , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/metabolismo , Ativação Enzimática , Oxirredução , Prenilação
16.
ACS Synth Biol ; 10(2): 228-235, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33535752

RESUMO

Conjugated alkenes such as dienes and polyenes have a range of applications as pharmaceutical agents and valuable building blocks in the polymer industry. Development of a renewable route to these compounds provides an alternative to fossil fuel derived production. The enzyme family of the UbiD decarboxylases offers substantial scope for alkene production, readily converting poly unsaturated acids. However, biochemical pathways producing the required substrates are poorly characterized, and UbiD-application has hitherto been limited to biological styrene production. Herein, we present a proof-of-principle study for microbial production of polyenes using a bioinspired strategy employing a polyketide synthase (PKS) in combination with a UbiD-enzyme. Deconstructing a bacterial iterative type II PKS enabled repurposing the broad-spectrum antibiotic andrimid biosynthesis pathway to access the metabolic intermediate 2,4,6-octatrienoic acid, a valuable chemical for material and pharmaceutical industry. Combination with the fungal ferulic acid decarboxylase (Fdc1) led to a biocatalytic cascade-type reaction for the production of hepta-1,3,5-triene in vivo. Our approach provides a novel route to generate unsaturated hydrocarbons and related chemicals and provides a blue-print for future development and application.


Assuntos
Antibacterianos/metabolismo , Aspergillus niger/enzimologia , Carboxiliases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas Fúngicas/metabolismo , Engenharia Metabólica/métodos , Biocatálise , Escherichia coli/genética , Ácidos Graxos Insaturados/metabolismo , Polienos/metabolismo , Policetídeo Sintases/metabolismo , Pirróis/metabolismo
17.
J Clin Invest ; 131(4)2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33586680

RESUMO

Tauopathies display a spectrum of phenotypes from cognitive to affective behavioral impairments; however, mechanisms promoting tau pathology and how tau elicits behavioral impairment remain unclear. We report a unique interaction between polyamine metabolism, behavioral impairment, and tau fate. Polyamines are ubiquitous aliphatic molecules that support neuronal function, axonal integrity, and cognitive processing. Transient increases in polyamine metabolism hallmark the cell's response to various insults, known as the polyamine stress response (PSR). Dysregulation of gene transcripts associated with polyamine metabolism in Alzheimer's disease (AD) brains were observed, and we found that ornithine decarboxylase antizyme inhibitor 2 (AZIN2) increased to the greatest extent. We showed that sustained AZIN2 overexpression elicited a maladaptive PSR in mice with underlying tauopathy (MAPT P301S; PS19). AZIN2 also increased acetylpolyamines, augmented tau deposition, and promoted cognitive and affective behavioral impairments. Higher-order polyamines displaced microtubule-associated tau to facilitate polymerization but also decreased tau seeding and oligomerization. Conversely, acetylpolyamines promoted tau seeding and oligomers. These data suggest that tauopathies launch an altered enzymatic signature that endorses a feed-forward cycle of disease progression. Taken together, the tau-induced PSR affects behavior and disease continuance, but may also position the polyamine pathway as a potential entry point for plausible targets and treatments of tauopathy, including AD.


Assuntos
Doença de Alzheimer/metabolismo , Poliaminas Biogênicas/metabolismo , Carboxiliases/metabolismo , Proteínas de Transporte/metabolismo , Hipocampo/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Carboxiliases/genética , Proteínas de Transporte/genética , Feminino , Hipocampo/patologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas tau/genética , Proteínas tau/metabolismo
18.
ChemSusChem ; 14(8): 1781-1804, 2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33631048

RESUMO

In recent years, (de)carboxylases that catalyze reversible (de)carboxylation have been targeted for application as carboxylation catalysts. This has led to the development of proof-of-concept (bio)synthetic CO2 fixation routes for chemical production. However, further progress towards industrial application has been hampered by the thermodynamic constraint that accompanies fixing CO2 to organic molecules. In this Review, biocatalytic carboxylation methods are discussed with emphases on the diverse strategies devised to alleviate the inherent thermodynamic constraints and their application in synthetic CO2 -fixation cascades.


Assuntos
Dióxido de Carbono/química , Carboxiliases/química , Carboxiliases/metabolismo , Biocatálise , Biotina/química , Dinitrocresóis/química , Metais/química , Estrutura Molecular , Piridoxal/química , Relação Estrutura-Atividade , Termodinâmica , Tiamina Pirofosfato/química
19.
Microb Cell Fact ; 20(1): 49, 2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33596923

RESUMO

BACKGROUND: In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escherichia coli. The resulting CatIBs are known for their high stability, easy and cost efficient production, and recyclability and thus provide an interesting alternative to conventionally immobilized enzymes. RESULTS: Here, we present the construction and characterization of a CatIB set of the lysine decarboxylase from Escherichia coli (EcLDCc), constructed via Golden Gate Assembly. A total of ten EcLDCc variants consisting of combinations of two linker and five aggregation inducing tag sequences were generated. A flexible Serine/Glycine (SG)- as well as a rigid Proline/Threonine (PT)-Linker were tested in combination with the artificial peptides (18AWT, L6KD and GFIL8) or the coiled-coil domains (TDoT and 3HAMP) as aggregation inducing tags. The linkers were fused to the C-terminus of the EcLDCc to form a linkage between the enzyme and the aggregation inducing tags. Comprehensive morphology and enzymatic activity analyses were performed for the ten EcLDCc-CatIB variants and a wild type EcLDCc control to identify the CatIB variant with the highest activity for the decarboxylation of L-lysine to 1,5-diaminopentane. Interestingly, all of the CatIB variants possessed at least some activity, whilst most of the combinations with the rigid PT-Linker showed the highest conversion rates. EcLDCc-PT-L6KD was identified as the best of all variants allowing a volumetric productivity of 457 g L- 1 d- 1 and a specific volumetric productivity of 256 g L- 1 d- 1 gCatIB-1. Noteworthy, wild type EcLDCc, without specific aggregation inducing tags, also partially formed CatIBs, which, however showed lower activity compared to most of the newly constructed CatIB variants (volumetric productivity: 219 g L- 1 d- 1, specific volumetric activity: 106 g L- 1 d- 1 gCatIB- 1). Furthermore, we demonstrate that microscopic analysis can serve as a tool to find CatIB producing strains and thus allow for prescreening at an early stage to save time and resources. CONCLUSIONS: Our results clearly show that the choice of linker and aggregation inducing tag has a strong influence on the morphology and the enzymatic activity of the CatIBs. Strikingly, the linker had the most pronounced influence on these characteristics.


Assuntos
Carboxiliases/metabolismo , Escherichia coli/metabolismo , Corpos de Inclusão/metabolismo
20.
Sci Rep ; 11(1): 972, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441661

RESUMO

Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. While the mechanisms underlying F. tularensis pathogenesis are largely unknown, previous studies have shown that a F. novicida transposon mutant with insertions in a gene coding for a putative lysine decarboxylase was attenuated in mouse spleen, suggesting a possible role of its protein product as a virulence factor. Therefore, we set out to structurally and functionally characterize the F. novicida lysine decarboxylase, which we termed LdcF. Here, we investigate the genetic environment of ldcF as well as its evolutionary relationships with other basic AAT-fold amino acid decarboxylase superfamily members, known as key actors in bacterial adaptative stress response and polyamine biosynthesis. We determine the crystal structure of LdcF and compare it with the most thoroughly studied lysine decarboxylase, E. coli LdcI. We analyze the influence of ldcF deletion on bacterial growth under different stress conditions in dedicated growth media, as well as in infected macrophages, and demonstrate its involvement in oxidative stress resistance. Finally, our mass spectrometry-based quantitative proteomic analysis enables identification of 80 proteins with expression levels significantly affected by ldcF deletion, including several DNA repair proteins potentially involved in the diminished capacity of the F. novicida mutant to deal with oxidative stress. Taken together, we uncover an important role of LdcF in F. novicida survival in host cells through participation in oxidative stress response, thereby singling out this previously uncharacterized protein as a potential drug target.


Assuntos
Proteínas de Bactérias/metabolismo , Carboxiliases/metabolismo , Francisella tularensis/metabolismo , Estresse Oxidativo/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Reparo do DNA/fisiologia , Escherichia coli/metabolismo , Macrófagos/metabolismo , Camundongos , Proteômica/métodos , Alinhamento de Sequência , Tularemia/microbiologia , Virulência/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...