Oligomeric State in the Crystal Structure of Modular FAD Synthetase Provides Insights into Its Sequential Catalysis in Prokaryotes

The crystal structure of the modular flavin adenine dinucleotide (FAD) synthetase from Corynebacterium ammoniagenes has been solved at 1.95 Å resolution. The structure of C. ammoniagenes FAD synthetase presents two catalytic modules—a C-terminus with ATP-riboflavin kinase activity and an N-terminus with ATP-flavin mononucleotide (FMN) adenylyltransferase activity—that are responsible for the synthesis of FAD from riboflavin in two sequential steps. In the monomeric structure, the active sites from both modules are placed 40 Å away, preventing the direct transfer of the product from the first reaction (FMN) to the second catalytic site, where it acts as substrate. Crystallographic and biophysical studies revealed a hexameric assembly formed by the interaction of two trimers. Each trimer presents a head-tail configuration, with FMN adenylyltransferase and riboflavin kinase modules from different protomers approaching the active sites and allowing the direct transfer of FMN. Experimental results provide molecular-level evidences of the mechanism of the synthesis of FMN and FAD in prokaryotes in which the oligomeric state could be involved in the regulation of the catalytic efficiency of the modular enzyme.     

Crystal Structure of Yeast FAD Synthetase (Fad1) in Complex with FAD

Flavin adenine dinucleotide (FAD) synthetase is an essential enzyme responsible for the synthesis of FAD by adenylation of riboflavin monophosphate (FMN). We have solved the 1.9 Å resolution structure of Fad1, the yeast FAD synthetase, in complex with the FAD product in the active site. The structure of Fad1 shows it to be a member of the PP-ATPase superfamily. Important conformational differences in the two motifs involved in binding the phosphate moieties of FAD compared to the Candida glabrata FMNT ortholog suggests that this loop is dynamic and undergoes substantial conformational changes during its catalytic cycle.     

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.     

Effect of sex hormones on n-3 polyunsaturated fatty acid biosynthesis in HepG2 cells and in human primary hepatocytes

Female humans and rodents have been shown to have higher 22:6n-3 status and synthesis than males. It is unclear which sex hormone is involved. We investigated the specificity of the effects of physiological concentrations of sex hormones in vitro on the mRNA expression of genes involved in polyunsaturated fatty acid (PUFA) biosynthesis and on the conversion of [d₅]-18:3n-3 to longer chain fatty acids. Progesterone, but not 17α-ethynylestradiol or testosterone, increased FADS2, FADS1, ELOVl 5 and ELOVl 2 mRNA expression in HepG2 cells, but only FADS2 in primary human hepatocytes. In HepG2 cells, these changes were accompanied by hypomethylation of specific CpG loci in the FADS2 promoter. Progesterone, not 17α-ethynylestradiol or testosterone, increased conversion of [d₅]-18:3n-3 to 20:5n-3, 22:5n-3 and 22:6n-3. These findings show that progesterone increases n-3 PUFA biosynthesis by up-regulating the mRNA expression of genes involved in this pathway, possibly via changes in the epigenetic regulation of FADS2.     

Deep-lipidotyping by mass spectrometry: recent technical advances and applications

In-depth structural characterization of lipids is an essential component of lipidomics. There has been a rapid expansion of mass spectrometry methods that are capable of resolving lipid isomers at various structural levels over the past decade. These developments finally make deep-lipidotyping possible, which provides new means to study lipid metabolism and discover new lipid biomarkers. In this review, we discuss recent advancements in tandem mass spectrometry (MS/MS) methods for identification of complex lipids beyond the species (known headgroup information) and molecular species (known chain composition) levels. These include identification at the levels of carbon-carbon double bond (C=C) location and sn-position, as well as characterization of acyl chain modifications. We also discuss the integration of isomer-resolving MS/MS methods with different lipid analysis workflows and their applications in lipidomics. The results showcase the distinct capabilities of deep-lipidotyping in untangling the metabolism of individual isomers and sensitive phenotyping by using relative fractional quantitation of the isomers.     

Single-Cell Droplet Microfluidic Screening for Antibodies Specifically Binding to Target Cells

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Detection of a quaternary organization into dimer of trimers of Corynebacterium ammoniagenes FAD synthetase at the single-molecule level and at the in cell level

Biochemical characterization of Corynebacterium ammoniagenes FADS (CaFADS) pointed to certain confusion about the stoichiometry of this bifunctional enzyme involved in the production of FMN and FAD in prokaryotes. Resolution of its crystal structure suggested that it might produce a hexameric ensemble formed by a dimer of trimers. We used atomic force microscopy (AFM) to direct imaging single CaFADS molecules bound to mica surfaces, while preserving their catalytic properties. AFM allowed solving individual CaFADS monomers, for which it was even possible to distinguish their sub-molecular individual N- and C-terminal modules in the elongated enzyme. Differences between monomers and higher stoichiometries were easily imaged, enabling us to detect formation of oligomeric species induced by ligand binding. The presence of ATP:Mg^2 + particularly induced the appearance of the hexameric assembly whose mean molecular volume resembles the crystallographic dimer of trimers. Finally, the AFM results are confirmed in cross-linking solution, and the presence of such oligomeric CaFADS species detected in cell extracts. All these results are consistent with the formation of a dimer of trimers during the enzyme catalytic cycle that might bear biological relevance.     

The polypyrimidine tract binding protein regulates desaturase alternative splicing and PUFA composition

The Δ6 desaturase, encoded by FADS2, plays a crucial role in omega-3 and omega-6 fatty acid synthesis. These fatty acids are essential components of the central nervous system, and they act as precursors for eicosanoid signaling molecules and as direct modulators of gene expression. The polypyrimidine tract binding protein (PTB or hnRNP I) is a splicing factor that regulates alternative pre-mRNA splicing. Here, PTB is shown to bind an exonic splicing silencer element and repress alternative splicing of FADS2 into FADS2 AT1. PTB and FADS2AT1 were inversely correlated in neonatal baboon tissues, implicating PTB as a major regulator of tissue-specific FADS2 splicing. In HepG2 cells, PTB knockdown modulated alternative splicing of FADS2, as well as FADS3, a putative desaturase of unknown function. Omega-3 fatty acids decreased by nearly one half relative to omega-6 fatty acids in PTB knockdown cells compared with controls, with a particularly strong decrease in eicosapentaenoic acid (EPA) concentration and its ratio to arachidonic acid (ARA). This is a rare demonstration of a mechanism specifically altering the cellular omega-3 to omega-6 fatty acid ratio without any change in diet/media. These findings reveal a novel role for PTB, regulating availability of membrane components and eicosanoid precursors for cell signaling.     

Delta-6-desaturase (FADS2) inhibition and omega-3 fatty acids in skeletal muscle protein turnover

Polyunsaturated fatty acids (PUFAs) are essential dietary components. They are not only used for energy, but also act as signaling molecules. The delta-6 desaturase (D6D) enzyme, encoded by the FADS2 gene, is one of two rate limiting enzymes that convert the PUFA precursors – α-linolenic (n-3) and linoleic acid (n-6) to their respective metabolites. Alterations in the D6D enzyme activity alters fatty acid profiles and are associated with metabolic and inflammatory diseases including cardiovascular disease and type 2 diabetes. Omega-3 PUFAs, specifically its constituent fatty acids DHA and EPA, are known for their anti-inflammatory ability and are also beneficial in the prevention of skeletal muscle wasting, however the mechanism for muscle preservation is not well understood. Moreover, little is known of the effects of altering the n-6/n-3 ratio in the context of a high-fat diet, which is known to downregulate protein synthesis. Twenty C57BL6 male mice were fed a high-fat lard (HFL, 45% fat (mostly lard), 35% carbohydrate and 20% protein, n-6:n-3 PUFA, 13:1) diet for 6 weeks. Mice were then divided into 4 groups (n = 5 per group): HFL– , high-fat oil– (HFO, 45% fat (mostly Menhaden oil), 35% carbohydrate and 20% protein, n-6:n-3 PUFA, 1:3), HFL+ (HFL diet plus an orally administered FADS2 inhibitor, 100 mg/kg/day), and HFO+ (HFO diet plus an orally administered FADS2 inhibitor, 100 mg/kg/day). After 2 weeks on their respective diets and treatments, animals were sacrificed and gastrocnemius muscle harvested. Protein turnover signaling were analyzed via Western Blot. 4-EBP1 and ribosomal protein S6 expression were measured. A two-way ANOVA revealed no significant change in the phosphorylation of both 4EBP-1 and ribosomal protein S6 with diet or inhibitor. There was a significant reduction in STAT3 phosphorylation with the inhibition of FADS2 (p = 0.03). Additionally, we measured markers of protein degradation through levels of FOXO phosphorylation, ubiquitin, and LC3B expression; there was a trend towards increased phosphorylation of FOXO (p = 0.08) and ubiquitinated proteins (p = 0.05) with FADS2 inhibition. LC3B expression, a marker of autophagy, was significantly higher in the HFL plus FADS2 inhibition group from all other comparisons. Lastly, we analyzed activation of mitochondrial biogenesis which is closely linked with protein synthesis through PGC1-α and Cytochrome-C expression, however no significant differences were associated with either marker across all groups. Collectively, these data suggest that the protective effects of muscle mass by omega-3 fatty acids are from inhibition of protein degradation. Our aim was to determine the role of PUFA metabolites, DHA and EPA, in skeletal muscle protein turnover and assess the effects of n-3s independently. We observed that by inhibiting the FADS2 enzyme, the protective effect of n-3s on protein synthesis and proliferation was lost; concomitantly, protein degradation was increased with FADS2 inhibition regardless of diet.