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

Glutamate decarboxylase (GadB), a pyridoxal 5'-phosphate (PLP)-dependent enzyme from *Escherichia coli*, is highly specific for L-glutamate and is used for GABA production. This research explores the enzyme's activity with a phosphinic analog of glutamate, D,L-2-amino-4-(hydroxyphosphinyl)butyric acid (D,L-Glu-γ-PH), also known as desmethylphosphinothricin (DMPT). DMPT is a key intermediate in the biosynthesis of the herbicide bialaphos and has shown antibacterial activity. Little is known about its biological activity and its interaction with glutamate-metabolizing enzymes. This study hypothesizes that the phosphinic group in DMPT could mimic the carboxyl group of glutamate, allowing EcGadB to recognize and process it. This investigation aims to determine if EcGadB can decarboxylate D,L-Glu-γ-PH and to explore the metabolic fate of the resulting product. The study's importance lies in understanding the metabolic versatility of EcGadB and exploring the potential of phosphinic compounds as bioisosteres for drug design. The study also explores the potential for using EcGadB's stereospecificity for the kinetic resolution of D,L-Glu-γ-PH to yield both the L- and D- isomers, providing access to an otherwise challenging compound for further investigation. Existing literature supports the idea that certain phosphonate and phosphinate compounds can mimic phosphate monoesters or tetrahedral intermediates of carboxyl group transformations. However, the substrate properties of phosphinic analogs of glutamate on glutamate-metabolizing enzymes, especially bacterial glutamate decarboxylase, haven't been explored thoroughly. This research addresses this gap in knowledge.

Previous research has extensively characterized EcGadB's structure and biochemical properties, including its high specificity for L-glutamate and its efficient immobilization for GABA production. Several studies have explored EcGadB's activity with various glutamate analogs, although phosphinic analogs have received scant attention. The literature indicates that phosphonic and phosphinic compounds—containing a carbon-phosphorus bond—occur naturally and are used in medicine and agriculture. Notable examples include the antibiotic fosfomycin and the herbicide bialaphos (containing phosphinothricin, PT), which are known to interact with glutamate-metabolizing enzymes. PT, for instance, inhibits glutamine synthetase. DMPT, a phosphinic analogue of glutamate, has shown some in vitro activity with metabotropic glutamate receptors and antibacterial properties in recent work. However, its behavior as a substrate or inhibitor for glutamate decarboxylases has not yet been systematically investigated.

The study employed a multifaceted approach including enzymatic assays, NMR spectroscopy (¹H, ¹³C, ³¹P NMR), molecular dynamics (MD) simulations, and antimicrobial assays. Initially, thin-layer chromatography (TLC) and ¹H NMR were used to investigate the reaction of D,L-Glu-γ-PH with EcGadB. MD simulations were performed using the AMBER 12 suite to compare the binding modes of L-Glu and L-Glu-γ-PH to EcGadB's active site. Kinetic parameters (kcat and Km) for EcGadB's reaction with L-Glu-γ-PH were determined using a coupled assay with GABase, a preparation containing GABA-transaminase and succinic semialdehyde dehydrogenase. This allowed for spectrophotometric detection of NADPH produced from the oxidation of the carbonyl group in the metabolic product. ³¹P NMR spectroscopy was used to confirm the metabolic pathway of GABA-PH through GABase. To synthesize D-Glu-γ-PH, a kinetic resolution approach was employed. The racemic D,L-Glu-γ-PH was subjected to EcGadB reaction, leaving the D-isomer unreacted and allowing the isolation of both D-Glu-γ-PH and GABA-PH after ultrafiltration and ion-exchange chromatography using Dowex 50WX8 resin. The purification involved the use of aminooxyethyl putrescine (AOEPUT) to separate D-Glu-γ-PH from PLP. The enantiomers were then characterized using NMR spectroscopy and optical rotation. Finally, the minimum inhibitory concentrations (MIC) of D,L-Glu-γ-PH, L-Glu-γ-PH, and D-Glu-γ-PH against E. coli K12 strain MG1655 were determined using the broth dilution method to assess their antibacterial activity.

The key findings demonstrate that EcGadB quantitatively decarboxylates L-Glu-γ-PH to GABA-PH. The molecular modeling studies revealed similar binding modes for L-Glu and L-Glu-γ-PH in the EcGadB active site, indicating that the phosphinic group acts as a bioisostere for the carboxyl group. Kinetic analysis showed that while EcGadB accepts L-Glu-γ-PH as a substrate, its efficiency is significantly lower than with L-Glu (over 200-fold). This is attributed to differences in the binding affinities of the carboxyl and phosphinic groups in the active site. Further experimentation confirmed that GABase can metabolize GABA-PH to Succinate-PH. This represents the first direct evidence of NAD(P)+-dependent enzymatic oxidation of a functional group in an alkylphosphinic acid's side chain, leaving the P-H bond intact. Crucially, the kinetic resolution of D,L-Glu-γ-PH using EcGadB successfully yielded preparative amounts of both GABA-PH and D-Glu-γ-PH. The antimicrobial assays showed that only L-Glu-γ-PH exhibits significant antibacterial activity, with a MIC90 comparable to ampicillin; D-Glu-γ-PH showed minimal activity.

The findings strongly support the hypothesis that the phosphinic group can act as a bioisostere for the carboxyl group in certain enzymatic reactions. This expands the understanding of enzyme substrate specificity and metabolic pathways involving phosphinic compounds. The successful kinetic resolution of D,L-Glu-γ-PH using EcGadB provides a valuable method for preparing both GABA-PH and the previously unknown D-Glu-γ-PH. The observation that L-Glu-γ-PH exhibits antibacterial activity while the D-isomer is inactive highlights the importance of stereochemistry in biological activity and opens avenues for drug design involving phosphinic acid analogs. The efficient conversion of GABA-PH to Succinate-PH by GABase expands knowledge of enzymatic transformations of phosphinic compounds, revealing the potential for their intracellular metabolism to target different enzymes.

This study demonstrates that the phosphinic group can function as a bioisostere of the carboxyl group in enzymatic reactions involving EcGadB and GABase. The enzymatic kinetic resolution of D,L-Glu-γ-PH provides an efficient method for preparing GABA-PH and D-Glu-γ-PH. Further research could explore the potential of phosphinic compounds as prodrugs, exploiting their metabolic transformations to target specific enzymes. Investigation into other phosphinic analogs and enzymes is warranted to expand the scope of bioisosteric replacements.

The study primarily focuses on EcGadB and GABase from *E. coli* and *P. fluorescens*, respectively. Generalizing the findings to other enzymes and organisms might require further investigation. The antimicrobial activity assessments were conducted using a single *E. coli* strain. Expanding the scope of antimicrobial testing to other bacterial species and strains would strengthen the results. The molecular modeling study used a simplified approach, and more sophisticated simulations might reveal additional insights into the interactions between the enzymes and substrates.