Enzymatic Synthesis of Biologically Active H-Phosphinic Analogue of α-Ketoglutarate

Amino acid analogues with a phosphorus-containing moiety replacing the carboxylic group are promising sources of biologically active compounds. The H-phosphinic group, with hydrogen-phosphorus-carbon (H-P-C) bonds and a flattened tetrahedral configuration, is a bioisostere of the carboxylic group. Consequently, amino-H-phosphinic acids undergo substrate-like enzymatic transformations, leading to new biologically active metabolites. Previous studies employing NMR-based metabolomic and proteomic analyses show that in Escherichia coli, α-KG-γ-P_H (the distal H-phosphinic analogue of α-ketoglutarate) can be converted into L-Glu-γ-P_H. Notably, α-KG-γ-P_H and L-Glu-γ-P_H are antibacterial compounds, but their intracellular targets only partially overlap. L-Glu-γ-P_H is known to be a substrate of aspartate transaminase and glutamate decarboxylase, but its substrate properties with NAD⁺-dependent glutamate dehydrogenase (GDH) have never been investigated. Compounds containing P-H bonds are strong reducing agents; therefore, enzymatic NAD⁺-dependent oxidation is not self-evident. Herein, we demonstrate that L-Glu-γ-P_H is a substrate of eukaryotic GDH and that the pH optimum of L-Glu-γ-P_H NAD⁺-dependent oxidative deamination is shifted to a slightly alkaline pH range compared to L-glutamate. By ³¹P NMR, we observe that α-KG-γ-P_H exists in a pH-dependent equilibrium of keto and germinal diol forms. Furthermore, the stereospecific enzymatic synthesis of α-KG-γ-P_H from L-Glu-γ-P_H using GDH is a possible route for its bio-based synthesis.