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1.
Gene ; 762: 145104, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32889060

RESUMO

Chalcone synthase (CHS, EC 2.3.1.74) is one of the key and rate-limiting enzymes of phenylpropanoid pathway which plays superior roles in the production of secondary metabolites. In the present study a full-length cDNA of CHS gene was isolated and characterized from Coelogyne ovalis, an orchid of ornamental and medicinal importance. The CHS gene sequence from C. ovalis (CoCHS) was found to be 1445 bp and comprised an open reading frame of 1182 bp, encoding for 394 amino acid residues. Further, the sequence alignment and phylogenetic analysis revealed that CoCHS protein shared high degree of similarity with CHS protein of other orchid species. It also confirmed that it contained all four motifs (I to IV) and signature sequence for the functionality of this gene. Structural modeling of CoCHS based on the crystallographic structure of Freesia hybrida indicated that CoCHS had a similar structure. Quantitative polymerase chain reaction (qPCR) disclosed that CoCHS was expressed in all tissues examined, with the highest transcript being in leaves, followed by pseudobulbs and roots. CoCHS expression was also evaluated in the in vitro-raised plantlets under the abiotic stress (dark, cold, UV-B, wounding, salinity). mRNA transcript expression of CHS gene was found to be positively enhanced and regulated by the different stress types. A correlation between the CoCHS transcript expression with flavonoid and anthocyanin contents revealed that a positive correlation existed between metabolites' content and CoCHS expression within the in vivo as well as in the in vitro-raised plant parts.


Assuntos
Aciltransferases/genética , Regulação da Expressão Gênica de Plantas , Orchidaceae/genética , Proteínas de Plantas/genética , Aciltransferases/química , Aciltransferases/metabolismo , Clonagem Molecular , Orchidaceae/classificação , Orchidaceae/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estresse Fisiológico
2.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(10): 1158-1161, 2020 Oct 10.
Artigo em Chinês | MEDLINE | ID: mdl-32924125

RESUMO

OBJECTIVE: To explore the genetic basis for an infant with congenital generalized lipodystrophy. METHODS: Peripheral blood samples of the child and her parents were collected for the extraction of genomic DNA. All exons and flanking sequences of the AGPAT2 gene were subjected to Sanger sequencing. RESULTS: The child was found to harbor compound heterozygous c.792_805delGGAGAACGGGGCCA (p.Gln264Hisfs*208) and c.335C>T (p.P112L) variants in exons 6 and 3 of the AGPAT2 gene, which were respectively inherited from her mother and father. c.792_805delGGAGAACGGGGCCA (p.Gln264Hisfs*208) was previously unreported, while c.335C>T (p.P112L) was known to be pathogenic. CONCLUSION: The compound heterozygous variants of the AGPAT2 gene probably underlie the disease in this child.


Assuntos
Aciltransferases/genética , Lipodistrofia Generalizada Congênita , Éxons , Feminino , Humanos , Lactente , Lipodistrofia Generalizada Congênita/genética , Mutação
3.
Nature ; 585(7826): 614-619, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879484

RESUMO

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.


Assuntos
Alcaloides/biossíntese , Alcaloides/provisão & distribução , Hiosciamina/biossíntese , Saccharomyces cerevisiae/metabolismo , Escopolamina/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Atropa belladonna/enzimologia , Derivados da Atropina/metabolismo , Transporte Biológico , Datura/enzimologia , Glucosídeos/biossíntese , Glucosídeos/metabolismo , Hiosciamina/provisão & distribução , Lactatos/metabolismo , Ligases/genética , Ligases/metabolismo , Modelos Moleculares , Doenças do Sistema Nervoso/tratamento farmacológico , Oxirredutases/genética , Oxirredutases/metabolismo , Engenharia de Proteínas , Saccharomyces cerevisiae/genética , Escopolamina/provisão & distribução , Vacúolos/metabolismo
4.
Food Chem ; 331: 127365, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32619909

RESUMO

Phenolic acids from Salvia miltiorrhiza have been widely used in nutritious, health-promoting products with an increasing demand. In the current study, two biosynthetic genes RAS (rosmarinic acid synthase) and CYP98A14 (a cytochrome P450-dependent monooxygenase) were successfully introduced into S. miltiorrhiza hairy roots. Overexpression of RAS and CYP98A14 resulted in higher content of phenolic acids (up to over 3-fold) in transgenic lines compared to the control. Meanwhile, DPPH results revealed that engineered S. miltiorrhiza hairy roots had stronger antioxidant activities than the control. In addition, phenolic acid crude extracts of the engineered hairy root lines overexpressing RAS or CYP98A14 showed improved antibacterial activities compared to the control lines. Our work exhibits a useful strategy for enhancement of phenolic acid production and bioactivities of S. miltiorrhiza hairy roots by genetic manipulation of RAS and CYP98A14, and also provides a new resource material to obtain active phenolic acids for food and healthy products.


Assuntos
Aciltransferases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxibenzoatos/metabolismo , Raízes de Plantas/química , Salvia miltiorrhiza/genética , Aciltransferases/genética , Antibacterianos/química , Antibacterianos/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Salmonella/efeitos dos fármacos , Salvia miltiorrhiza/química , Salvia miltiorrhiza/metabolismo
5.
DNA Cell Biol ; 39(9): 1639-1648, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32552000

RESUMO

Colorectal cancer (CRC) patients with KRAS mutation are refractory and usually have poor prognosis. We aimed to identify the hub gene associated with KRAS mutant CRCs. Weighted gene coexpression network analysis (WGCNA) was used to calculate the key module and the hub genes in GSE39582. Combined with the protein-protein interaction (PPI) network and survival analysis, the real hub gene was identified and further validated. With the highest module significance value and correlation coefficient, the blue module was selected as the key module, 19 genes were identified as the hub gene candidates. The above genes were significantly downregulated in KRAS mutant CRCs compared with the wild type. Four genes (AAR2, PSMA7, NELFCD, and PIGU) were further screened as the potential hub genes by the PPI network. Low expression of PIGU for KRAS mutant patients had a poor prognosis. Therefore, PIGU was identified as the hub gene. PIGU expression was also downregulated in other two CRC datasets. "MAPK SIGNALING PATHWAY" was enriched in PIGU lowly expressed samples. PIGU was identified and validated to be closely related to KRAS mutation. It could be a potential prognosis biomarker and a novel treatment target for KRAS mutant CRC patients.


Assuntos
Aciltransferases/genética , Neoplasias Colorretais/genética , Redes Reguladoras de Genes , Proteínas Proto-Oncogênicas p21(ras)/genética , Aciltransferases/metabolismo , Idoso , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação
6.
Hum Genet ; 139(12): 1499-1511, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32533362

RESUMO

Vascular anomalies (VAs), comprising wide subtypes of tumors and malformations, are often caused by variants in multiple tyrosine kinase (TK) receptor signaling pathways including TIE2, PIK3CA and GNAQ/11. Yet, a portion of individuals with clinical features of VA do not have variants in these genes, suggesting that there are undiscovered pathogenic factors underlying these patients and possibly with overlapping phenotypes. Here, we identified one rare non-synonymous variant (c.968A > G) in the seventh exon of GPAA1 (Glycosylphosphatidylinositol Anchor Attachment Protein 1), shared by the four affected members of a large pedigree with multiple types of VA using whole-exome sequencing. GPAA1 encodes a glycosylphosphatidylinositol (GPI) transamidase complex protein. This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum (ER). We showed such variant led to scarce expression of GPAA1 protein in vascular endothelium and induced a localization change from ER membrane to cytoplasm and nucleus. In addition, expressing wild-type GPAA1 in endothelial cells had an effect to inhibit cell proliferation and migration, while expressing variant GPAA1 led to overgrowth and overmigration, indicating a loss of the quiescent status. Finally, a gpaa1-deficient zebrafish model displayed several types of developmental defects as well as vascular dysplasia, demonstrating that GPAA1 is involved in angiogenesis and vascular remodeling. Altogether, our results indicate that the rare coding variant in GPAA1 (c.968A > G) is causally related to familial forms of VAs.


Assuntos
Exoftalmia/genética , Glicoproteínas de Membrana/genética , Complexos Multiproteicos/genética , Malformações Vasculares/genética , Aciltransferases/genética , Adulto , Animais , Sistemas CRISPR-Cas/genética , Movimento Celular/genética , Proliferação de Células/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Éxons/genética , Exoftalmia/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Malformações Vasculares/patologia , Sequenciamento Completo do Exoma , Peixe-Zebra/genética
7.
PLoS One ; 15(4): e0226537, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302307

RESUMO

Enzymes of the chalcone synthase (CHS) family participate in the synthesis of multiple secondary metabolites in plants, fungi and bacteria. CHS showed a significant correlation with the accumulation patterns of anthocyanin. The peel color, which is primarily determined by the content of anthocyanin, is an economically important trait for eggplants that is affected by heat stress. A total of 7 CHS (SmCHS1-7) putative genes were identified in a genome-wide analysis of eggplants (S. melongena L.). The SmCHS genes were distributed on 7 scaffolds and were classified into 3 clusters. Phylogenetic relationship analysis showed that 73 CHS genes from 7 Solanaceae species were classified into 10 groups. SmCHS5, SmCHS6 and SmCHS7 were continuously down-regulated under 38°C and 45°C treatment, while SmCHS4 was up-regulated under 38°C but showed little change at 45°C in peel. Expression profiles of key anthocyanin biosynthesis gene families showed that the PAL, 4CL and AN11 genes were primarily expressed in all five tissues. The CHI, F3H, F3'5'H, DFR, 3GT and bHLH1 genes were expressed in flower and peel. Under heat stress, the expression level of 52 key genes were reduced. In contrast, the expression patterns of eight key genes similar to SmCHS4 were up-regulated at a treatment of 38°C for 3 hour. Comparative analysis of putative CHS protein evolutionary relationships, cis-regulatory elements, and regulatory networks indicated that SmCHS gene family has a conserved gene structure and functional diversification. SmCHS showed two or more expression patterns, these results of this study may facilitate further research to understand the regulatory mechanism governing peel color in eggplants.


Assuntos
Aciltransferases/genética , Flavonoides/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Solanum melongena/genética , Aciltransferases/metabolismo , Antocianinas/genética , Antocianinas/metabolismo , Vias Biossintéticas , Flavonoides/metabolismo , Resposta ao Choque Térmico , Família Multigênica , Filogenia , Proteínas de Plantas/metabolismo , Solanum melongena/fisiologia , Transcriptoma
8.
Artigo em Inglês | MEDLINE | ID: mdl-32244096

RESUMO

Lipoic acid (LA) and its reduced form (dihydrolipoic acid, DHLA) have unique antioxidant properties among such molecules. Moreover, after a process termed lipoylation, LA is an essential prosthetic group covalently-attached to several key multi-subunit enzymatic complexes involved in primary metabolism, including E2 subunits of pyruvate dehydrogenase (PDH). The metabolic pathway of lipoylation has been extensively studied in Escherichia coli and Arabidopsis thaliana in which protein modification occurs via two routes: de novo synthesis and salvage. Common to both pathways, lipoyl synthase (LIP1 in plants, LipA in bacteria, EC 2.8.1.8) inserts sulphur atoms into the molecule in a final, activating step. However, despite the detection of LA and DHLA in other plant species, including tomato (Solanum lycopersicum), no plant LIP1s have been characterised to date from species other than Arabidopsis. In this work, we present the identification and characterisation of two LIPs from tomato, SlLIP1 and SlLIP1p. Consistent with in silico data, both are widely-expressed, particularly in reproductive organs. In line with bioinformatic predictions, we determine that yellow fluorescent protein tagged versions of SlLIP1 and SlLIP1p are mitochondrially- and plastidially-localised, respectively. Both possess the molecular hallmarks and domains of well-characterised bacterial LipAs. When heterologously-expressed in an E. coli lipA mutant, both are capable of complementing specific growth phenotypes and increasing lipoylation levels of E2 subunits of PDH in vivo, demonstrating that they do indeed function as lipoyl synthases.


Assuntos
Aciltransferases , Lipoilação , Lycopersicon esculentum , Mitocôndrias , Plastídeos , Aciltransferases/genética , Aciltransferases/metabolismo , Escherichia coli/genética , Lycopersicon esculentum/enzimologia , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Plastídeos/enzimologia , Ácido Tióctico/metabolismo
9.
Nat Commun ; 11(1): 1266, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152276

RESUMO

Endophilins-A are conserved endocytic adaptors with membrane curvature-sensing and -inducing properties. We show here that, independently of their role in endocytosis, endophilin-A1 and endophilin-A2 regulate exocytosis of neurosecretory vesicles. The number and distribution of neurosecretory vesicles were not changed in chromaffin cells lacking endophilin-A, yet fast capacitance and amperometry measurements revealed reduced exocytosis, smaller vesicle pools and altered fusion kinetics. The levels and distributions of the main exocytic and endocytic factors were unchanged, and slow compensatory endocytosis was not robustly affected. Endophilin-A's role in exocytosis is mediated through its SH3-domain, specifically via a direct interaction with intersectin-1, a coordinator of exocytic and endocytic traffic. Endophilin-A not able to bind intersectin-1, and intersectin-1 not able to bind endophilin-A, resulted in similar exocytic defects in chromaffin cells. Altogether, we report that two endocytic proteins, endophilin-A and intersectin-1, are enriched on neurosecretory vesicles and regulate exocytosis by coordinating neurosecretory vesicle priming and fusion.


Assuntos
Aciltransferases/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Endocitose/fisiologia , Sistemas Neurossecretores/metabolismo , Aciltransferases/genética , Animais , Células Cromafins/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sistemas Neurossecretores/citologia
10.
Nat Commun ; 11(1): 1132, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111831

RESUMO

The promising drug target N-myristoyltransferase (NMT) catalyses an essential protein modification thought to occur exclusively at N-terminal glycines (Gly). Here, we present high-resolution human NMT1 structures co-crystallised with reactive cognate lipid and peptide substrates, revealing high-resolution snapshots of the entire catalytic mechanism from the initial to final reaction states. Structural comparisons, together with biochemical analysis, provide unforeseen details about how NMT1 reaches a catalytically competent conformation in which the reactive groups are brought into close proximity to enable catalysis. We demonstrate that this mechanism further supports efficient and unprecedented myristoylation of an N-terminal lysine side chain, providing evidence that NMT acts both as N-terminal-lysine and glycine myristoyltransferase.


Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Glicina/metabolismo , Lisina/metabolismo , Aciltransferases/genética , Catálise , Domínio Catalítico , Coenzima A/química , Coenzima A/genética , Coenzima A/metabolismo , Cristalografia por Raios X , Humanos , Cinética , Mutação , Ácido Mirístico/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
11.
Food Chem ; 318: 126483, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32126468

RESUMO

In this study, the antioxidant activity of germinating Chinese wild rice was found to decline initially, after which it increased. The largest difference in antioxidant activity was observed between the 36-h (G36) and the 120-h germination (G120) stage. We further assessed the dynamic changes in metabolites, phenolic acids, flavonoids, and phenolic biosynthetic genes in germinating Chinese wild rice. Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry revealed that 315 metabolites were up-regulated and 28 were down-regulated between G36 and G120. Levels of p-hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillin, p-coumaric acid, ferulic acid, and epigallocatechin increased significantly during germination. Gene expression of four phenylalanine ammonia-lyases, one 4-coumarate-CoA ligase, one cinnamoyl-CoA reductase, two cinnamyl alcohol dehydrogenases, one chalcone synthase, and one chalcone isomerase was significantly higher at G120 than at G36 and promoted phenolics accumulation. This study elucidated the biochemical mechanisms involved in antioxidant activity and phenolic profile changes during Chinese wild rice germination.


Assuntos
Antioxidantes/metabolismo , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Fenóis/metabolismo , Proteínas de Plantas/genética , Poaceae/fisiologia , Aciltransferases/genética , Cromatografia Líquida de Alta Pressão , Coenzima A Ligases/genética , Germinação , Hidroxibenzoatos/metabolismo , Liases Intramoleculares/genética , Espectrometria de Massas , Oxirredutases/genética , Fenilalanina Amônia-Liase/genética , Poaceae/química , Poaceae/genética , Sementes/química , Sementes/genética , Sementes/fisiologia
12.
Am J Hum Genet ; 106(4): 484-495, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32220290

RESUMO

Glycosylphosphatidylinositol (GPI)-anchored proteins are critical for embryogenesis, neurogenesis, and cell signaling. Variants in several genes participating in GPI biosynthesis and processing lead to decreased cell surface presence of GPI-anchored proteins (GPI-APs) and cause inherited GPI deficiency disorders (IGDs). In this report, we describe 12 individuals from nine unrelated families with 10 different bi-allelic PIGK variants. PIGK encodes a component of the GPI transamidase complex, which attaches the GPI anchor to proteins. Clinical features found in most individuals include global developmental delay and/or intellectual disability, hypotonia, cerebellar ataxia, cerebellar atrophy, and facial dysmorphisms. The majority of the individuals have epilepsy. Two individuals have slightly decreased levels of serum alkaline phosphatase, while eight do not. Flow cytometric analysis of blood and fibroblasts from affected individuals showed decreased cell surface presence of GPI-APs. The overexpression of wild-type (WT) PIGK in fibroblasts rescued the levels of cell surface GPI-APs. In a knockout cell line, transfection with WT PIGK also rescued the GPI-AP levels, but transfection with the two tested mutant variants did not. Our study not only expands the clinical and known genetic spectrum of IGDs, but it also expands the genetic differential diagnosis for cerebellar atrophy. Given the fact that cerebellar atrophy is seen in other IGDs, flow cytometry for GPI-APs should be considered in the work-ups of individuals presenting this feature.


Assuntos
Aciltransferases/genética , Moléculas de Adesão Celular/genética , Doenças Cerebelares/genética , Epilepsia/genética , Variação Genética/genética , Hipotonia Muscular/genética , Transtornos do Neurodesenvolvimento/genética , Anormalidades Múltiplas/genética , Alelos , Feminino , Humanos , Deficiência Intelectual/genética , Masculino , Malformações do Sistema Nervoso/genética , Linhagem , Síndrome
13.
Parasit Vectors ; 13(1): 132, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32171322

RESUMO

BACKGROUND: Proving that specific genes are essential for the intracellular viability of Leishmania parasites within macrophages remains a challenge for the identification of suitable targets for drug development. This is especially evident in the absence of a robust inducible expression system or functioning RNAi machinery that works in all Leishmania species. Currently, if a target gene of interest in extracellular parasites can only be deleted from its genomic locus in the presence of ectopic expression from a wild type copy, it is assumed that this gene will also be essential for viability in disease-promoting intracellular parasites. However, functional essentiality must be proven independently in both life-cycle stages for robust validation of the gene of interest as a putative target for chemical intervention. METHODS: Here, we have used plasmid shuffle methods in vivo to provide supportive genetic evidence that N-myristoyltransferase (NMT) is essential for Leishmania viability throughout the parasite life-cycle. Following confirmation of NMT essentiality in vector-transmitted promastigotes, a range of mutant parasites were used to infect mice prior to negative selection pressure to test the hypothesis that NMT is also essential for parasite viability in an established infection. RESULTS: Ectopically-expressed NMT was only dispensable under negative selection in the presence of another copy. Total parasite burdens in animals subjected to negative selection were comparable to control groups only if an additional NMT copy, not affected by the negative selection, was expressed. CONCLUSIONS: NMT is an essential gene in all parasite life-cycle stages, confirming its role as a genetically-validated target for drug development.


Assuntos
Aciltransferases/genética , Genes Essenciais , Leishmania/genética , Leishmania/fisiologia , Estágios do Ciclo de Vida/genética , Proteínas de Protozoários/genética , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Genoma de Protozoário , Leishmania donovani/genética , Leishmania donovani/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Transcriptoma
14.
J Biotechnol ; 313: 18-28, 2020 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-32171790

RESUMO

The polyhydroxyalkanoate (PHA) producing capability of four bacterial strains isolated from Antarctica was reported in a previous study. This study analyzed the PHA synthase genes and the PHA-associated gene clusters from the two antarctic Pseudomonas isolates (UMAB-08 and UMAB-40) and the two antarctic Janthinobacterium isolates (UMAB-56 and UMAB-60) through whole-genome sequence analysis. The Pseudomonas isolates were found to carry PHA synthase genes which fall into two different PHA gene clusters, namely Class I and Class II, which are involved in the biosynthesis of short-chain-length-PHA (SCL-PHA) and medium-chain-length-PHA (MCL-PHA), respectively. On the other hand, the Janthinobacterium isolates carry a Class I and an uncharacterized putative PHA synthase genes. No other gene involved in PHA synthesis was detected in close proximity to the uncharacterized putative PHA synthase gene in the Janthinobacterium isolates, therefore it falls into a separate clade from the ordinary Class I, II, III and IV clades of PHA synthase (PhaC) phylogenetic tree. Multiple sequence alignment showed that the uncharacterized putative PHA synthase gene contains all the highly conserved amino acid residues and the proposed catalytic triad of PHA synthase. PHA biosynthesis and in vitro PhaC enzymatic assay results showed that this uncharacterized putative PHA synthase from Janthinobacterium sp. UMAB-60 is funtional. This report adds new knowledge to the PHA synthase database as we describe scarce information of PHA synthase genes and PHA-associated gene clusters from the antarctic bacterial isolates (extreme and geographically isolated environment) and comparing with those from non-antarctic PHA-producing bacteria.


Assuntos
Aciltransferases/genética , Genoma Bacteriano/genética , Família Multigênica , Oxalobacteraceae/enzimologia , Poli-Hidroxialcanoatos/metabolismo , Pseudomonas/enzimologia , Regiões Antárticas , Oxalobacteraceae/genética , Filogenia , Pseudomonas/genética , Alinhamento de Sequência , Sequenciamento Completo do Genoma
15.
Microb Cell Fact ; 19(1): 73, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32197639

RESUMO

BACKGROUND: Acridone alkaloids are heterocyclic compounds that exhibit a broad-range of pharmaceutical and chemotherapeutic activities, including anticancer, antiviral, anti-inflammatory, antimalarial, and antimicrobial effects. Certain plant species such as Citrus microcarpa, Ruta graveolens, and Toddaliopsis bremekampii synthesize acridone alkaloids from anthranilate and malonyl-CoA. RESULTS: We synthesized two acridones in Escherichia coli. Acridone synthase (ACS) and anthraniloyl-CoA ligase genes were transformed into E. coli, and the synthesis of acridone was examined. To increase the levels of endogenous anthranilate, we tested several constructs expressing proteins involved in the shikimate pathway and selected the best construct. To boost the supply of malonyl-CoA, genes coding for acetyl-coenzyme A carboxylase (ACC) from Photorhabdus luminescens were overexpressed in E. coli. For the synthesis of 1,3-dihydroxy-10-methylacridone, we utilized an N-methyltransferase gene (NMT) to supply N-methylanthranilate and a new N-methylanthraniloyl-CoA ligase. After selecting the best combination of genes, approximately 17.3 mg/L of 1,3-dihydroxy-9(10H)-acridone (DHA) and 26.0 mg/L of 1,3-dihydroxy-10-methylacridone (NMA) were synthesized. CONCLUSIONS: Two bioactive acridone derivatives were synthesized by expressing type III plant polyketide synthases and other genes in E. coli, which increased the supplement of substrates. This study showed that is possible to synthesize diverse polyketides in E. coli using plant polyketide synthases.


Assuntos
Acridonas/metabolismo , Escherichia coli , Aciltransferases/genética , Proteínas de Bactérias/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Microrganismos Geneticamente Modificados/metabolismo , Photorhabdus/enzimologia , Proteínas de Plantas/genética , Policetídeo Sintases/genética , Proteínas Recombinantes/genética
16.
Sci Rep ; 10(1): 3749, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111914

RESUMO

Lipoyl synthases are key enzymes in lipoic acid biosynthesis, a co-factor of several enzyme complexes involved in central metabolism. Plant pyruvate dehydrogenase complex (PDH), located in mitochondria and plastids, catalyses the first step of fatty acid biosynthesis in these organelles. Among their different components, the E2 subunit requires the lipoic acid prosthetic group to be active. De novo lipoic acid biosynthesis is achieved by the successive action of two enzymes on octanoyl-ACP: octanoyltransferase (LIP2) and lipoyl synthase (LIP1). In this study, two plastidial lipoyl synthase genes from sunflower (Helianthus annuus L.) were identified (HaLIP1p1 and HaLIP1p2), sequenced and cloned in a heterologous production system (Escherichia coli). Gene expression studies revealed similar expression patterns for both isoforms, with a slight predominance of HaLIP1p1 in vegetative tissues and mature seeds. Tertiary structural models for these enzymes indicate they both have the same theoretical catalytic sites, using lipoyl-lys and 5-deoxyadenosine as docking substrates. The fatty acid profile of E. coli cells overexpressing HaLIP1p1 and HaLIP1p2 did not present major differences, and the in vivo activity of both proteins was confirmed by complementation of an E. coli JW0623 mutant in which lipoyl synthase is defective. Although no significant differences were detected in the total fatty acid composition of transgenic Arabidopsis thaliana seeds overexpressing any of both proteins, a lipidomic analysis revealed a redistribution of the glycerolipid species, accompanied with increased phosphatidylethanolamine (PE) content and a decrease in diacyglycerols (DAG) and phosphatidylcholine (PC). Depletion of the SAM co-factor caused by HaLIP1p1 and HaLIP1p2 overexpression in transgenic plants could explain this remodelling through its effects on PC synthesis.


Assuntos
Aciltransferases , Arabidopsis , Ácidos Graxos , Helianthus/genética , Proteínas de Plantas , Plantas Geneticamente Modificadas , Sulfurtransferases , Aciltransferases/biossíntese , Aciltransferases/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/genética , Helianthus/enzimologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/genética , Sementes/metabolismo , Sulfurtransferases/biossíntese , Sulfurtransferases/genética
17.
Am J Pathol ; 190(5): 1059-1067, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32084365

RESUMO

Mutations in retinoid isomerase (RPE65) or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA). Despite the success of recent RPE65 gene therapy, follow-up studies show that patients continue to experience photoreceptor degeneration and lose vision benefit over time. In Lrat-/- mouse model, mislocalized medium (M)-wavelength opsin was degraded, whereas mislocalized short (S)-wavelength opsin accumulated before the onset of cone degeneration. The mechanism for the foveal M/long-wavelength cone photoreceptor degeneration in LCA is unknown. By crossing Lrat-/- mice with a proteasome reporter mouse strain, this study showed that M-opsin-enriched dorsal cones in Lrat-/- mice exhibit proteasome stress because of the degradation of large amounts of M-opsin. Deletion of M-opsin relieves the proteasome stress and completely prevents M cone degeneration in Lrat-/-Opn1sw-/- mice (a pure M cone LCA model, Opn1sw encoding S-opsin) for at least 12 months. These results suggest that M-opsin degradation-associated proteasome stress plays a major role in M cone degeneration in Lrat-/- model. This finding may represent a general mechanism for M cone degeneration in multiple forms of cone degeneration because of M-opsin mislocalization and degradation. These results have important implications for the current gene therapy strategy for LCA that emphasizes the need for combinatorial therapies to both improve vision and slow photoreceptor degeneration.


Assuntos
Opsinas dos Cones/metabolismo , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/patologia , Degeneração Neural/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Aciltransferases/deficiência , Aciltransferases/genética , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Células Fotorreceptoras Retinianas Cones/metabolismo
18.
Nat Commun ; 11(1): 870, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054839

RESUMO

Land plants produce diverse flavonoids for growth, survival, and reproduction. Chalcone synthase is the first committed enzyme of the flavonoid biosynthetic pathway and catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC). However, it also produces other polyketides, including p-coumaroyltriacetic acid lactone (CTAL), because of the derailment of the chalcone-producing pathway. This promiscuity of CHS catalysis adversely affects the efficiency of flavonoid biosynthesis, although it is also believed to have led to the evolution of stilbene synthase and p-coumaroyltriacetic acid synthase. In this study, we establish that chalcone isomerase-like proteins (CHILs), which are encoded by genes that are ubiquitous in land plant genomes, bind to CHS to enhance THC production and decrease CTAL formation, thereby rectifying the promiscuous CHS catalysis. This CHIL function has been confirmed in diverse land plant species, and represents a conserved strategy facilitating the efficient influx of substrates from the phenylpropanoid pathway to the flavonoid pathway.


Assuntos
Aciltransferases/metabolismo , Embriófitas/metabolismo , Liases Intramoleculares/metabolismo , Proteínas de Plantas/metabolismo , Aciltransferases/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Biocatálise , Vias Biossintéticas/genética , Chalconas/biossíntese , Embriófitas/genética , Evolução Molecular , Flavonoides/biossíntese , Genes de Plantas , Teste de Complementação Genética , Liases Intramoleculares/genética , Cinética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Policetídeos/metabolismo , Especificidade por Substrato
19.
Nat Commun ; 11(1): 860, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054864

RESUMO

Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with each other in the mammalian plasma membranes, forming dynamic microdomains. How their interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi apparatus, we report that ß1,3-galactosyltransferase 4 (B3GALT4), the previously characterized GM1 ganglioside synthase, additionally functions in transferring galactose to the N-acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously unexpected functional relationships between GPI-anchored proteins and glycosphingolipids in the Golgi. Through the same screening, we also show that GPI biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated degradation to prevent GPI accumulation when the transfer of synthesized GPI to proteins is defective. Our data demonstrates cross-talks of GPI biosynthesis with glycosphingolipid biosynthesis and the ER quality control system.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Glicoesfingolipídeos/biossíntese , Glicosilfosfatidilinositóis/biossíntese , Aciltransferases/deficiência , Aciltransferases/genética , Aciltransferases/metabolismo , Sistemas CRISPR-Cas , Degradação Associada com o Retículo Endoplasmático/genética , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Técnicas de Inativação de Genes , Glicoesfingolipídeos/genética , Glicosilfosfatidilinositóis/genética , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(8): 4109-4116, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32041866

RESUMO

The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is an essential lipid A biosynthetic enzyme that is conserved in the majority of gram-negative bacteria. It has emerged as an attractive novel antibiotic target due to the recent discovery of an LpxH-targeting sulfonyl piperazine compound (referred to as AZ1) by AstraZeneca. However, the molecular details of AZ1 inhibition have remained unresolved, stymieing further development of this class of antibiotics. Here we report the crystal structure of Klebsiella pneumoniae LpxH in complex with AZ1. We show that AZ1 fits snugly into the L-shaped acyl chain-binding chamber of LpxH with its indoline ring situating adjacent to the active site, its sulfonyl group adopting a sharp kink, and its N-CF3-phenyl substituted piperazine group reaching out to the far side of the LpxH acyl chain-binding chamber. Intriguingly, despite the observation of a single AZ1 conformation in the crystal structure, our solution NMR investigation has revealed the presence of a second ligand conformation invisible in the crystalline state. Together, these distinct ligand conformations delineate a cryptic inhibitor envelope that expands the observed footprint of AZ1 in the LpxH-bound crystal structure and enables the design of AZ1 analogs with enhanced potency in enzymatic assays. These designed compounds display striking improvement in antibiotic activity over AZ1 against wild-type K. pneumoniae, and coadministration with outer membrane permeability enhancers profoundly sensitizes Escherichia coli to designed LpxH inhibitors. Remarkably, none of the sulfonyl piperazine compounds occupies the active site of LpxH, foretelling a straightforward path for rapid optimization of this class of antibiotics.


Assuntos
Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/metabolismo , Aciltransferases/genética , Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Metabolismo dos Lipídeos , Testes de Sensibilidade Microbiana , Mutação , Piperazinas/química , Piperazinas/farmacologia , Conformação Proteica , Pirofosfatases/genética
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