Differential Response of the Proteins Involved in Amino Acid Metabolism in Two Saccharomyces cerevisiae Strains during the Second Fermentation in a Sealed Bottle

The traditional method for sparkling wine making consists of a second fermentation of a base wine followed by ageing in the same bottle that reaches the consumers. Nitrogen metabolism is the second most important process after carbon and takes place during wine fermentation by yeast. Amino acids are the most numerous nitrogen compounds released by this process. They contribute to the organoleptic properties of the wines and, therefore, to their sensory quality. The main objective of this study is to compare the differential proteomic response of amino acid metabolism, specifically their proteins and their interactions in the G1 strain (unconventional yeast) during sparkling wine production versus the conventional P29 strain. One of the new trends in winemaking is the improvement of the organoleptic diversity of wine. We propose the use of unconventional yeast that shows desirable characteristics for the industry. For this purpose, these two yeasts were grown at sealed bottle conditions for the second fermentation (Champenoise method). No differences were obtained in the middle of fermentation between the yeast strains. The number of proteins identified, and the relationships established, were similar, highlighting lysine metabolism. At the end of the second fermentation, the difference between each strain was remarkable. Hardly any proteins were identified in unconventional versus conventional yeast. However, in both strains, the metabolism of sulfur amino acids, methionine, and cysteine obtained a greater number of proteins involved in these processes. The release of these amino acids to the medium would allow the yeast to balance the internal redox potential by reoxidation of NADPH. This study is focused on the search for a more complete knowledge of yeast metabolism, specifically the metabolism of amino acids, which are key compounds during the second fermentation.

Pyridoxine Therapy: Not Just the Dose, the Duration Matters Too

Pyridoxine-dependent epilepsy (PDE) (OMIM 266100) is an autosomal recessive disorder of lysine metabolism secondary to antiquitin deficiency. The prototypical presentation is intractable neonatal seizures that do not respond to conventional antiseizure medication but are well controlled by pyridoxine supplementation. Atypical forms account for one-third of the PDE spectrum and may escape early diagnosis. The common atypical presentations include the prenatal onset of seizures, seizures onset as delayed as 3 years of age, autism, arrested hydrocephalus, and fetal ventriculomegaly. Herein, we describe a 9-month-old child with neonatal-onset refractory seizures who failed two short trials of pyridoxine therapy and was later diagnosed with PDE by molecular studies. Regardless of the therapeutic response, a prolonged course of pyridoxine therapy is justified to identify delayed responders in infants with drug-refractory epilepsy of no apparent etiology.

Lysine metabolism conveys kidney protection in hypertension

Hypertension and kidney disease, two related, common, and severe disease entities, have been repeatedly associated with genomic variants and metabolic alterations of lysine metabolism. Here, we developed a stable isotope labeling strategy compatible with untargeted metabolomics acquisition to investigate the physiology and molecular spectrum of lysine’s metabolic fate in vivo. Mice received 13C6 labeled lysine through the diet over two months to track more than 100 lysine metabolites across various organs and body fluids. Lysine reacts rapidly with molecules of the central carbon metabolism, as opposed to slow or incorporation into proteins and metabolization into acylcarnitines. The kidney rapidly forms these lysine conjugates and lysine metabolism is decreased in early stages of hypertension. Lysine administration completely diminished the development of salt-sensitive hypertension and kidney injury in the Dahl salt-sensitive rat model. Administration of lysine leads to diuresis, acceleration of 13C6 lysine conjugate formation, and inhibition of albumin uptake, thereby protecting from nephron injury and metabolic stress. Lysine conjugates with malonyl-CoA to form a novel metabolite Ne-malonyl-lysine to inhibit fatty acid synthesis. Formation of Ne-malonyl-Lysine, and acetyl-Lysine during lysine treatment depletes malonyl-CoA and acetyl-CoA, respectively, a process that occurs at the expense of protein malonylation and acetylation. A significant fraction of lysine molecules was metabolized in the kidney and excreted as fructoselysine and saccharopine, via the urine, leading to an overall depletion of central carbon metabolites from the organism. A ketogenic diet also ameliorated hypertension, yet to a lower extent, and increased several lysine conjugates, including Ne-malonyl-lysine. In conclusion, isotope tracing of orally administered lysine illuminated lysine metabolism, and L-lysine protects the kidneys in metabolically defined subtypes of kidney disease.

Review of Lysine Metabolism with a Focus on Humans

ABSTRACT Lysine cannot be synthesized by most higher organisms and, therefore, is an indispensable amino acid (IAA) that must be consumed in adequate amounts to maintain protein synthesis. Although lysine is an abundant amino acid in body proteins, lysine is limited in abundance in many important food sources (e.g. grains). Older observations assigned importance to lysine because animals fed a lysine-deficient diet did not lose weight as fast as animals placed upon other IAA-deficient diets, leading to the theory that there may be a special pool of lysine or metabolites that could be converted to lysine. The first step in the lysine catabolic pathway is the formation of saccharopine and then 2-aminoadipic acid, processes that are mitochondrial. The catabolism of 2-aminoadipic acid proceeds via decarboxylation to a series of CoA esters ending in acetyl-CoA. In mammals, the liver appears to be the primary site of lysine catabolism. In humans, the metabolic and oxidative response of lysine to diets either restricted in protein or in lysine is consistent with what has been measured for other IAAs with isotopically labeled tracers. Intestinal microflora are known to metabolize urea to ammonia and scavenge nitrogen (N) for the synthesis of amino acids. Studies feeding 15N-ammonium chloride or 15N-urea to animals and to humans, demonstrate the appearance of 15N-lysine in gut microbial lysine and in host lysine. However, the amount of 15N-lysine transferred to the host is difficult to assess directly using current methods. It is important to understand the role of the gut microflora in human lysine metabolism, especially in conditions where dietary lysine intake may be limited, but better methods need to be devised.

Deglutarylation of GCDH by SIRT5 controls lysine metabolism in mice

ABSTRACTA wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be non-enzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways. To better understand the biology of protein glutarylation, we studied the relationship between enzymes within the lysine/tryptophan catabolic pathways, protein glutarylation, and regulation by the deglutarylating enzyme Sirtuin 5 (SIRT5). Here, we identify glutarylation on the lysine oxidation pathway enzyme glutaryl-CoA dehydrogenase (GCDH). We show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We then demonstrate the ability of SIRT5 to deglutarylate GCDH, restoring its enzymatic activity. Finally, metabolomic and bioinformatic analyses indicate an expanded role for SIRT5 in regulating amino acid metabolism. Together, these data support a model whereby a feedback loop exists within the lysine/tryptophan oxidation pathway, in which glutaryl-CoA is produced, in turn inhibiting GCDH function via glutaryl modification of GCDH lysine residues, and can be relieved by SIRT5 deacylation activity.

Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in Pseudomonas putida Lysine Metabolism

P. putidalysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism inP. putidaremained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism inP. putida. We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing proteinP. putida5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA tod-2-hydroxyglutarate (d-2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.