The energetics and evolution of oxidoreductases in deep time.

The core metabolic reactions of life drive electrons through a class of redox protein enzymes, the oxidoreductases. The energetics of electron flow is determined by the redox potentials of organic and inorganic cofactors as tuned by the protein environment. Understanding how protein structure affects oxidation-reduction energetics is crucial for studying metabolism, creating bioelectronic systems, and tracing the history of biological energy utilization on Earth. We constructed ProtReDox (https://protein-redox-potential.web.app), a manually curated database of experimentally determined redox potentials. With over 500 measurements, we can begin to identify how proteins modulate oxidation-reduction energetics across the tree of life. By mapping redox potentials onto networks of oxidoreductase fold evolution, we can infer the evolution of electron transfer energetics over deep time. ProtReDox is designed to include user-contributed submissions with the intention of making it a valuable resource for researchers in this field.

Synthesis of [1-13 C-5-12 C]-alpha-ketoglutarate enables noninvasive detection of 2-hydroxyglutarate.

Isocitrate dehydrogenase 1 (IDH1) mutations that generate the oncometabolite 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG) have been identified in many types of tumors and are an important prognostic factor in gliomas. 2-HG production can be determined by hyperpolarized carbon-13 magnetic resonance spectroscopy (HP-13 C-MRS) using [1-13 C]-α-KG as a probe, but peak contamination from naturally occurring [5-13 C]-α-KG overlaps with the [1-13 C]-2-HG peak. Via a newly developed oxidative-Stetter reaction, [1-13 C-5-12 C]-α-KG was synthesized. α-KG metabolism was measured via HP-13 C-MRS using [1-13 C-5-12 C]-α-KG as a probe. [1-13 C-5-12 C]-α-KG was synthesized in high yields, and successfully eliminated the signal from C5 of α-KG in the HP-13 C-MRS spectra. In HCT116 IDH1 R132H cells, [1-13 C-5-12 C]-α-KG allowed for unimpeded detection of [1-13 C]-2-HG. 12 C-enrichment represents a novel method to circumvent spectral overlap, and [1-13 C-5-12 C]-α-KG shows promise as a probe to study IDH1 mutant tumors and α-KG metabolism.

Sulfur Amino Acid Restriction Enhances Exercise Capacity in Mice by Boosting Fat Oxidation in Muscle.

Dietary restriction of the sulfur-containing amino acids methionine and cysteine (SAAR) improves body composition, enhances insulin sensitivity, and extends lifespan; benefits seen also with endurance exercise. Yet, the impact of SAAR on skeletal muscle remains largely unexplored. Here we demonstrate that one week of SAAR in sedentary, young, male mice increases endurance exercise capacity. Indirect calorimetry showed that SAAR increased lipid oxidation at rest and delayed the onset of carbohydrate utilization during exercise. Transcriptomic analysis revealed increased expression of genes involved in fatty acid catabolism especially in glycolytic muscle following SAAR. These findings were functionally supported by increased fatty acid circulatory turnover flux and muscle ß-oxidation. Reducing lipid uptake from circulation through endothelial cell (EC)-specific CD36 deletion attenuated the running phenotype. Mechanistically, VEGF-signaling inhibition prevented exercise increases following SAAR, without affecting angiogenesis, implicating noncanonical VEGF signaling and EC CD36-dependent fatty acid transport in regulating exercise capacity by influencing muscle substrate availability.

Simple Esterification of [1-13C]-Alpha-Ketoglutarate Enhances Membrane Permeability and Allows for Noninvasive Tracing of Glutamate and Glutamine Production.

Alpha-ketoglutarate (α-KG) is a key metabolite and signaling molecule in cancer cells, but the low permeability of α-KG limits the study of α-KG mediated effects in vivo. Recently, cell-permeable monoester and diester α-KG derivatives have been synthesized for use in vivo, but many of these derivatives are not compatible for use in hyperpolarized carbon-13 nuclear magnetic resonance spectroscopy (HP-13C-MRS). HP-13C-MRS is a powerful technique that has been used to noninvasively trace labeled metabolites in real time. Here, we show that using diethyl-[1-13C]-α-KG as a probe in HP-13C-MRS allows for noninvasive tracing of α-KG metabolism in vivo.