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

Abstract

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.

Keywords: H-phosphinic analogue of α-ketoglutarate; H-phosphinic analogues of glutamate; glutamate dehydrogenase; glutamate metabolism.

Figure 1

Chemical structure of the compounds containing phosphonic and phosphinic groups. (a) Well-known phosphorus-containing compounds with pharmacological or herbicidal activity. (b) Compounds of interest in this study, i.e., H-phosphinic analogues of L-glutamate and α-ketoglutarate (L-Glu-γ-P_H and α-KG-γ-P_H), respectively, and phosphonic analogue of glutamate (D,L-Glu-γ-P₅).

Figure 2

L-Glu-γ-P_H is a low-affinity substrate of GDH. (a) Schematic representation of the oxidative deamination of L-Glu-γ-P_H catalyzed by GDH, yielding α-KG-γ-P_H. (b) Reactions (500 µL) were performed in Tris–HCl buffer (100 mM, pH 7.5) at 25 °C containing L-glutamate (10 mM) or L-Glu-γ-P_H (10 mM) and NAD⁺ (5 mM) and initiated by addition of GDH (14 µg). Data are from a representative experiment.

Figure 3

pH dependence of the initial reaction rates of GDH reaction with L-glutamate, rac-L-Glu-γ-P_H and L-Glu-γ-P_H as substrates. (a) Comparison of L-glutamate with rac-L-Glu-γ-P_H and L-Glu-γ-P_H as GDH substrates at different pH values. Reactions (500 µL) were performed in Tris–HCl buffer (100 mM, pH 6.5–9.0) and Gly-NaOH buffer (100 mM, pH 9.0–11.0) at 25 °C containing L-glutamate (10 mM), rac-Glu-γ-P_H (10 mM) or L-Glu-γ-P_H (10 mM) and NAD⁺ (5 mM) and initiated by addition of GDH (14 µg). (b) pH dependence of the GDH reaction rate for rac-L-Glu-γ-P_H and L-Glu-γ-P_H (in detail). Results are means ± SD of n = 3 independent assays, representative of n = 3 independent experiments.

Figure 4

Enzymatic synthesis of α-KG-γ-P_H from L-Glu-γ-P_H using GDH. (a) The percentage of L-glutamate and L-Glu-γ-P_H conversion into α-KG and α-KG-γ-P_H, respectively, with/without buffer. Reactions (500 µL) were performed without buffer at pH 9.0 in a mixture containing L-glutamate (10 mM) or L-Glu-γ-P_H (10 mM) and NAD⁺ (5 mM) at 25 °C, initiated by the addition of GDH (14 µg). A similar mixture in Tris–HCl buffer (100 mM, pH 9.0) was used as a control. Results are shown as means ± SD of n = 3 independent assays, representative of n = 3 independent experiments. (b) Accumulation of α-KG-γ-P_H over time in preparative synthesis. Reaction (100 mL) was performed at 25 °C in a buffer-free system at pH 9.0 in a mixture containing L-Glu-γ-P_H (10 mM) and NAD⁺ (5 mM) and initiated by addition of GDH (5 mg).

Figure 5

NMR spectra of α-KG-γ-P_H existing in aqueous solution as an equilibrium mixture of keto (I) and dihydroxy (II) forms (equation on the top). Panels (A1–A4): ¹H-³¹P HMBC spectrum cut-away views containing ³¹P correlation cross-peaks with phosphorus-bound proton highlighted yellow in formulas above. Panel (B): ¹H-decoupled ³¹P-³¹P NOESY spectrum displaying strong (I) and (II) exchange peaks (pointed by arrow marks). Note: vertical scale is common for all panels.

Figure 6

Chemical structures of α-amino-H-phosphinic (III) and α-aminophosphonic acids (IV) confronted with amino acids (in the middle). The H-phosphinic group possesses a hydrogen atom (highlighted in yellow) that allows this group to acquire a flattened tetrahedral geometry making it bioisostere of the carboxyl group.