Synthetic analogues of 2-oxo acids discriminate metabolic contribution of the 2-oxoglutarate and 2-oxoadipate dehydrogenases in mammalian cells and tissues

Abstract

The biological significance of the DHTKD1-encoded 2-oxoadipate dehydrogenase (OADH) remains obscure due to its catalytic redundancy with the ubiquitous OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH). In this work, metabolic contributions of OADH and OGDH are discriminated by exposure of cells/tissues with different DHTKD1 expression to the synthesized phosphonate analogues of homologous 2-oxodicarboxylates. The saccharopine pathway intermediates and phosphorylated sugars are abundant when cellular expressions of DHTKD1 and OGDH are comparable, while nicotinate and non-phosphorylated sugars are when DHTKD1 expression is order(s) of magnitude lower than that of OGDH. Using succinyl, glutaryl and adipoyl phosphonates on the enzyme preparations from tissues with varied DHTKD1 expression reveals the contributions of OADH and OGDH to oxidation of 2-oxoadipate and 2-oxoglutarate in vitro. In the phosphonates-treated cells with the high and low DHTKD1 expression, adipate or glutarate, correspondingly, are the most affected metabolites. the marker of fatty acid β-oxidation, adipate, is mostly decreased by the shorter, OGDH-preferring, phosphonate, in agreement with the known OGDH dependence of β-oxidation. The longest, OADHpreferring, phosphonate mostly affects the glutarate level. Coupled decreases in sugars and nicotinate upon the OADH inhibition link the perturbation in glucose homeostasis, known in OADH mutants, to the nicotinate-dependent NAD metabolism. 2-Oxo acid dehydrogenase complexes comprise a family of multimeric enzymes functioning at the intersections of metabolic pathways involving carbohydrates, lipids and amino acids 1. The family includes the well-characterized 2-oxoglutarate dehydrogenase complex (OGDHC), which couples the tricarboxylic acid (TCA) cycle with degradation of amino acids of the 2-oxoglutarate group, namely, glutamate, glutamine, arginine, histidine, and proline. The substrate-specific 2-oxoglutarate dehydrogenase (OGDH, EC 1.2.4.1, encoded by the OGDH gene, also known as E1o component of the complex), is a well-known and rate-limiting component of OGDHC. The complex also comprises two other types of enzymes: E2o (EC 2.3.1.61) and E3 (EC 1.8.1.4), encoded by the dihydrolipoamide succinyltransferase (DLST) and dihydrolipoamide dehydrogenase (DLD) genes, respectively. Multiple copies of the E1, E2 and E3 component enzymes form multienzyme complexes schematically exemplified in Fig. 1A. The multimeric structure allows effective coupling of the 2-oxoglutarate oxidative decarboxylation

Selective Inhibition of 2-Oxoglutarate and 2-Oxoadipate Dehydrogenases by the Phosphonate Analogs of Their 2-Oxo Acid Substrates

“…In order to differentiate the contributions of OADH and OGDH to the reactions with OA and OG in such preparations, the enzyme complexes from the rat tissues with different expression of the isoenzymes are used. According to the transcriptomics data (Artiukhov et al, 2020 ), the rat heart possesses mainly OGDH ( DHTKD1 / OGDH mRNA ratio ≈ 0.01), while the liver contains comparable levels of the two isoenzymes ( DHTKD1 / OGDH ≈ 1.6). In view of the similar substrate specificity of OGDH and another, the OGDHL -encoded isoenzymes (Bunik et al, 2008 ), added by a minor content of the OGDHL transcripts (≤ 1% of OGDH ) in either the heart or liver (Bunik et al, 2008 ; Artiukhov et al, 2020 ), the kinetic contribution of the OGDHL -encoded OGDH-like isoenzyme in these tissues is neglected.…”

“…Trisodium salts of the succinyl, glutaryl, and adipoyl phosphonates and their ester precursors were synthesized as described elsewhere (Bunik et al, 2005 ; Artiukhov et al, 2020 ). Briefly, the trialkyl ester precursors of the phosphonates were prepared by standard Arbuzov reaction of trialkyl phosphite with the corresponding monoalkyl ester chlorides of succinic, glutaric and adipic acids.…”

“…Enzymes catalyzing OGDH and OADH reactions were enriched from rat hearts and livers using polyethylene glycol precipitation, as previously described (Artiukhov et al, 2020 ).…”

“…In order to discriminate physiological significance of the reactions catalyzed by the isoenzymes of 2-oxoglutarate dehydrogenase, encoded by the OGDH and DHTKD1 genes, in vivo , a homologous series of the phosphonate analogs of dicarboxylic 2-oxo acids ( Figure 1B ) has been synthesized (Artiukhov et al, 2020 ), namely the succinyl (SP), glutaryl (GP), and adipoyl (AP) phosphonates. Cellular experiments have shown specific action of the compounds on metabolomes, coinciding with their inhibition of the OGDH and OADH reactions, catalyzed by the partially purified OGDH and OADH.…”

“…Cellular experiments have shown specific action of the compounds on metabolomes, coinciding with their inhibition of the OGDH and OADH reactions, catalyzed by the partially purified OGDH and OADH. A number of other enzymatic reactions, employing 2-oxo acids, or their structural analogs, are not affected by the phosphonates (Artiukhov et al, 2020 ).…”