Enzymatic kinetic resolution of desmethylphosphinothricin indicates that phosphinic group is a bioisostere of carboxyl group

Abstract

Escherichia coli glutamate decarboxylase (EcGadB), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, is highly specific for L-glutamate and was demonstrated to be effectively immobilised for the production of γ-aminobutyric acid (GABA), its decarboxylation product. Herein we show that EcGadB quantitatively decarboxylates the L-isomer of D,L-2-amino-4-(hydroxyphosphinyl)butyric acid (D,L-Glu-γ-P_H), a phosphinic analogue of glutamate containing C-P-H bonds. This yields 3-aminopropylphosphinic acid (GABA-P_H), a known GABA_B receptor agonist and provides previously unknown D-Glu-γ-P_H, allowing us to demonstrate that L-Glu-γ-P_H, but not D-Glu-γ-P_H, is responsible for D,L-Glu-γ-P_H antibacterial activity. Furthermore, using GABase, a preparation of GABA-transaminase and succinic semialdehyde dehydrogenase, we show that GABA-P_H is converted to 3-(hydroxyphosphinyl)propionic acid (Succinate-P_H). Hence, PLP-dependent and NADP⁺-dependent enzymes are herein shown to recognise and metabolise phosphinic compounds, leaving unaffected the P-H bond. We therefore suggest that the phosphinic group is a bioisostere of the carboxyl group and the metabolic transformations of phosphinic compounds may offer a ground for prodrug design.

Fig. 1. Chemical structures of DMPT and its metabolically related derivatives.

The herbicide bialaphos (PTT), produced by S. viridochromogenes and S. hygroscopicus, is a tripeptide containing L-phosphinothricin (PT) followed by alanyl-alanine (L-Ala-LAla) residues. In the biosynthetic pathway leading to PTT, desmethylphosphinothricin (DMPT; L-Glu-γ-P_H) is a key intermediate, from which 3-aminopropylphosphinic acid (3-APPA; GABA-P_H) and Succinate-P_H can be derived following in order DMPT decarboxylation and oxidation, respectively.

Fig. 2. Retrosynthetic analysis of the D-Glu-γ-P_H.

(A) From racemic D,L-Glu-γ-P_H (this work, in blue) in one enzymatic step; (B) from D-2-aminobutyrolactone hydrochloride (3) via o-nitrophenyl-sulfoxide (5) in six steps; (C) from D-2-aminobutyrolactone hydrochloride (3) via phenylselenide (4) in six steps; (D) from D-methionine via sulfoxide (6) in three steps. Pathways (B)–(D) lead to the same key precursor methyl N-Cbz-D-vinylglycine (2).

Fig. 3. Proposed binding mode for studied adducts.

Snapshots of the molecular dynamic simulations. a Proposed binding mode for PLP-L-Glu adduct (cyan). b Proposed binding mode for PLP-L-Glu-γ-P_H adduct (cyan). Residues involved in interaction are reported as stick, yellow for chain B and green for chain A of the functional dimer. PLP and acetic acid (ACY) of 1PMM are also reported as magenta lines. The enzyme is depicted as grey cartoon and the H-bonds are reported as yellow dotted lines. Active site residues contributed by the other subunit in the functional dimer are also labelled with an asterisk.

Fig. 4. Enzymatic reactions that yield Succinate-P_H and NADPH starting from D,L-Glu-γ- P_H.

The EcGadB (0.09 mg ml⁻¹) decarboxylation reaction of D,L-Glu-γ- P_H (18 mM) was carried out at pH 4.6, in 10 mM sodium acetate buffer (pH adjusted with small additions of diluted HCl) and then halted after 4 h by removing EcGadB by ultrafiltration. Half of the flow-through was dried and resuspended in an isovolume of GABase assay solution where the two indicated enzymatic reactions were run one-pot, with succinic semialdehyde dehydrogenase activity making the whole process irreversible. ³¹P NMR spectra of: (a) EcGadB reaction mixture at time zero; (b) EcGadB reaction mixture after interruption by enzyme removal; (c) reaction mixture after GABase treatment; (d) model mixture of L-Glu-γ-P_H, GABA-P_H, and Succinate-P_H.

Fig. 5. ³¹P NMR spectroscopy analysis.

The kinetics of the decarboxylation of D,L-Glu-γ-P_H by EcGadB yielding GABA-P_H are shown. The time point (in minutes) at which each measurement was done is shown on the right and next to it (in brackets) is provided the Glu-γ-P_H/GABA-P_H ratio obtained by peak integration. Fragments of the ³¹P NMR spectra are shown as a stack.

Fig. 6. Phosphinic compounds as substrates of bacterial enzymes.

EcGadB converts at mildly acidic pH (yellow background) the L-isomer of D,L-Glu-γ-P_H (carbon skeleton in yellow on the outmost right), quantitatively yielding GABA-P_H (carbon skeleton in yellow on the bottom left) and leaving D-Glu-γ-P_H unmodified (carbon skeleton in cyan on the outmost right). The former becomes a substrate of GABase that yield 3-(hydroxyphosphinyl)propionic acid (i.e., Succinate-P_H shown with carbon skeleton in yellow on the top centre) in a coupled reaction that occurs at mildly alkaline pH (blue background).