A Methylidene Group in the Phosphonic Acid Analogue of Phenylalanine Reverses the Enantiopreference of Binding to Phenylalanine Ammonia‐Lyases

Abstract

Aromatic amino acid ammonia‐lyases and aromatic amino acid 2,3‐aminomutases contain the post‐translationally formed prosthetic 3,5‐dihydro‐4‐methylidene‐5H‐imidazol‐5‐one (MIO) group. MIO enzymes catalyze the stereoselective synthesis of α‐ or β‐amino acid enantiomers, making these chemical processes environmentally friendly and affordable. Characterization of novel inhibitors enables structural understanding of enzyme mechanism and recognizes promising herbicide candidates as well. The present study found that both enantiomers of the aminophosphonic acid analogue of the natural substrate phenylalanine and a novel derivative bearing a methylidene at the β‐position inhibited phenylalanine ammonia‐lyases (PAL), representing MIO enzymes. X‐ray methods unambiguously determined the absolute configuration of all tested enantiomers during their synthesis. Enzyme kinetic measurements revealed the enantiomer of the methylidene‐substituted substrate analogue as being a mirror image relation to the natural l‐phenylalanine as the strongest inhibitor. Isothermal titration calorimetry (ITC) confirmed the binding constants and provided a detailed analysis of the thermodynamic driving forces of ligand binding. Molecular docking suggested that binding of the (R)‐ and (S)‐enantiomers is possible by a mirror image packing.

Substrate Tunnel Engineering Aided by X-ray Crystallography and Functional Dynamics Swaps the Function of MIO-Enzymes

“…The clear-cut electron densities in the structure complexed with the inhibitors confirm the absolute configuration determination of the (S)-APPA inhibitor (Figure 3b) and reveal a surprising enantiomer preference switch generated by the methylidene group in (R)-APEP at the β-position (Figure 3b). 37 Inferring D-Phe binding from this structure, we propose that the positioning of the β-carbon atom compared to Tyr A (Y110, Figure 3c) is the most important structural feature determining enantioselectivity.…”

“…The crystal structures of PcPAL have been solved in apo form (PDB ID: 6H2O) and in complex with previously characterized potent phosphonic acid inhibitors ( R )-(1-amino-2-phenylethyl)­phosphonic acid [( R )-APEP, PDB ID: 6HQF] and ( S )-(1-amino-2-phenylallyl)­phosphonic acid [( S )-APPA, PDB ID: 6F6T] (Figure ). Table S3 lists the details of data collection and structure refinement.…”

“…58 Our previous study showed that the methylidene group at the β-position in APPA altered the enantiopreference of binding of this phosphonic acid analogue of Phe in PALs. 37 Our recent set of experiments introducing the binding pathway-blocking residues from the (S)-selective PaPAM to the (R)-selective TcPAM indicated the potential of structure-based enzyme access tunnel engineering to alter the enantioselectivity of MIO-enzymes (Figure 6). The strict enantiomer preference of the (R)-selective TcPAM altered significantly in both the ligand access tunnel mutants of TcPAM, whereas the double mutant became an (S)-selective β-AL.…”

Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity

“…The rabbit skeletal myosin subfragment-S1 was a kind gift of Máté Gyimesi, Eötvös University, Budapest, Hungary. These proteins were expressed and purified as described previously [23,24,25]. The proteins were dialyzed against a buffer pH 7.5 comprising 20 mM HEPES, 100 mM NaCl and 1 mM TCEP.…”

Synthesis of α-Aminophosphonic Acid Derivatives Through the Addition of O- and S-Nucleophiles to 2H-Azirines and Their Antiproliferative Effect on A549 Human Lung Adenocarcinoma Cells

“…Within this class of drugs, hybrids molecules introducing two potentially pharmacophores, including allylic amine moieties and α-aminophosphonic acid functional groups, such as allylic α-aminophosphonic acid derivatives ( IV and V ), have attracted scarce attention since only a few examples have been reported in the literature. For instance, (1-amino-2-propenyl)phosphonic acid ( IV ) inhibit alanine racemase and D-alanine:D-alanine ligase [ 30 , 31 ], while α-aminophosphonic acid analogue ( V ) of the natural phenylalanine bearing a methylidene at the β-position acts as an inhibitor of phenylalanine ammonia-lyases (PAL) [ 32 ] ( Figure 1 ).…”