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1.
J Inorg Biochem ; 240: 112085, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36640554

RESUMO

Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni2+-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17α-hydroxylation and 17,20-lyase reactions were studied using ultra-performance liquid chromatography (UPLC)-mass spectrometry analysis. Mouse Cyp17a1 showed cytochrome b5 stimulation in catalysis. In comparison to human enzyme, much higher specificity constants (kcat/Km) were observed with mouse Cyp17a1. In the reactions of Δ4-steroids (progesterone and 17α-OH progesterone), the specificity constants were 2100 times higher than the human enzyme. The addition of cytochrome b5 produced significant stimulation of 17,20-lyase activities of mouse Cyp17a1. Two Arg mutants of mouse Cyp17a1 (R347H and R358Q) displayed a larger decrease in 17,20-lyase reaction (from 17α-OH pregnenolone to dehydroepiandrosterone, DHEA) than 17α-hydroxylation, indicating that -as in human CYP17A1-these basic residues in mouse Cyp17a1 are important in interacting with the cytochrome b5 protein in the lyase reactions.


Assuntos
Liases , Progesterona , Humanos , Camundongos , Animais , Progesterona/química , Progesterona/metabolismo , Esteroide 17-alfa-Hidroxilase/química , Liases/metabolismo , Citocromos b/metabolismo , Hidroxilação , Esteroides , Pregnenolona/química , Pregnenolona/metabolismo , Catálise
2.
Proc Natl Acad Sci U S A ; 120(4): e2212246120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36652470

RESUMO

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived ß-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.


Assuntos
Liases , Lignina/metabolismo , Proteínas de Bactérias/metabolismo , Oxirredutases/metabolismo , Estereoisomerismo
3.
Int J Biol Macromol ; 226: 608-617, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36521700

RESUMO

Vanillin (3-methoxy-4-hydroxybenzaldehyde) is one of the most important flavoring substances used in the cosmetic and food industries. Feruloyl-CoA hydratase/lyase (FCHL) is an enzyme that catalyzes the production of vanillin from feruloyl-CoA. In this study, we report kinetic parameters and biochemical properties of FCHL from Sphingomonas paucimobilis SYK-6 (SpFCHL). Also, the crystal structures of an apo-form of SpFCHL and two complexed forms with acetyl-CoA and vanillin/CoA was present. Comparing the apo structure to its complexed forms of SpFCHL, a gate loop with an "open and closed" role was observed at the entrance of the substrate-binding site. With vanillin and CoA complexed to SpFCHL, we captured a conformational change in the feruloyl moiety-binding pocket that repositions the catalytic SpFCHLE146 and other key residues. This binding pocket does not tightly fit the vanillin structure, suggesting substrate promiscuity of this enzyme. This observation is in good agreement with assay results for phenylpropanoid-CoAs and indicates important physicochemical properties of the substrate for the hydratase/lyase reaction mechanism. In addition, we showed that various phenolic aldehydes could be produced using the 4CL-FCHL biosynthesis platform.


Assuntos
Liases , Aldeídos , Acil Coenzima A/química
4.
Proc Natl Acad Sci U S A ; 119(43): e2207955119, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36215519

RESUMO

Oxygen plays a key role in supporting life on our planet. It is particularly important in higher eukaryotes where it boosts bioenergetics as a thermodynamically favorable terminal electron acceptor and has important roles in cell signaling and development. Many human diseases stem from either insufficient or excessive oxygen. Despite its fundamental importance, we lack methods with which to manipulate the supply of oxygen with high spatiotemporal resolution in cells and in organisms. Here, we introduce a genetic system, SupplemeNtal Oxygen Released from ChLorite (SNORCL), for on-demand local generation of molecular oxygen in living cells, by harnessing prokaryotic chlorite O2-lyase (Cld) enzymes that convert chlorite (ClO2-) into molecular oxygen (O2) and chloride (Cl-). We show that active Cld enzymes can be targeted to either the cytosol or mitochondria of human cells, and that coexpressing a chlorite transporter results in molecular oxygen production inside cells in response to externally added chlorite. This first-generation system allows fine temporal and spatial control of oxygen production, with immediate research applications. In the future, we anticipate that technologies based on SNORCL will have additional widespread applications in research, biotechnology, and medicine.


Assuntos
Cloretos , Liases , Humanos , Oxirredutases/genética , Oxigênio
5.
Front Endocrinol (Lausanne) ; 13: 970190, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36187111

RESUMO

Background: 17α-hydroxylase/17, 20-lyase deficiency (17-OHD) is caused by the mutations of the CYP17A1 gene. The classical phenotype of 17-OHD includes hypertension, hypokalemia, and abnormal sexual development, with partial 17-OHD typically less severe than the complete deficiency. Infertility is always one of the main clinical manifestations of partial 17-OHD. However, to date, the pregnancy potentials of partial 17-OHD female patients have rarely been investigated, and few live-birth cases have been reported among them. Moreover, the reproductive endocrine characteristics of partial 17-OHD female patients have not been completely clarified and the treatment skills of in vitro fertilization and embryo transfer (IVF-ET) have not been well summarized yet. Methods: Two Chinese infertile female patients clinically diagnosed as partial 17-OHD were enrolled and their pedigree investigations were performed. Hormones were determined to depict the endocrine conditions of partial 17-OHD female patients. The adrenocorticotropic hormone (ACTH) stimulation test was performed to evaluate the functions of the adrenal cortex. Genotype analysis was conducted by next-generation sequencing (NGS) and Sanger sequencing was used to verify the results. IVF-ET was performed for the treatment of their infertility. Specifically, the progestin-primed ovarian stimulation (PPOS) protocol was chosen for the controlled ovarian hyperstimulation (COH) cycles, and the hormone replacement treatment (HRT) protocol was adopted for the endometrial preparation in frozen-thawed embryo transfer (FET) cycles. Results: Hormone assays revealed a reduced estradiol (E2) and testosterone (T) level, and an elevated progesterone (P4) level. The classic ACTH stimulating test evidenced a suboptimal response of cortisol to ACTH. Genotype analysis demonstrated that the proband1 carried two variants: c.1459_1467del (p.Asp487_Phe489del)het and c.995T>C (p.lle332Thr)het. The proband2 was found to be a homozygote with the mutation of c.1358T>A (p.Phe453Ser)hom. The two female patients both succeeded in pregnancy and delivery of healthy babies through IVF-ET, with the usage of PPOS, HRT, and low-dose glucocorticoids. Conclusions: Partial 17-OHD female patients manifested menstrual cycle disorders and infertility clinically; displayed high P4 and low E2 and T; showed sparse pubic hair in physical examinations; and revealed multiple ovarian cysts in ultrasonic visualization. Moreover, the pregnancy potentials of infertile partial 17-OHD women seemed to increase with the adoption of IVF-ET. Considering the sustained elevated P4 level, PPOS is a feasible protocol for them in COH.


Assuntos
Infertilidade Feminina , Liases , Síndrome de Hiperestimulação Ovariana , Hormônio Adrenocorticotrópico , Estradiol , Feminino , Fertilização In Vitro/métodos , Humanos , Hidrocortisona , Infertilidade Feminina/genética , Infertilidade Feminina/terapia , Oxigenases de Função Mista , Linhagem , Gravidez , Progesterona , Progestinas , Testosterona
6.
Womens Health (Lond) ; 18: 17455057221122597, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36129002

RESUMO

Here, we reported a case of a 16-year-old Chinese female patient (46, XX) diagnosed as 17α-hydroxylase/17, 20-lyase deficiency (17-OHD) in June 2018 and over 3 years follow-up outcomes; 17-OHD is a rare form of congenital adrenal hyperplasia. The patient presented with primary amenorrhea, underdeveloped secondary sexual characteristics, hypertension and hypokalemia. Hormonal findings revealed decreased estrogen and androgen, increased progesterone, low cortisol concentration and compensatory high adrenocorticotropic hormone level. Mutation analysis of the CYP17A1 gene identified the c.1459_1467del GACTCTTTC homozygous deletion in exon 8, namely, D487_F489del mutation, resulting in the deletion of Aspartate-Serine-Phenylalanine amino acids. The patient's father and mother were all heterozygous carriers of this mutation. The diagnosis and follow-up outcomes provided useful insights to support clinical decision-making and appropriate treatment.


Assuntos
Liases , Esteroide 17-alfa-Hidroxilase , Adolescente , Hormônio Adrenocorticotrópico/genética , Androgênios , Ácido Aspártico/genética , Estrogênios , Feminino , Seguimentos , Homozigoto , Humanos , Hidrocortisona , Liases/genética , Oxigenases de Função Mista/genética , Fenilalanina/genética , Progesterona , Deleção de Sequência , Serina/genética , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo
7.
Molecules ; 27(18)2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36144762

RESUMO

Chondroitin sulfate (CS) and dermatan sulfate (DS) are found in nature linked to proteoglycans, most often as hybrid CS/DS chains. In the extracellular matrix, where they are highly expressed, CS/DS are involved in fundamental processes and various pathologies. The structural diversity of CS/DS domains gave rise to efforts for the development of efficient analytical methods, among which is mass spectrometry (MS), one of the most resourceful techniques for the identification of novel species and their structure elucidation. In this context, we report here on the introduction of a fast, sensitive, and reliable approach based on ion mobility separation (IMS) MS and MS/MS by collision-induced dissociation (CID), for the profiling and structural analysis of CS/DS hexasaccharide domains in human embryonic kidney HEK293 cells decorin (DCN), obtained after CS/DS chain releasing by ß-elimination, depolymerization using chondroitin AC I lyase, and fractionation by size-exclusion chromatography. By IMS MS, we were able to find novel CS/DS species, i.e., under- and oversulfated hexasaccharide domains in the released CS/DS chain. In the last stage of analysis, the optimized IMS CID MS/MS provided a series of diagnostic fragment ions crucial for the characterization of the misregulations, which occurred in the sulfation code of the trisulfated-4,5-Δ-GlcAGalNAc[IdoAGalNAc]2 sequence, due to the unusual sulfation sites.


Assuntos
Sulfatos de Condroitina , Liases , Sulfatos de Condroitina/química , Decorina , Dermatan Sulfato/química , Células HEK293 , Humanos , Proteoglicanas/química , Espectrometria de Massas em Tandem/métodos
8.
Chemosphere ; 309(Pt 1): 136535, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36150484

RESUMO

The biogas production (BP), volatile fatty acids (VFAs), microbial communities, and microbes' active enzymes were studied upon the addition of biochar (0-1.5%) at 6% and 8% slaughterhouse waste (SHW) loadings. The 0.5% biochar enhanced BP by 1.5- and 1.6-folds in 6% and 8% SHW-loaded reactors, respectively. Increasing the biochar up to 1.5% caused a reduction in BP at 6% SHW. However, the BP from 8% of SHW was enhanced by 1.4-folds at 1.5% biochar. The VFAs production in all 0.5% biochar amended reactors was highly significant compared to control (p-value < 0.05). The biochar addition increased the bacterial and archaeal diversity at both 6% and 8% SHW loadings. The highest number of OTUs at 0.5% biochar were 567 and 525 in 6% and 8% SHW, respectively. Biochar prompted the Clostridium abundance and increased the lyases and transaminases involved in the degradation of lipids and protein, respectively. Biochar addition improved the Methanosaeta and Methanosphaera abundance in which the major enzymes were reductase and hydrogenase. The archaeal enzymes showed mixed acetoclastic and hydrogenotrophic methanogenesis.


Assuntos
Hidrogenase , Liases , Microbiota , Archaea/metabolismo , Biocombustíveis , Anaerobiose , Reatores Biológicos , Matadouros , Metano/metabolismo , Hidrogenase/metabolismo , Bactérias/metabolismo , Ácidos Graxos Voláteis/metabolismo , Liases/metabolismo , Transaminases , Digestão
9.
Microbiol Spectr ; 10(5): e0249622, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36106896

RESUMO

The opportunistic pathogen Streptococcus pneumoniae (pneumococcus) is a human nasopharyngeal commensal, and host N-glycan metabolism promotes its colonization and invasion. It has been reported that glucose represses, while fetuin, a glycoconjugated model protein, induces, the genes involved in N-glycan degradation through the two-component system TCS07. However, the mechanisms of glucose repression and TCS07 induction remain unknown. Previously, we found that the pneumococcal aquaglyceroporin Pn-AqpC facilitates oxygen uptake, thereby contributing to the antioxidant potential and virulence. In this study, through Tandem Mass Tag (TMT) quantitative proteomics, we found that the deletion of Pn-aqpC caused a marked upregulation of 11 proteins involved in N-glycan degradation in glucose-grown pneumococcus R6. Both quantitative RT-PCR and GFP fluorescence reporters revealed that the upregulation of N-glycan genes was completely dependent on response regulator (RR) 07, but not on the histidine kinase HK07 of TCS07 or the phosphoryl-receiving aspartate residue of RR07 in ΔPn-aqpC, indicating that RR07 was activated in an HK07-independent manner when Pn-AqpC was absent. The deletion of Pn-aqpC also enhanced the expression of pyruvate formate lyase and increased formate production, probably due to reduced cellular oxygen content, indicating that a shunt of glucose catabolism to mixed acid fermentation occurs. Notably, formate induced the N-glycan degradation genes in glucose-grown R6, but the deletion of rr07 abolished this induction, indicating that formate activates RR07. However, the induction of N-glycan degradation proteins reduced the intraspecies competition of R6 in glucose. Therefore, although N-glycan degradation promotes pneumococcal pathogenesis, the glucose metabolites-based RR07 regulation reported here is of importance for balancing growth fitness and the pathogenicity of pneumococcus. IMPORTANCE Pneumococcus, a human opportunistic pathogen, is capable of metabolizing host complex N-glycans. N-glycan degradation promotes pneumococcus colonization in the nasopharynx as well as invasion into deeper tissues, thus significantly contributing to pathogenesis. It is known that the two-component system 07 induces the N-glycan metabolizing genes; however, how TCS07 is activated remains unknown. This study reveals that formate, the anaerobic fermentation metabolite of pneumococcus, is a novel activator of the response regulator (RR) 07. Although the high expression of N-glycan degradation genes promotes pneumococcal colonization in the nasopharynx and pathogenesis, this reduces pneumococcal growth fitness in glucose as indicated in this work. Notably, the presence of Pn-AqpC, an oxygen-transporting aquaglyceroporin, enables pneumococcus to maintain glucose homolactic acid fermentation, thus reducing formate production, maintaining RR07 inactivation, and controlling N-glycan degrading genes at a non-induced status. Thus, this study highlights a novel fermentation metabolism pattern linking TCS-regulated carbohydrate utilization strategies as a trade-off between the fitness and the pathogenicity of pneumococcus.


Assuntos
Aquagliceroporinas , Liases , Humanos , Streptococcus pneumoniae/metabolismo , Fermentação , Histidina Quinase/metabolismo , Ácido Aspártico/metabolismo , Antioxidantes/metabolismo , Polissacarídeos , Formiatos/metabolismo , Glucose/metabolismo , Fetuínas/metabolismo , Aquagliceroporinas/metabolismo , Piruvatos/metabolismo , Liases/metabolismo , Oxigênio/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
10.
PLoS One ; 17(9): e0272904, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36112631

RESUMO

Advanced periodontitis has been shown to have strong association with the residence of the bacterial consortia known as the red complex comprised by Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. T. forsythia shares a distant genetic linkage to Bacteroidetes thetaiotaomicron and may therefore produce analogous polysaccharide utilization loci (PUL) which enable complex carbohydrate degradation, import, and use, although this capacity has yet to be demonstrated. Chondroitin sulfate A is a linear, sulfated carbohydrate linked to periodontal disease as the principal species of glycosaminoglycan appended on the surface of cortical bone of teeth and in supporting dental ligaments. Through genomic comparisons with B. thetaiotaomicron, a new PUL-like operon (Bfo2285-Bfo2295, and Bfo3043) was identified in T. forsythia and the crystal structure of two proteins from this PUL-like operon, Bfo2290 and Bfo2294, were reported using X-ray crystallography. Enzyme kinetics for Bfo2290 were reported using a pH-dependent assay and suggested a Km of 0.75 mg/ml ± 0.60 mg/ml, Kcat of 3.74 min-1 ± 0.88 min-1, and Vmax of 7.48 µM/min ± 1.76 µM/min with partially degraded chondroitin sulfate A. Fluorophore-assisted carbohydrate electrophoresis was used to show the processive degradation of chondroitin sulfate A by the proteins encoded in T. forsythia PUL-like operon, and revealed Bfo2291 and Bfo2290 to be an endolytic chondroitin sulfate A lyase and exolytic ΔDi-4S chondroitin sulfate A sulfatase, respectively.


Assuntos
Liases , Tannerella forsythia , Sulfatos de Condroitina/metabolismo , Óperon/genética , Sulfatases , Tannerella forsythia/genética , Tannerella forsythia/metabolismo
11.
Mol Hum Reprod ; 28(10)2022 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-36069625

RESUMO

Follicles are the functional unit of the ovary and several methods have been developed to grow follicles ex vivo, which recapitulate key events of oogenesis and folliculogenesis. Enzymatic digestion protocols are often used to increase the yield of follicles from the ovary. However, the impact of these protocols on the outermost theca and granulosa cells, and thereby follicle function, is not well defined. To investigate the impact of enzymatic digestion on follicle function, we collected preantral follicles from CD1 mice either by enzymatic digestion (Enzy-FL) or mechanical isolation (Mech-FL) and compared follicle growth, steroidogenesis and cell differentiation within an encapsulated in vitro follicle growth system which maintains the 3D architecture of the oocyte and its surrounding somatic cells. Follicles were encapsulated in 0.5% alginate and cultured for 8 days. Compared with Enzy-FL, Mech-FL grew more rapidly and produced significantly higher levels of androstenedione, estradiol and progesterone. The expression of theca-interstitial cell marker genes, Cyp17a1, which encodes 17-hydroxylase/17, 20-lyase and catalyzes the hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, and the conversion of these products into dehydroepiandrosterone and androstenedione, and Star, which encodes a transport protein essential for cholesterol entry into mitochondria, were also higher in Mech-FL than in Enzy-FL. Mech-FL maintained an intact theca-interstitial layer on the outer edge of the follicle that phenocopied in vivo patterns as confirmed by alkaline phosphatase staining, whereas theca-interstitial cells were absent from Enzy-FL from the onset of culture. Therefore, preservation of the theca cell layer at the onset of culture better supports follicle growth and function. Interestingly, granulosa cells in the outermost layers of Enzy-FL expressed CYP17A1 by Day 4 of culture while maintaining inhibin α-subunit expression and a cuboidal nucleus. Thus, in the absence of theca-interstitial cells, granulosa cells have the potential to differentiate into androgen-producing cells. This work may have implications for human follicle culture, where enzymatic isolation is required owing to the density of the ovarian cortex.


Assuntos
Liases , Progesterona , 17-alfa-Hidroxipregnenolona/metabolismo , 17-alfa-Hidroxiprogesterona/metabolismo , Alginatos/metabolismo , Fosfatase Alcalina/metabolismo , Androgênios/metabolismo , Androstenodiona/metabolismo , Animais , Proteínas de Transporte/metabolismo , Desidroepiandrosterona/metabolismo , Estradiol/metabolismo , Feminino , Células da Granulosa/metabolismo , Humanos , Inibinas/metabolismo , Liases/metabolismo , Camundongos , Pregnenolona/metabolismo , Progesterona/metabolismo , Células Tecais
12.
Microbiology (Reading) ; 168(9)2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36129827

RESUMO

This study aimed to reveal the physicochemical and organoleptic effects of three functional lactic acid bacteria (LAB) isolates in a milk medium: Lacticaseibacillus paracasei subsp. tolerans NOC-122, Levilactobacillus parabrevis NOC-111 and Latilactobacillus curvatus NOC-110. A total of 200 indigenous LAB strains isolated from artisanal tulum cheeses were screened for potential proteolytic and lipolytic activity, citrate-lyase-synthesizing and exopolysaccharide-producing ability. Furthermore, a total of six fermented products were produced using these strains as a single culture or as a co-culture. The physicochemical and microbiological properties, angiotensin-converting-enzyme (ACE) inhibitor activity, and the amino acid and volatile aroma compound profiles were determined. According to the results, NOC-110 and NOC-122 were effective in increasing ACE-inhibitory activity. On the other hand, NOC-122 was responsible for a fresh cheesy, slightly oily flavour when used as a single culture. NOC-111 gave a fresh, fruity and slightly herbal flavour; NOC-110 gave a flavour similar to that of NOC-122 when they were used as a single culture. Also, co-cultures of the strains were investigated. The results of the study provide a guide to the usability of these isolates as single or co-cultures in the production of dairy-based food. These findings can be of value for many future studies and innovative food products.


Assuntos
Lactobacillales , Liases , Aminoácidos , Angiotensinas , Citratos , Microbiologia de Alimentos , Lactobacillus
13.
Biosci Biotechnol Biochem ; 86(11): 1536-1542, 2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36085174

RESUMO

Various d-amino acids play important physiological roles in mammals, but the pathways of their production remain unknown except for d-serine, which is generated by serine racemase. Previously, we found that Escherichia coli cystathionine ß-lyase possesses amino acid racemase activity in addition to ß-lyase activity. In the present work, we evaluated the enzymatic activities of human cystathionine γ-lyase, which shares a relatively high amino acid sequence identity with cystathionine ß-lyase. The enzyme did not show racemase activity toward various amino acids including alanine and lyase and dehydratase activities were highest toward l-cystathionine and l-homoserine, respectively. The enzyme also showed weak activity toward l-cysteine and l-serine but no activity toward d-amino acids. Intriguingly, the pH and temperature profiles of lyase activity were distinct from those of dehydratase activity. Catalytic efficiency was higher for lyase activity than for dehydratase activity.


Assuntos
Isomerases de Aminoácido , Liases , Humanos , Animais , Cistationina gama-Liase/química , Cistationina gama-Liase/metabolismo , Aminoácidos , Cistationina , Cisteína , Homosserina , Liases/metabolismo , Escherichia coli/metabolismo , Serina , Racemases e Epimerases , Alanina , Hidroliases , Mamíferos/metabolismo
14.
J Biotechnol ; 358: 67-75, 2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36087783

RESUMO

As a valuable platform chemical, 2,3-Butanediol (2,3-BDO) has a variety of industrial applications, and its microbial production is particularly attractive as an alternative to petroleum-based production. In this study, the regulation of intracellular carbon flux and NADH/NAD+ was used to increase the 2,3-BDO production of Enterobacter aerogenes. The genes encoding lactate dehydrogenase (ldh) and pyruvate formate lyase (pfl) were disrupted using the λ-Red recombination method and CRISPR-Cas9 to reduce the production of several byproducts and the consumption of NADH. Knockout of ldh or pfl increased intracellular NADH/NAD+ by 111 % and 113 %, respectively. Moreover, two important genes in the 2,3-BDO biosynthesis pathway, acetolactate synthase (budB) and acetoin reductase (budC), were overexpressed in E. aerogenes to further amply the metabolic flux toward 2,3-BDO production. And the overexpression of budB or budC increased intracellular NADH/NAD+ by 46 % and 57 %, respectively. In shake-flask cultivation with sucrose as carbon source, the 2,3-BDO titer of the IAM1183-LPBC was 3.55 times that of the wild type. In the 5-L fermenter, the maximal 2,3-BDO production produced by the IAM1183-LPBC was 2.88 times that of the original strain. This work offers new ideas for promoting the biosynthesis of 2,3-BDO for industrial applications.


Assuntos
Acetolactato Sintase , Enterobacter aerogenes , Liases , Petróleo , Acetolactato Sintase/metabolismo , Butileno Glicóis/metabolismo , Carbono/metabolismo , Ciclo do Carbono , Enterobacter aerogenes/genética , Enterobacter aerogenes/metabolismo , Fermentação , Formiatos , L-Lactato Desidrogenase/genética , Engenharia Metabólica/métodos , NAD/metabolismo , Piruvatos , Sacarose
15.
Plant Sci ; 325: 111466, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36174799

RESUMO

Chlorophylls are ubiquitous pigments responsible for the green color in plants. Changes in the chlorophyll content have a significant impact on photosynthesis, plant growth and development. In this study, we used a yellow stigma mutant (ys) generated from a green stigma tomato WT by using ethylmethylsulfone (EMS)-induced mutagenesis. Compared with WT, the stigma of ys shows low chlorophyll content and impaired chloroplast ultrastructure. Through map-based cloning, the ys gene is localized to a 100 kb region on chromosome 4 between dCAPS596 and dCAPS606. Gene expression analysis and nonsynonymous SNP determination identified the Solyc04g015750, as the potential candidate gene, which encodes a magnesium chelatase H subunit (CHLH). In ys mutant, a single base C to T substitution in the SlCHLH gene results in the conversion of Serine into Leucine (Ser92Leu) at the N-terminal region. The functional complementation test shows that the SlCHLH from WT can rescue the green stigma phenotype of ys. In contrast, knockdown of SlCHLH in green stigma tomato AC, observed the yellow stigma phenotype at the stigma development stage. Overexpression of the mutant gene Slys in green stigma tomato AC results in the light green stigma. These results indicate that the mutation of the N-terminal S92 to Leu in SlCHLH is the main reason for the formation of the yellow stigma phenotype. Characterization of the ys mutant enriches the current knowledge of the tomato chlorophyll mutant library and provides a novel and effective tool for understanding the function of CHLH in tomato.


Assuntos
Liases , /genética , Liases/genética , Clorofila/metabolismo , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas
16.
J Appl Physiol (1985) ; 133(4): 798-813, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35952350

RESUMO

Consumption of a Western-style diet (WD; high fat, high sugar, low fiber) is associated with impaired vascular function and increased risk of cardiovascular diseases (CVD), which could be mediated partly by increased circulating concentrations of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO). We investigated if suppression of TMAO with 3,3-dimethyl-1-butanol (DMB; inhibitor of microbial TMA lyase) in mice could prevent: 1) WD-induced vascular endothelial dysfunction and aortic stiffening and 2) WD-induced reductions in endurance exercise tolerance and increases in frailty, as both are linked to WD, vascular dysfunction, and increased CVD risk. C57BL/6N mice were fed standard chow or WD (41% fat, ∼25% sugar, 4% fiber) for 5 mo beginning at ∼2 mo of age. Within each diet, mice randomly received (n = 11-13/group) normal drinking water (control) or 1% DMB in drinking water for the last 8 wk (from 5 to 7 mo of age). Plasma TMAO was increased in WD-fed mice but suppressed by DMB. WD induced endothelial dysfunction, assessed as carotid artery endothelium-dependent dilation to acetylcholine, and progressive increases in aortic stiffness (measured serially in vivo as pulse wave velocity), both of which were fully prevented by supplementation with DMB. Endurance exercise tolerance, assessed as time to fatigue on a rotarod test, was impaired in WD-fed mice but partially recovered by DMB. Lastly, WD-induced increases in frailty (31-point index) were prevented by DMB. Our findings indicate DMB or other TMAO-lowering therapies may be promising for mitigating the adverse effects of WD on physiological function, and thereby reducing risk of chronic diseases.NEW & NOTEWORTHY We provide novel evidence that increased circulating concentrations of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) contribute to vascular dysfunction associated with consumption of a Western-style diet and that this dysfunction can be prevented by suppressing TMAO with DMB, thereby supporting translation of this compound to humans. Furthermore, to our knowledge, we present the first evidence of the role of TMAO in mediating impairments in endurance exercise tolerance and increased frailty in any context.


Assuntos
Água Potável , Fragilidade , Liases , Doenças Vasculares , Acetilcolina , Animais , Dieta Ocidental/efeitos adversos , Humanos , Metilaminas , Camundongos , Camundongos Endogâmicos C57BL , Análise de Onda de Pulso , Açúcares , Doenças Vasculares/etiologia , Doenças Vasculares/prevenção & controle
17.
Andrologia ; 54(10): e14545, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35942817

RESUMO

Adrenomedullin (ADM) has beneficial effects on Leydig cells under pathological conditions, including lipopolysaccharide (LPS)-induced orchitis. Our previous studies demonstrated that ADM exerts a restorative effect on steroidogenesis in LPS-treated primary rat Leydig cells by attenuating oxidative stress, inflammation and apoptosis. In this study, we aim to investigate whether ADM inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo. Rats were administered with LPS and injected with Ad-ADM, an adeno-associated virus vector that expressed ADM. Then, rat testes were collected for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) immunofluorescence staining. Steroidogenic enzymes or steroidogenic regulatory factors or protein, including steroidogenic factor-1 (SF-1), liver receptor homologue-1 (LRH1), Nur77, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), 3ß-HSD, cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD), were detected via gene expression profiling and western blot analysis. Plasma testosterone concentrations were measured. Results showed that ADM may inhibit Leydig cell dysfunction by rescuing steroidogenic enzymes and steroidogenic regulatory factors in vivo. The reduction in the number of Leydig cells after LPS exposure was reversed by ADM. ADM rescued the gene or protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD and plasma testosterone concentrations. To summarize ADM could rescue some important steroidogenic enzymes, steroidogenic regulatory factors and testosterone production in Leydig cells in vivo.


Assuntos
Células Intersticiais do Testículo , Liases , 3-Hidroxiesteroide Desidrogenases/metabolismo , Adrenomedulina/genética , Adrenomedulina/metabolismo , Adrenomedulina/farmacologia , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Liases/metabolismo , Liases/farmacologia , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroide 17-alfa-Hidroxilase/farmacologia , Testosterona
18.
Am J Med Genet A ; 188(11): 3312-3317, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35972040

RESUMO

Sphingosine Lyase Insufficiency Syndrome (SPLIS) or SGPL1 Deficiency is a newly described entity that is characterized by steroid-resistant nephrotic syndrome, primary adrenal insufficiency, lymphopenia, ichthyosis, and/or endocrine and neurologic abnormalities. The earliest identification of SGPL1 pathogenic variants in association with this syndrome was reported in 2017. Since then, at least 36 patients have been reported with this pediatric syndrome. Here, we report a new patient with SPLIS who had a prenatal finding of adrenal calcifications, congenital nephrotic syndrome, and abnormal newborn screening concerning for Severe Combined Immunodeficiency. We conclude that SPLIS is a clinically recognizable condition with prenatal onset. This case should increase awareness of SPLIS in the differential diagnosis for adrenal calcifications. We present a case on the severe end of the clinical spectrum of SPLIS, and a review of the literature.


Assuntos
Doenças das Glândulas Suprarrenais , Insuficiência Adrenal , Calcinose , Liases , Síndrome Nefrótica , Insuficiência Adrenal/diagnóstico , Insuficiência Adrenal/genética , Aldeído Liases/genética , Calcinose/diagnóstico , Calcinose/genética , Criança , Feminino , Humanos , Recém-Nascido , Síndrome Nefrótica/patologia , Gravidez , Esfingosina , Esteroides , Síndrome
19.
Bioorg Med Chem Lett ; 75: 128951, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36031020

RESUMO

We report herein, the discovery of BMS-737 (compound 33) as a potent, non-steroidal, reversible small molecule inhibitor demonstrating 11-fold selectivity for CYP17 lyase over CYP17 hydroxylase, as well as a clean xenobiotic CYP profile for the treatment of castration-resistant prostate cancer (CRPC). Extensive SAR studies on the initial lead 1 at three different regions of the molecule resulted in the identification of BMS-737, which demonstrated a robust 83% lowering of testosterone without any significant perturbation of the mineralocorticoid and glucocorticoid levels in cynomologous monkeys in a 1-day PK/PD study.


Assuntos
Liases , Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Antagonistas de Androgênios , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Glucocorticoides , Humanos , Masculino , Mineralocorticoides , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Esteroide 17-alfa-Hidroxilase , Testosterona , Xenobióticos
20.
ChemSusChem ; 15(20): e202201147, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-35917230

RESUMO

A laccase-Lig multienzymatic multistep system for lignin depolymerization was designed and developed. Studies were performed on pristine and fractionated lignins (Kraft and Organosolv) using a specific cascade of enzymes, that is, laccases from Bacillus licheniformis and from Funalia trogii, respectively for Kraft and Organosolv lignin, followed by the Lig system from Sphingobium sp. SYK-6 (ß-etherases Lig E and Lig F, glutathione lyase Lig G). Careful elucidation of the structural modifications occurring in the residual lignins associated with the identification and quantification of the generated low-molecular-weight compounds showed that (i) the laccase-Lig system cleaves non-phenolic aryl glycerol ß-O-4 aryl ether bonds, and (ii) the overall reactivity is heavily dependent on the individual lignin structure. More specifically, samples with low phenolic/aliphatic OH groups ratio undergo net depolymerization, while an increased phenolic/aliphatic OH ratio results in the polymerization of the residual lignin irrespective of its botanical origin and isolation process.


Assuntos
Lignina , Liases , Lignina/química , Lacase/química , Glicerol , Éteres , Glutationa
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