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1.
Sheng Wu Gong Cheng Xue Bao ; 37(6): 1827-1844, 2021 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-34227279

RESUMO

Vitamin C is an essential vitamin for human beings. It has a huge market in the fields of food and pharmaceuticals. 2-keto-L-gulonic acid is an important precursor to produce vitamin C by microbial fermentation in industrial. In microbial fermentations, the L-sorbose pathway and the D-gluconate pathway have been the focus of research because of high yield. This article aims at stating recent research progress in dehydrogenases related to biosynthesis of vitamin C in the L-sorbose pathway and the D-gluconate pathway. The properties of dehydrogenase in terms of localization, substrate specificity, cofactors, and electron transport carrier are elaborated. And then, the main problems and strategies are reviewed in the L-sorbose pathway and in the D-gluconate pathway. Finally, future research on the dehydrogenases in the biosynthesis of vitamin C through L-sorbose pathway and D-gluconate pathway is discussed.


Assuntos
Ácido Ascórbico , Sorbose , Fermentação , Humanos , Oxirredutases/genética
2.
Nat Commun ; 12(1): 3619, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131130

RESUMO

L-2-Hydroxyglutarate (L-2-HG) plays important roles in diverse physiological processes, such as carbon starvation response, tumorigenesis, and hypoxic adaptation. Despite its importance and intensively studied metabolism, regulation of L-2-HG metabolism remains poorly understood and none of regulator specifically responded to L-2-HG has been identified. Based on bacterial genomic neighborhood analysis of the gene encoding L-2-HG oxidase (LhgO), LhgR, which represses the transcription of lhgO in Pseudomonas putida W619, is identified in this study. LhgR is demonstrated to recognize L-2-HG as its specific effector molecule, and this allosteric transcription factor is then used as a biorecognition element to construct an L-2-HG-sensing FRET sensor. The L-2-HG sensor is able to conveniently monitor the concentrations of L-2-HG in various biological samples. In addition to bacterial L-2-HG generation during carbon starvation, biological function of the L-2-HG dehydrogenase and hypoxia induced L-2-HG accumulation are also revealed by using the L-2-HG sensor in human cells.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Regulação da Expressão Gênica , Glutaratos/metabolismo , Proteínas de Bactérias/genética , Líquidos Corporais , Escherichia coli , Células HEK293 , Humanos , Oxirredutases/genética , Oxirredutases/metabolismo , Pseudomonas putida/genética , Fatores de Transcrição/metabolismo
3.
Nat Commun ; 12(1): 3268, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075034

RESUMO

Halocyclization of alkenes is a powerful bond-forming tool in synthetic organic chemistry and a key step in natural product biosynthesis, but catalyzing halocyclization with high enantioselectivity remains a challenging task. Identifying suitable enzymes that catalyze enantioselective halocyclization of simple olefins would therefore have significant synthetic value. Flavin-dependent halogenases (FDHs) catalyze halogenation of arene and enol(ate) substrates. Herein, we reveal that FDHs engineered to catalyze site-selective aromatic halogenation also catalyze non-native bromolactonization of olefins with high enantioselectivity and near-native catalytic proficiency. Highly selective halocyclization is achieved by characterizing and mitigating the release of HOBr from the FDH active site using a combination of reaction optimization and protein engineering. The structural origins of improvements imparted by mutations responsible for the emergence of halocyclase activity are discussed. This expansion of FDH catalytic activity presages the development of a wide range of biocatalytic halogenation reactions.


Assuntos
Alcenos/metabolismo , Biocatálise , Flavinas/metabolismo , Oxirredutases/metabolismo , Alcenos/química , Domínio Catalítico/genética , Ciclização , Ensaios Enzimáticos , Flavinas/química , Halogenação , Cinética , Simulação de Acoplamento Molecular , Mutagênese , Mutação , Oxirredutases/química , Oxirredutases/genética , Engenharia de Proteínas , Estereoisomerismo
4.
Hum Genet ; 140(8): 1157-1168, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33959807

RESUMO

Infantile nystagmus syndrome (INS) denominates early-onset, involuntary oscillatory eye movements with different etiologies. Nystagmus is also one of the symptoms in oculocutaneus albinism (OCA), a heterogeneous disease mainly caused by defects in melanin synthesis or melanosome biogenesis. Dopachrome tautomerase (DCT, also called TYRP2) together with tyrosinase (TYR) and tyrosin-related protein 1 (TYRP1) is one of the key enzymes in melanin synthesis. Although DCT´s role in pigmentation has been proven in different species, until now only mutations in TYR and TYRP1 have been found in patients with OCA. Detailed ophthalmological and orthoptic investigations identified a consanguineous family with two individuals with isolated infantile nystagmus and one family member with subtle signs of albinism. By whole-exome sequencing and segregation analysis, we identified the missense mutation c.176G > T (p.Gly59Val) in DCT in a homozygous state in all three affected family members. We show that this mutation results in incomplete protein maturation and targeting in vitro compatible with a partial or total loss of function. Subsequent screening of a cohort of patients with OCA (n = 85) and INS (n = 25) revealed two heterozygous truncating mutations, namely c.876C > A (p.Tyr292*) and c.1407G > A (p.Trp469*), in an independent patient with OCA. Taken together, our data suggest that mutations in DCT can cause a phenotypic spectrum ranging from isolated infantile nystagmus to oculocutaneous albinism.


Assuntos
Albinismo Oculocutâneo/genética , Oxirredutases Intramoleculares/genética , Melaninas/biossíntese , Mutação de Sentido Incorreto , Nistagmo Congênito/genética , Adolescente , Albinismo Oculocutâneo/diagnóstico , Albinismo Oculocutâneo/enzimologia , Albinismo Oculocutâneo/patologia , Sequência de Bases , Calnexina/genética , Calnexina/metabolismo , Criança , Estudos de Coortes , Consanguinidade , Feminino , Regulação da Expressão Gênica , Células HEK293 , Homozigoto , Humanos , Oxirredutases Intramoleculares/deficiência , Masculino , Melaninas/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Nistagmo Congênito/diagnóstico , Nistagmo Congênito/enzimologia , Nistagmo Congênito/patologia , Oxirredutases/genética , Oxirredutases/metabolismo , Linhagem , Sequenciamento Completo do Exoma , Adulto Jovem
5.
Plant Sci ; 308: 110930, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34034878

RESUMO

Huanglongbing (HLB) is one of the most destructive diseases in citrus worldwide. Unfortunately, HLB has no cure and management relies on insecticides to reduce populations of the vector, Diaphorina citri Kuwayama (Hemiptera: Liviidae). We propose an attract-and-kill strategy using a trap crop as an alternative to vector control to reduce transmission of the pathogen, 'Candidatus Liberibacter asiaticus'. We evaluated vector response to phytoene desaturase-silenced citrus trees using virus-induced gene silencing technology. Citrus tristeza virus (CTV) was used to produce a phytoene desaturase-silenced citrus (CTV-tPDS) that expresses visual, olfactory, and gustatory cues to attract D. citri. We found that D. citri were more attracted to CTV-tPDS plants with noticeably better fecundity and overall population fitness than on control plants. Moreover, rearing D. citri on CTV-tPDS plants significantly increased their survival probability compared with those reared on control plants. CTV-tPDS plants possessed reduced content of both carotenoid and chlorophyll pigments resulting in a consistent photobleached phenotype on citrus leaves which provided a sufficient close-range visual attractant to stimulate D. citri landing. Additionally, CTV-tPDS plants exhibited an enriched profile of volatile organic compounds (VOCs), which offered adequate olfactory cues to attract psyllid from long-range. Finally, CTV-tPDS plants exhibited an enriched metabolite content of phloem sap and leaves which offered appropriate gustatory cues that influenced probing/feeding behavior. We believe that introducing CTV-tPDS plants (as a trap crop) to D. citri-infested orchards will attract and congregate psyllids to facilitate their removal from the target crop with insecticides or by other means. This new strategy could be deployed relatively quickly and economically to HLB-impacted citrus industries. Moreover, it is an eco-friendly strategy because it should partially reduce the input of chemical insecticides ameliorating the indirect cost of HLB infection.


Assuntos
Vetores Artrópodes/fisiologia , Citrus/fisiologia , Inativação Gênica , Hemípteros/fisiologia , Oxirredutases/genética , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/genética , Animais , Citrus/genética , Controle de Insetos , Oxirredutases/metabolismo , Controle Biológico de Vetores , Proteínas de Plantas/metabolismo
6.
Nat Commun ; 12(1): 2828, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990581

RESUMO

Pinoresinol-lariciresinol reductases (PLRs) are enzymes involved in the lignan biosynthesis after the initial dimerization of two monolignols, and this represents the entry point for the synthesis of 8-8' lignans and contributes greatly to their structural diversity. Of particular interest has been the determination of how differing substrate specificities are achieved with these enzymes. Here, we present crystal structures of IiPLR1 from Isatis indigotica and pinoresinol reductases (PrRs) AtPrR1 and AtPrR2 from Arabidopsis thaliana, in the apo, substrate-bound and product-bound states. Each structure contains a head-to-tail homodimer, and the catalytic pocket comprises structural elements from both monomers. ß4 loop covers the top of the pocket, and residue 98 from the loop governs catalytic specificity. The substrate specificities of IiPLR1 and AtPrR2 can be switched via structure-guided mutagenesis. Our study provides insight into the molecular mechanism underlying the substrate specificity of PLRs/PrRs and suggests an efficient strategy for the large-scale commercial production of the pharmaceutically valuable compound lariciresinol.


Assuntos
Oxirredutases/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Butileno Glicóis , Domínio Catalítico/genética , Cristalografia por Raios X , Furanos/metabolismo , Isatis/genética , Isatis/metabolismo , Lignanas/biossíntese , Lignanas/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredutases/química , Oxirredutases/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Engenharia de Proteínas , Multimerização Proteica , Eletricidade Estática , Especificidade por Substrato
7.
Food Funct ; 12(9): 3978-3991, 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33977989

RESUMO

Tyrosinase is considered a molecular marker of melanoma, and few natural antitumor drugs targeting tyrosinase have been identified. In this study, proanthocyanidins (PAs) were isolated from the leaves of Photinia × fraseri and their structures were characterized by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the effects of antityrosinase activity were investigated. The results showed that the basic structural units of PAs are composed of catechin and epicatechin and that oligomer is the main component. PAs exhibited better antityrosinase activity via chelation of copper ions and by disturbing o-quinone production. Furthermore, analyses of the cell cycle, apoptosis rate, and regulation of melanin protein expression revealed preliminarily that PAs could affect melanin production by downregulating microphthalmia transcription factor (MITF) expression and by inhibiting the activities of tyrosinase and tyrosinase related protein 1 (TRP-1), leading to cell cycle arrest and apoptosis of melanoma cells. Collectively, our study demonstrated that PAs are potential tyrosinase inhibitors and have good antimelanoma effects. These findings provide a theoretical support for the application of tyrosinase inhibitors and for further drug development.


Assuntos
Apoptose , Ciclo Celular/efeitos dos fármacos , Melanoma Experimental/patologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Photinia/química , Proantocianidinas/farmacologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Levodopa/química , Levodopa/metabolismo , Melaninas/biossíntese , Melaninas/genética , Melanoma Experimental/enzimologia , Melanoma Experimental/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Estrutura Molecular , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Ácido Periódico , Folhas de Planta/química , Proantocianidinas/química , Proantocianidinas/isolamento & purificação
8.
Biomed Res Int ; 2021: 5568845, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33981770

RESUMO

The flora compositions of nitrogen-fixing bacteria in roots of Pennisetum giganteum z.x.lin at different growth stages and the expression and copy number of nitrogen-fixing gene nifH were studied by Illumina Miseq second-generation sequencing technology and qRT-PCR. The results showed that there were more than 40,000~50,000 effective sequences in 5 samples from the roots of P. giganteum, with Proteobacteria and Cyanobacteria as the dominant nitrogen-fixing bacteria based on the OTU species annotations for each sample and Bradyrhizobium as the core bacterial genera. The relative expression and quantitative change of nifH gene in roots of P. giganteum at different growth stages were consistent with the changes in the flora compositions of nitrogen-fixing microbia. Both revealed a changing trend with an initial increase and a sequential decrease, as well as changing order as jointing stage>maturation stage>tillering stage>seedling stage>dying stage. The relative expression and copy number of nifH gene were different in different growth stages, and the difference among groups basically reached a significant level (p < 0.05). The relative expression and copy number of nifH gene at the jointing stage were the highest, and the 2-△△CT value was 4.43 folds higher than that at the seedling stage, with a copy number of 1.32 × 107/g. While at the dying stage, it was the lowest, and the 2-△△CT value was 0.67 folds, with a copy number of 0.31 × 107/g.


Assuntos
Proteínas de Bactérias , Bactérias Fixadoras de Nitrogênio , Oxirredutases , Pennisetum/microbiologia , Raízes de Plantas/microbiologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dosagem de Genes/genética , Genes Bacterianos/genética , Bactérias Fixadoras de Nitrogênio/classificação , Bactérias Fixadoras de Nitrogênio/genética , Bactérias Fixadoras de Nitrogênio/metabolismo , Oxirredutases/análise , Oxirredutases/genética , Oxirredutases/metabolismo , Microbiologia do Solo
9.
Microbes Environ ; 36(2)2021.
Artigo em Inglês | MEDLINE | ID: mdl-33907062

RESUMO

Malodorous emissions are a crucial and inevitable issue during the decomposition of biological waste and contain a high concentration of ammonia. Biofiltration technology is a feasible, low-cost, energy-saving method that reduces and eliminates malodors without environmental impact. In the present study, we evaluated the effectiveness of compost from cattle manure and food waste as deodorizing media based on their removal of ammonia and the expression of ammonia-oxidizing genes, and identified the bacterial and archaeal communities in these media. Ammonia was removed by cattle manure compost, but not by food waste compost. The next-generation sequencing of 16S ribosomal RNA obtained from cattle manure compost revealed the presence of ammonia-oxidizing bacteria (AOB), including Cytophagia, Alphaproteobacteria, and Gammaproteobacteria, and ammonia-oxidizing archaea (AOA), such as Thaumarchaeota. In cattle manure compost, the bacterial and archaeal ammonia monooxygenase A (amoA) genes were both up-regulated after exposure to ammonia (fold ratio of 14.2±11.8 after/before), and the bacterial and archaeal communities were more homologous after than before exposure to ammonia, which indicates the adaptation of these communities to ammonia. These results suggest the potential of cattle manure compost as an efficient biological deodorization medium due to the activation of ammonia-oxidizing microbes, such as AOB and AOA, and the up-regulation of their amoA genes.


Assuntos
Archaea/enzimologia , Proteínas Arqueais/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Esterco/microbiologia , Oxirredutases/metabolismo , Amônia/metabolismo , Animais , Archaea/classificação , Archaea/genética , Archaea/metabolismo , Proteínas Arqueais/genética , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Bovinos , Compostagem , Filtração , Esterco/análise , Oxirredução , Oxirredutases/genética , Filogenia
10.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804237

RESUMO

Ataxia in children is a common clinical sign of numerous neurological disorders consisting of impaired coordination of voluntary muscle movement. Its most common form, cerebellar ataxia, describes a heterogeneous array of neurologic conditions with uncountable causes broadly divided as acquired or genetic. Numerous genetic disorders are associated with chronic progressive ataxia, which complicates clinical management, particularly on the diagnostic stage. Advances in omics technologies enable improvements in clinical practice and research, so we proposed a multi-omics approach to aid in the genetic diagnosis and molecular elucidation of an undiagnosed infantile condition of chronic progressive cerebellar ataxia. Using whole-exome sequencing, RNA-seq, and untargeted metabolomics, we identified three clinically relevant mutations (rs141471029, rs191582628 and rs398124292) and an altered metabolic profile in our patient. Two POLR1C diagnostic variants already classified as pathogenic were found, and a diagnosis of hypomyelinating leukodystrophy was achieved. A mutation on the MMACHC gene, known to be associated with methylmalonic aciduria and homocystinuria cblC type, was also found. Additionally, preliminary metabolome analysis revealed alterations in our patient's amino acid, fatty acid and carbohydrate metabolism. Our findings provided a definitive genetic diagnosis reinforcing the association between POLR1C mutations and hypomyelinating leukodystrophy and highlighted the relevance of multi-omics approaches to the disease.


Assuntos
Ataxia Cerebelar/diagnóstico , RNA Polimerases Dirigidas por DNA/genética , Genoma/genética , Oxirredutases/genética , Transcriptoma/genética , Adolescente , Adulto , Ataxia Cerebelar/genética , Ataxia Cerebelar/patologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Metaboloma/genética , Mutação/genética , Linhagem , RNA-Seq , Deficiência de Vitamina B 12/genética , Sequenciamento Completo do Exoma/métodos , Adulto Jovem
11.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810284

RESUMO

There is an increasing interest in polyphenols, plant secondary metabolites, in terms of fruit quality and diet, mainly due to their antioxidant effect. However, the identification of key gene enzymes and their roles in the phenylpropanoid pathway in temperate fruits species remains uncertain. Apricot (Prunus armeniaca) is a Mediterranean fruit with high diversity and fruit quality properties, being an excellent source of polyphenol compounds. For a better understanding of the phenolic pathway in these fruits, we selected a set of accessions with genetic-based differences in phenolic compounds accumulation. HPLC analysis of the main phenolic compounds and transcriptional analysis of the genes involved in key steps of the polyphenol network were carried out. Phenylalanine ammonia-lyase (PAL), dihydroflavonol-4-reductase (DFR) and flavonol synthase (FLS) were the key enzymes selected. Orthologous of the genes involved in transcription of these enzymes were identified in apricot: ParPAL1, ParPAL2, ParDFR, ParFLS1 and ParFLS2. Transcriptional data of the genes involved in those critical points and their relationships with the polyphenol compounds were analyzed. Higher expression of ParDFR and ParPAL2 has been associated with red-blushed accessions. Differences in expression between paralogues could be related to the presence of a BOXCOREDCPAL cis-acting element related to the genes involved in anthocyanin synthesis ParFLS2, ParDFR and ParPAL2.


Assuntos
Metaboloma , Polifenóis/biossíntese , Prunus/metabolismo , Transcriptoma , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polifenóis/genética , Prunus/genética
12.
Nat Commun ; 12(1): 2132, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837197

RESUMO

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, reveals a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes. Expression of five fungal enzymes, including three from unexplored clades, expands the AA7-substrate range and unveils a cellooligosaccharide dehydrogenase activity, previously unknown within AA7. Sequence and structural analyses identify unique signatures distinguishing the strict dehydrogenase clade from canonical AA7 oxidases. The discovered dehydrogenase directly is able to transfer electrons to an AA9 lytic polysaccharide monooxygenase (LPMO) and fuel cellulose degradation by LPMOs without exogenous reductants. The expansion of redox-profiles and substrate range highlights the functional diversity within AA7 and sets the stage for harnessing AA7 dehydrogenases to fine-tune LPMO activity in biotechnological conversion of plant feedstocks.


Assuntos
Celulose/metabolismo , Proteínas Fúngicas/metabolismo , Oomicetos/enzimologia , Oxirredutases/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Cristalografia por Raios X , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Flavoproteínas Transferidoras de Elétrons/metabolismo , Ensaios Enzimáticos , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Microbiologia Industrial/métodos , Espectroscopia de Ressonância Magnética , Oomicetos/genética , Oxirredução , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Oxirredutases/ultraestrutura , Filogenia , Análise de Sequência de DNA , Especificidade por Substrato
13.
Nat Commun ; 12(1): 2092, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33828077

RESUMO

Streptoseomycin (STM, 1) is a bacterial macrolactone that has a unique 5/14/10/6/6-pentacyclic ring with an ether bridge. We have previously identified the biosynthetic gene cluster for 1 and characterized StmD as [6 + 4]- and [4 + 2]-bispericyclase that catalyze a reaction leading to both 6/10/6- and 10/6/6-tricyclic adducts (6 and 7). The remaining steps, especially how to install and stabilize the required 10/6/6-tricyclic core for downstream modifications, remain unknown. In this work, we have identified three oxidoreductases that fix the required 10/6/6-tryciclic core. A pair of flavin-dependent oxidoreductases, StmO1 and StmO2, catalyze the direct hydroxylation at [6 + 4]-adduct (6). Subsequently, a spontaneous [3,3]-Cope rearrangement and an enol-ketone tautomerization result in the formation of 10/6/6-tricyclic intermediate 12b, which can be further converted to a stable 10/6/6-tricyclic alcohol 11 through a ketoreduction by StmK. Crystal structure of the heterodimeric complex NtfO1-NtfO2, homologues of StmO1-StmO2 with equivalent function, reveals protein-protein interactions. Our results demonstrate that the [6 + 4]-adduct instead of [4 + 2]-adduct is the bona fide biosynthetic intermediate.


Assuntos
Reação de Cicloadição/métodos , Lactonas/química , Lactonas/metabolismo , Catálise , Fermentação , Flavinas , Hidroxilação , Cetonas , Modelos Moleculares , Oxirredutases/genética
15.
Biochem Biophys Res Commun ; 551: 107-113, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33725571

RESUMO

Site-directed mutagenesis (SDM), an indispensable method in molecular biology and protein engineering, is rather time-consuming and laborious. Protein engineering, especially that of enzymes, nowadays increasingly relies on rational design approaches in which both SDM and protein expression are the bottlenecks because they are generally based on the recombinant DNA technology. Here, we developed a new PCR-based mutagenesis method, DiRect, that achieves high performance in product quality (≥99% substitution) without recombinant DNA technology. We applied DiRect in combination with a cell-free protein expression system to an industrially relevant enzyme, nicotinamide adenine dinucleotide phosphate-dependent 3-quinuclidinone reductase from Rhodotorula rubra. In a single round of screening, 90 newly designed mutant proteins were produced within two days, and an unreported mutant (Q135I) exhibiting much higher thermostability than the wild-type enzyme was successfully identified within one extra day. Thus, DiRect is a simple, efficient, and potentially scalable SDM method.


Assuntos
Mutagênese Sítio-Dirigida/métodos , Engenharia de Proteínas/métodos , Sistema Livre de Células , Estabilidade Enzimática , Mutação , NADP/metabolismo , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Rhodotorula/enzimologia
16.
J Chem Ecol ; 47(3): 248-264, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33779878

RESUMO

The European grapevine moth, Lobesia botrana, uses (E,Z)-7,9-dodecadienyl acetate as its major sex pheromone component. Through in vivo labeling experiments we demonstrated that the doubly unsaturated pheromone component is produced by ∆11 desaturation of tetradecanoic acid, followed by chain shortening of (Z)-11-tetradecenoic acid to (Z)-9-dodecenoic acid, and subsequently introduction of the second double bond by an unknown ∆7 desaturase, before final reduction and acetylation. By sequencing and analyzing the transcriptome of female pheromone glands of L. botrana, we obtained 41 candidate genes that may be involved in sex pheromone production, including the genes encoding 17 fatty acyl desaturases, 13 fatty acyl reductases, 1 fatty acid synthase, 3 acyl-CoA oxidases, 1 acetyl-CoA carboxylase, 4 fatty acid transport proteins and 2 acyl-CoA binding proteins. A functional assay of desaturase and acyl-CoA oxidase gene candidates in yeast and insect cell (Sf9) heterologous expression systems revealed that Lbo_PPTQ encodes a ∆11 desaturase producing (Z)-11-tetradecenoic acid from tetradecanoic acid. Further, Lbo_31670 and Lbo_49602 encode two acyl-CoA oxidases that may produce (Z)-9-dodecenoic acid by chain shortening (Z)-11-tetradecenoic acid. The gene encoding the enzyme introducing the E7 double bond into (Z)-9-dodecenoic acid remains elusive even though we assayed 17 candidate desaturases in the two heterologous systems.


Assuntos
Dodecanol/análogos & derivados , Atrativos Sexuais/biossíntese , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Sequência de Aminoácidos , Animais , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Mariposas , Ácido Mirístico/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Saccharomyces cerevisiae/metabolismo , Células Sf9/metabolismo , Transcriptoma
17.
Nutrients ; 13(3)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673530

RESUMO

Anemia is a frequent finding in children with celiac disease but the detailed pathophysiological mechanisms in the intestine remain obscure. One possible explanation could be an abnormal expression of duodenal iron transport proteins. However, the results have so far been inconsistent. We investigated this issue by comparing immunohistochemical stainings of duodenal cytochrome B (DCYTB), divalent metal transporter 1 (DMT1), ferroportin, hephaestin and transferrin receptor 1 (TfR1) in duodenal biopsies between 27 children with celiac disease and duodenal atrophy, 10 celiac autoantibody-positive children with potential celiac disease and six autoantibody-negative control children. Twenty out of these 43 subjects had anemia. The expressions of the iron proteins were investigated with regard to saturation and the percentage of the stained area or stained membrane length of the enterocytes. The results showed the stained area of ferroportin to be increased and the saturation of hephaestin to be decreased in celiac disease patients compared with controls. There were no differences in the transporter protein expressions between anemic and non-anemic patients. The present results suggest an iron status-independent alteration of ferroportin and hephaestin proteins in children with histologically confirmed celiac disease.


Assuntos
Antígenos CD/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Doença Celíaca/metabolismo , Grupo dos Citocromos b/metabolismo , Proteínas de Membrana/metabolismo , Oxirredutases/metabolismo , Receptores da Transferrina/metabolismo , Fatores de Transcrição/metabolismo , Adolescente , Anemia Ferropriva/complicações , Anemia Ferropriva/metabolismo , Antígenos CD/genética , Proteínas de Transporte de Cátions/genética , Doença Celíaca/complicações , Criança , Pré-Escolar , Grupo dos Citocromos b/genética , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Proteínas de Membrana/genética , Oxirredutases/genética , Receptores da Transferrina/genética , Fatores de Transcrição/genética
18.
Nat Chem Biol ; 17(5): 585-592, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33707784

RESUMO

YcaO enzymes catalyze several post-translational modifications on peptide substrates, including thioamidation, which substitutes an amide oxygen with sulfur. Most predicted thioamide-forming YcaO enzymes are encoded adjacent to TfuA, which when present, is required for thioamidation. While activation of the peptide amide backbone is well established for YcaO enzymes, the function of TfuA has remained enigmatic. Here we characterize the TfuA protein involved in methyl-coenzyme M reductase thioamidation and demonstrate that TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO toward the thioamidation substrate. We also report a crystal structure of a TfuA, which displays a new protein fold. Our structural and mutational analyses of TfuA have uncovered conserved binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser-Lys catalytic pair.


Assuntos
Proteínas Arqueais/química , Euryarchaeota/enzimologia , Methanobacteriaceae/enzimologia , Methanocaldococcus/enzimologia , Oxirredutases/química , Tioamidas/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Euryarchaeota/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Cinética , Lectina de Ligação a Manose/química , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/metabolismo , Methanobacteriaceae/genética , Methanocaldococcus/genética , Modelos Moleculares , Mutação , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Tioamidas/metabolismo
19.
Plant Cell ; 33(1): 129-152, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33751095

RESUMO

Lignans/neolignans are generally synthesized from coniferyl alcohol (CA) in the cinnamate/monolignol pathway by oxidation to generate the corresponding radicals with subsequent stereoselective dimerization aided by dirigent proteins (DIRs). Genes encoding oxidases and DIRs for neolignan biosynthesis have not been identified previously. In Arabidopsis thaliana, the DIR AtDP1/AtDIR12 plays an essential role in the 8-O-4' coupling in neolignan biosynthesis by unequivocal structural determination of the compound missing in the atdp1 mutant as a sinapoylcholine (SC)-conjugated neolignan, erythro-3-{4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-hydroxymethylethoxy]-3,5-dimethoxyphenyl}acryloylcholine. Phylogenetic analyses showed that AtDP1/AtDIR12 belongs to the DIR-a subfamily composed of DIRs for 8-8' coupling of monolignol radicals. AtDP1/AtDIR12 is specifically expressed in outer integument 1 cells in developing seeds. As a putative oxidase for neolignan biosynthesis, we focused on AtLAC5, a laccase gene coexpressed with AtDP1/AtDIR12. In lac5 mutants, the abundance of feruloylcholine (FC)-conjugated neolignans decreased to a level comparable to those in the atdp1 mutant. In addition, SC/FC-conjugated neolignans were missing in the seeds of mutants defective in SCT/SCPL19, an enzyme that synthesizes SC. These results strongly suggest that AtDP1/AtDIR12 and AtLAC5 are involved in neolignan biosynthesis via SC/FC. A tetrazolium penetration assay showed that seed coat permeability increased in atdp1 mutants, suggesting a protective role of neolignans in A. thaliana seeds.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Lignanas/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Oxirredutases/genética , Oxirredutases/metabolismo
20.
Cell Mol Life Sci ; 78(7): 3691-3707, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33687500

RESUMO

Saccharomyces cerevisiae plays an important role in the heterologous expression of an array of proteins due to its easy manipulation, low requirements and ability for protein post-translational modifications. The implementation of the preproleader secretion signal of the α-factor mating pheromone from this yeast contributes to increase the production yields by targeting the foreign protein to the extracellular environment. The use of this signal peptide combined with enzyme-directed evolution allowed us to achieve the otherwise difficult functional expression of fungal laccases in S. cerevisiae, obtaining different evolved α-factor preproleader sequences that enhance laccase secretion. However, the design of a universal signal peptide to enhance the production of heterologous proteins in S. cerevisiae is a pending challenge. We describe here the optimisation of the α-factor preproleader to improve recombinant enzyme production in S. cerevisiae through two parallel engineering strategies: a bottom-up design over the native α-factor preproleader (αnat) and a top-down design over the fittest evolved signal peptide obtained in our lab (α9H2 leader). The goal was to analyse the effect of mutations accumulated in the signal sequence throughout iterations of directed evolution, or of other reported mutations, and their possible epistatic interactions. Both approaches agreed in the positive synergism of four mutations (Aα9D, Aα20T, Lα42S, Dα83E) contained in the final optimised leader (αOPT), which notably enhanced the secretion of several fungal oxidoreductases and hydrolases. Additionally, we suggest a guideline to further drive the heterologous production of a particular enzyme based on combinatorial saturation mutagenesis of positions 86th and 87th of the αOPT leader fused to the target protein.


Assuntos
Hidrolases/metabolismo , Fator de Acasalamento/metabolismo , Oxirredutases/metabolismo , Precursores de Proteínas/metabolismo , Sinais Direcionadores de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Hidrolases/genética , Fator de Acasalamento/genética , Oxirredutases/genética , Precursores de Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
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