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.

The fast spread of bacteria that are resistant to many classes of antibiotics (multidrug resistant) is a global threat to human and animal health with a worrisome scenario ahead. Novel therapeutical strategies are of crucial importance to combat this phenomenon. For this purpose, we investigated the antimicrobial properties of the naturally occurring tripeptide Bialaphos and a dipeptide L-leucyl-L-phosphinoithricin, the synthesis and diastereomers separation of which are herein described. We demonstrate that these compounds are effective on clinical isolates of the human pathogen Klebsiella pneumoniae, causing hospital-acquired and community-acquired infections. The tested isolates were remarkable for their resistance to more than 20 commercial antibiotics of different classes. Based on previous literature data and our experiments consisting of glutamine supplementation, we suggest that both compounds release phosphinothricin—a well-known nanomolar inhibitor of glutamine synthetase—after their penetration in the bacterial cells; and, in this way, exert their antibacterial effect by negatively affecting nitrogen assimilation in this pathogen.

Desmethylphosphinothricin (L-Glu-γ-P_H) is the H-phosphinic analog of glutamate with carbon-phosphorus-hydrogen (C-P-H) bonds. In L-Glu-γ-P_H the phosphinic group acts as a bioisostere of the glutamate γ-carboxyl group allowing the molecule to be a substrate of Escherichia coli glutamate decarboxylase, a pyridoxal 5'-phosphate-dependent α-decarboxylase. In addition, the L-Glu-γ-P_H decarboxylation product, GABA-P_H, is further metabolized by bacterial GABA-transaminase, another pyridoxal 5'-phosphate-dependent enzyme, and succinic semialdehyde dehydrogenase, a NADP⁺-dependent enzyme. The product of these consecutive reactions, the so-called GABA shunt, is succinate-P_H, the H-phosphinic analog of succinate, a tricarboxylic acid cycle intermediate. Notably, L-Glu-γ-P_H displays antibacterial activity in the same concentration range of well-established antibiotics in E. coli. The dipeptide L-Leu-Glu-γ-P_H was shown to display an even higher efficacy, likely as a consequence of an improved penetration into the bacteria. Herein, to further understand the intracellular effects of L-Glu-γ-P_H, ¹H NMR-based metabolomics, and LC-MS-based shotgun proteomics were used. This study included also the keto-derivative of L-Glu-γ-P_H, α-ketoglutarate-γ-P_H (α-KG-γ-P_H), which also exhibits antimicrobial activity. L-Glu-γ-P_H and α-KG-γ-P_H are found to similarly impact bacterial metabolism, although the overall effect of α-KG-γ-P_H is more pervasive. Notably, α-KG-γ-P_H is converted intracellularly into L-Glu-γ-P_H, but the opposite was not found. In general, both molecules impact the pathways where aspartate, glutamate, and glutamine are used as precursors for the biosynthesis of related metabolites, activate the acid stress response, and deprive cells of nitrogen. This work highlights the multi-target drug potential of L-Glu-γ-P_H and α-KG-γ-P_H and paves the way for their exploitation as antimicrobials.

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.

New antibiotics are unquestionably needed to fight the emergence and spread of multidrug-resistant bacteria. To date, antibiotics targeting bacterial central metabolism have been poorly investigated. By determining the minimal inhibitory concentration (MIC) of desmethylphosphinothricin (Glu-γ-P_H), an analogue of glutamate with a phosphinic moiety replacing the γ-carboxyl group, we previously showed its promising antibacterial activity on Escherichia coli. Herein, we synthetized and determined the growth inhibition exerted on E. coli by an L-Leu dipeptide derivative of Glu-γ-P_H (L-Leu-D,L-Glu-γ-P_H). Furthermore, we compared the growth inhibition obtained with this dipeptide with that exerted by the free amino acid, i.e., Glu-γ-P_H, and by their phosphonic and non-desmethylated analogues. All the tested compounds were more effective when assayed in a chemically-defined minimal medium. The dipeptide L-Leu-D,L-Glu-γ-P_H had a significantly improved antibacterial activity (2 μg/mL), at a concentration between the non-desmethytaled (0.1 μg/mL) and the phosphonic (80 μg/mL) analogues. Also, in Bacillus subtilis, the dipeptide L-Leu-D,L-Glu-γ-P_H displayed an activity comparable to that of the antibiotic amoxicillin. This work highlights the antibacterial relevance of the phosphinic pharmacophore and proposes new avenues for the development of novel antimicrobial drugs containing the phosphinic moiety.

l-Homocysteine, formed from S-adenosyl methionine following demethylation and adenosine release, accumulates when the methionine recycling pathway and other pathways become impaired, thus leading to hyperhomocysteinemia, a biomarker in cardiovascular diseases, neurological/psychiatric disorders, and cancer. The partial oxidation of the l-homocysteine thiol group and its decarboxylation on C-alpha lead to the formation of l-homocysteinesulfinic acid (l-HCSA) and homohypotaurine (HHT), respectively. Both compounds are not readily available from commercial suppliers, which hinders the investigation of their biological activities. Herein, the chemical synthesis of l-HCSA, from l-homocystine, was the starting point for establishing the bio-based synthesis of HHT using recombinant Escherichia coli glutamate decarboxylase (EcGadB), an enzyme already successfully employed for the bio-based synthesis of GABA and its phosphinic analog. Prior to HHT synthesis, k_cat (33.92 ± 1.07) and K_M (38.24 ± 3.45 mM) kinetic constants were determined for l-HCSA on EcGadB. The results of our study show that the EcGadB-mediated synthesis of HHT can be achieved with good yields (i.e., 40% following enzymatic synthesis and column chromatography). Purified HHT was tested in vitro on primary human umbilical vein endothelial cells and rat cardiomyoblasts and compared to the fully oxidized analog, homotaurine (OT, also known as tramiprosate), in widespread pharmaceutical use. The results show that both cell lines display statistically significant recovery from the cytotoxic effects induced by H₂O₂ in the presence of HHT.