Inactivation mechanism of N61S mutant of human FMO3 towards trimethylamine.
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ABSTRACT:
SUBMITTER: Gao C
PROVIDER: S-EPMC5676948 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Elevated levels of circulating pro-atherogenic uremic solutes, particularly trimethylamine N-oxide (TMAO), have been implicated in cardiovascular disease development in patients with chronic kidney disease (CKD). TMAO is generated from trimethylamine (TMA) via metabolism by hepatic flavin-containing monooxygenase isoform 3 (FMO3). We determined the functional effects of three common FMO3 variants at amino acids 158, 308, and 257 on TMAO concentrations in a prospective cohort study and evaluated associations of polymorphisms with CKD progression and mortality. Each additional minor allele at amino acid 158 was associated with a 0.38 μg/mL higher circulating TMAO (p = 0.01) and with faster rates of annualized relative eGFR decline. Participants with 0, 1 and 2 variant alleles averaged an eGFR loss of 8%, 12%, and 14% per year, respectively (p-for trend = 0.05). Compared to participants with the homozygous reference allele, heterozygous and homozygous variant participants had a 2.0-fold (95% CI: 0.85, 4.6) and 2.2-fold (95% CI: 0.89, 5.48) higher risk of mortality, respectively (p-for-trend = 0.04). No associations with clinical outcomes were observed for allelic variants at amino acids 257 or 308. Understanding the contribution of genetic variation of FMO3 to disease progression and all-cause mortality can guide recommendations for diet modification or pharmacotherapy in CKD patients at increased risk of adverse outcomes.
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Project description:C-type inactivation is of great physiological importance in voltage-activated K+ channels (Kv), but its structural basis remains unresolved. Knowledge about C-type inactivation has been largely deduced from the bacterial K+ channel KcsA, whose selectivity filter constricts under inactivating conditions. However, the filter is highly sensitive to its molecular environment, which is different in Kv channels than in KcsA. In particular, a glutamic acid residue at position 71 along the pore helix in KcsA is substituted by a valine conserved in most Kv channels, suggesting that this side chain is a molecular determinant of function. Here, a combination of X-ray crystallography, solid-state NMR and MD simulations of the E71V KcsA mutant is undertaken to explore inactivation in this Kv-like construct. X-ray and ssNMR data show that the filter of the Kv-like mutant does not constrict under inactivating conditions. Rather, the filter adopts a conformation that is slightly narrowed and rigidified. On the other hand, MD simulations indicate that the constricted conformation can nonetheless be stably established in the mutant channel. Together, these findings suggest that the Kv-like KcsA mutant may be associated with different modes of C-type inactivation, showing that distinct filter environments entail distinct C-type inactivation mechanisms.
| S-EPMC8943062 | biostudies-literature
Project description:High YAP activity is associated with poor prognosis human breast cancers, but its role during the initial stage of mammary cell transformation is unknown. To address this question, we designed experiments that exploit the ability of KRASG12D-transduced subsets of freshly isolated normal human mammary cells to form invasive tumors rapidly and efficiently when transplanted into immunodeficient mice. Initial examination of the newly developing tumors thus generated revealed a consistent marked loss of nuclear YAP, independent of the initial primary human mammary cell type transduced. Conversely, co-transduction of the same subsets of primary human mammary cells with KRASG12D plus the constitutively active YAPS127A prevented tumor formation. These findings contrast with the enhanced display of transformed properties obtained when the immortalized, but non-tumorigenic MCF10A cells are transduced just with YAPS127A. In addition, we show that YAPS127A-transduction of the human MDA-MB-231 breast cancer cell line (that carry a similar KRAS mutation) enhances their metastatic activity in vivo. We also discover that the KRASG12D-induced early loss of YAP in primary human mammary cells is associated with their induced secretion of amphiregulin. Collectively, these findings suggest that YAP can differentially affect the acquisition of malignant properties by human mammary cells at different stages of their transformation.
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Project description:BACKGROUND:In the course of drug discovery and development process, sufficient reference standards of drug metabolites are required, especially for preclinical/clinical or new therapeutic drugs. Whole-cell synthesis of drug metabolites is of great interest due to its low cost, low environmental impact and specificity of the enzymatic reaction compared to chemical synthesis. Here, Escherichia coli (E. coli) JM109 cells over-expressing the recombinant human FMO3 (flavin-containing monooxygenase isoform 3) were used for the conversions of clomiphene, dasatinib, GSK5182 and tozasertib to their corresponding N-oxide metabolites. RESULTS:The effects of NADPH regeneration, organic solvents as well as C-terminal truncations of human FMO3 were investigated. Under the optimized conditions, in excess of 200 mg/L of N-oxide metabolite of each of the four drugs could be produced by whole-cell catalysis within 24 h. Of these, more than 90% yield conversions were obtained for the N-oxidation of clomiphene and dasatinib. In addition, FMO3 shows high regio-selectivity in metabolizing GSK5182 where only the (Z) isomer is monooxygenated. CONCLUSIONS:The study shows the successful use of human FMO3-based whole-cell as a biocatalyst for the efficient synthesis of drug metabolites including regio-selective reactions involving GSK5182, a new candidate against type 2 diabetes mellitus.
| S-EPMC7085137 | biostudies-literature
Project description:The mitochondrial presequence protease (PreP) is a member of the pitrilysin class of metalloproteases. It degrades the mitochondrial targeting presequences of mitochondria-localized proteins as well as unstructured peptides such as amyloid-? peptide. The specific activity of PreP is reduced in Alzheimer patients and animal models of Alzheimer disease. The loss of activity can be mimicked in vitro by exposure to oxidizing conditions, and indirect evidence suggested that inactivation was due to methionine oxidation. We performed peptide mapping analyses to elucidate the mechanism of inactivation. None of the 24 methionine residues in recombinant human PreP was oxidized. We present evidence that inactivation is due to oxidation of cysteine residues and consequent oligomerization through intermolecular disulfide bonds. The most susceptible cysteine residues to oxidation are Cys34, Cys112, and Cys119. Most, but not all, of the activity loss is restored by the reducing agent dithiothreitol. These findings elucidate a redox mechanism for regulation of PreP and also provide a rational basis for therapeutic intervention in conditions characterized by excessive oxidation of PreP.
| S-EPMC4258540 | biostudies-literature
Project description:BACKGROUND:Sufficient reference standards of drug metabolites are required in the drug discovery and development process. However, such drug standards are often expensive or not commercially available. Chemical synthesis of drug metabolite is often difficulty due to the highly regio- and stereo-chemically demanding. The present work aims to construct stable and efficient biocatalysts for the generation of drug metabolites in vitro. RESULT:In this work, using benzydamine as a model drug, two easy-to-perform approaches (whole cell catalysis and enzyme immobilization) were investigated for the synthesis of FMO3-generated drug metabolites. The whole cell catalysis was carried out by using cell suspensions of E. coli JM109 harboring FMO3 and E. coli BL21 harboring GDH (glucose dehydrogenase), giving 1.2 g/L benzydamine N-oxide within 9 h under the optimized conditions. While for another approach, two HisTrap HP columns respectively carrying His6-GDH and His6-FMO3 were connected in series used for the biocatalysis. In this case, 0.47 g/L benzydamine N-oxide was generated within 2.5 h under the optimized conditions. In addition, FMO3 immobilization at the C-terminal (membrane anchor region) significantly improved its enzymatic thermostability by more than 10 times. Moreover, the high efficiency of these two biocatalytic approaches was also confirmed by the N-oxidation of tamoxifen. CONCLUSIONS:The results presented in this work provides new possibilities for the drug-metabolizing enzymes-mediated biocatalysis.
| S-EPMC6691536 | biostudies-literature
Project description:Impaired conversion of trimethylamine to trimethylamine N-oxide by human flavin containing monooxygenase 3 (FMO3) is strongly associated with primary trimethylaminuria, also known as 'fish-odor' syndrome. Numerous non-synonymous mutations in FMO3 have been identified in patients suffering from this metabolic disorder (e.g., N61S, M66I, P153L, and R492W), but the molecular mechanism(s) underlying the functional deficit attributed to these alleles has not been elucidated. The purpose of the present study was to determine the impact of these disease-associated genetic variants on FMO3 holoenzyme formation and on steady-state kinetic parameters for metabolism of several substrates, including trimethylamine. For comparative purposes, several common allelic variants not associated with primary trimethylaminuria (i.e., E158K, V257M, E308G, and the E158K/E308G haplotype) were also analyzed. When recombinantly expressed in insect cells, only the M66I and R492W mutants failed to incorporate/retain the FAD cofactor. Of the remaining mutant proteins P153L and N61S displayed substantially reduced (<10%) catalytic efficiencies for trimethylamine N-oxygenation relative to the wild-type enzyme. For N61S, reduced catalytic efficiency was solely a consequence of an increased K(m), whereas for P153L, both K(m) and k(cat) were altered. Similar results were obtained when benzydamine N-oxygenation was monitored. A homology model for FMO3 was constructed based on the crystal structure for yeast FMO which places the N61 residue alone, of the mutants analyzed here, in close proximity to the FAD catalytic center. These data demonstrate that primary trimethylaminuria is multifactorial in origin in that enzyme dysfunction can result from kinetic incompetencies as well as impaired assembly of holoprotein.
| S-EPMC2039921 | biostudies-literature
Project description:The etiology of cardiovascular disease (CVD) is impacted by multiple modifiable and non-modifiable risk factors including dietary choices, genetic predisposition, and environmental exposures. However, mechanisms linking diet, exposure to pollutants, and CVD risk are largely unclear. Recent studies identified a strong link between plasma levels of nutrient-derived Trimethylamine N-oxide (TMAO) and coronary artery disease. Dietary precursors of TMAO include carnitine and phosphatidylcholine, which are abundant in animal-derived foods. Dioxin-like pollutants can upregulate a critical enzyme responsible for TMAO formation, hepatic flavin containing monooxygenase 3 (FMO3), but a link between dioxin-like PCBs, upregulation of FMO3, and increased TMAO has not been reported. Here, we show that mice exposed acutely to dioxin-like PCBs exhibit increased hepatic FMO3 mRNA, protein, as well as an increase in circulating levels of TMAO following oral administration of its metabolic precursors. C57BL/6 mice were exposed to 5?mol PCB 126/kg mouse weight (1.63mg/kg). At 48h post-PCB exposure, mice were subsequently given a single gavage of phosphatidylcholine dissolved in corn oil. Exposure to 5 ?mole/kg PCB 126 resulted in greater than 100-fold increase in FMO3 mRNA expression, robust induction of FMO3 protein, and a 5-fold increase in TMAO levels compared with vehicle treated mice. We made similar observations in mice exposed to PCB 77 (49.6mg/kg twice); stable isotope tracer studies revealed increased formation of plasma TMAO from an orally administered precursor trimethylamine (TMA). Taken together, these observations suggest a novel diet-toxicant interaction that results in increased production of a circulating biomarker of cardiovascular disease risk.
| S-EPMC4893916 | biostudies-literature
Project description:Large size, protein binding and intracellular sequestration are well known to limit dialytic removal of compounds. In studying the normal renal and dialytic handling of trimethylamine oxide (TMAO), a molecule associated with cardiovascular disease in the general population, we discovered two largely unrecognized additional limitations to sustained reduction of a solute by chronic hemodialysis. We measured solute levels and handling in subjects on chronic hemodialysis (ESRD, n = 7) and compared these with levels and clearance in normal controls (NLS, n = 6). The ESRD patients had much higher peak predialysis plasma levels of TMAO than NLS (77 ± 26 vs 2±1 ?M, mean ± SD, p<0.05). For comparison, predialysis BUN levels in ESRD subjects were 45±11 mg/dl and 15±3 mg/dl in NLS. Thus TMAO levels in ESRD average about 40 fold those in NLS while BUN is 3 fold NLS. However, the fractional reduction of TMAO concentration during dialysis, was in fact greater than that of urea (86±3 vs 74±6%, TMAO vs urea, p < 0.05) and its dialytic clearance while somewhat lower than that of urea was comparable to creatinine's. Also production rates were similar (533±272 vs 606 ± 220 ? moles/day, ESRD vs NLS, p>0.05). However, TMAO has a volume of distribution about one half that of urea. Also in NLS the urinary clearance of TMAO was high (219±78 ml/min) compared to the urinary urea and creatinine clearances (55±14 and 119±21 ml/min, respectively). Thus, TMAO levels achieve multiples of normal much greater than those of urea due mainly to 1) TMAO's high clearance by the normal kidney relative to urea and 2) its smaller volume of distribution. Modelling suggests that only much more frequent dialysis would be required to lower levels Thus, additional strategies such as reducing production should be explored. Furthermore, using urea as the sole marker of dialysis adequacy may be misleading since a molecule, TMAO, that is dialyzed readily accumulates to much higher multiples of normal with urea based dialysis prescriptions.
| S-EPMC4674074 | biostudies-literature
Project description:Atherosclerosis is associated with various pathological manifestations, such as ischemic heart disease, ischemic stroke, and peripheral arterial disease, and remains a leading cause of public health concern. Atherosclerosis is an inflammatory disease characterized by endothelial dysfunction; vascular inflammation; and the deposition of lipids, cholesterol, calcium, and cellular debris within the vessel wall intima. In-depth studies of gut flora in recent years have shown that bacterial translocation and the existence of bacterial active products in blood circulation can affect the inflammatory state of the whole blood vessel. The gut flora is considered to be a large "secretory organ," which produces trimethylamine-N-oxide (TMAO), short-chain fatty acids and secondary bile acids by breaking down the ingested food. Studies have shown that TMAO is an independent risk factor for the occurrence of malignant adverse cardiovascular events, but whether it is harmful or beneficial to patients with cardiovascular diseases with mild or no clinical manifestations remains controversial. We review the relationship between TMAO and its precursor (L-carnitine) and coronary atherosclerosis and summarize the potential molecular mechanism and therapeutic measures of TMAO on coronary atherosclerosis.
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