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-PH (the distal H-phosphinic analogue of α-ketoglutarate) can be converted into L-Glu-γ-PH. Notably, α-KG-γ-PH and L-Glu-γ-PH are antibacterial compounds, but their intracellular targets only partially overlap. L-Glu-γ-PH 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-PH is a substrate of eukaryotic GDH and that the pH optimum of L-Glu-γ-PH 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-PH exists in a pH-dependent equilibrium of keto and germinal diol forms. Furthermore, the stereospecific enzymatic synthesis of α-KG-γ-PH from L-Glu-γ-PH using GDH is a possible route for its bio-based synthesis.