Abstract
α-Ketoglutaramate (KGM) is an underexamined metabolite of L-glutamine in the metabolic pathway of glutaminase II of α-ketoglutarate formation. Presumably, KGM may be a biomarker of hepatic encephalopathy and other hyperammonemic diseases. This metabolite is a substrate for the ω-amidase enzyme and is used to determine its activity in the study of the biochemistry of various types of cancer. However, the commercial unavailability of KGM hinders its widespread use. Methods for the preparative synthesis of KGM are known, but they either do not provide the proper yield or proper purity of the target product. In this work, a detailed description of the procedures is given that allows the production of KGM with a purity above 97% and a yield of the target product above 75% using L-amino acid oxidase from C. adamanteus as a catalyst of L-glutamine conversion. KGM can be obtained both in the form of a highly concentrated aqueous solution and in the form of crystals of sodium salt. The developed methods can be used both for scaling up the synthesis of KGM and for creating economical biocatalytic technologies for the production of other highly purified preparations.
Highlights
The cells of most cancers reprogram their metabolism to a greater extent to α-ketoglutarate, a Krebs cycle metabolite formed from L-glutamine, to produce high-energy succinyl-CoA through oxidative decarboxylation catalyzed by α-ketoglutarate dehydrogenase complex [1,2]
We have found that the use of lower concentrations of L-amino acid oxidase in the reaction mixture can significantly reduce the rate of accumulation of the α-ketoglutarate byproduct
The structure of the synthesized KGM was confirmed by mass spectrometry; we showed the quantitative conversion of the synthesized product to α-ketoglutarate by human ω-amidase (NIT2), for which KGM is a specific substrate
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. As the most abundant amino acid in the human body, L-glutamine is involved in many biochemical transformations. The cells of most cancers reprogram their metabolism to a greater extent to α-ketoglutarate, a Krebs cycle metabolite formed from L-glutamine, to produce high-energy succinyl-CoA through oxidative decarboxylation catalyzed by α-ketoglutarate dehydrogenase complex [1,2]. Cancer cells use NADPH-dependent pathways for carboxylation of α-ketoglutarate with the formation of isocitrate, which is a substrate for the production of nucleic acids, lipids, and other building blocks necessary for the rapid proliferation. Α-Ketoglutarate used in these processes is formed from Cancer cells use NADPH-dependent pathways for carboxylation of α-ketoglutarate with the formation of isocitrate, which is a substrate for the production of nucleic acids, lipids, and other building blocks necessary for the rapid proliferation. α-Ketoglutarate used in these processes is formed from
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