Antibiotics have saved millions of lives from infectious diseases over the last 70 years, and penicillin has been one of the most successful among them since its discovery. As a canonical β-lactam antibiotic, penicillin works as an inhibitor of transpeptidase, which is involved in cell wall synthesis, and causes cell autolysis. However, resistance emerged as the result of widespread usage of penicillin. Resistant bacteria express β-lactamases to hydrolyze the β-lactam ring in most of the β-lactam antibiotics. Unlike canonical β-lactam antibiotics, tabtoxinine-β-lactam (TβL), which is a natural product by plant pathogenic Pseudomonas syringae, does not inhibit transpeptidase. Instead, TβL is a potent inhibitor of glutamine synthetase (GS). GS is crucial for nitrogen metabolism. It catalyzes the conversion of L-Glu and NH3 to L-Gln. Inhibition of GS can cause accumulation of L-Glu and NH3 and blocks downstream production of amino acid, purines and amino sugars. GS inhibition by TβL is time- and ATP-dependent, but the mechanism of inhibition is still unknown. We hypothesized that GS catalyzes the ATP-dependent phosphorylation of TβL to generate a potent transition state inhibition complex (GS-ADP-TβL-Pi). To test our hypothesis, we detected TβL-Pi released from denatured GS-ADP-TβL-Pi complex by LC-mass spectrometry. Furthermore, we characterized the site of TβL phosphorylation within the intact GS-ADP-TβL-Pi complex using solid state NMR. Computational models show that phosphorylated TβL resembles GS tetrahedral intermediate. The potency of TβL was compared with two best-known GS inhibitors: MetSox and Glufos by using Kitz-Wilson model, in which GS from Homo sapiens, E. coli and S. aureus was used to test inhibitor specificity. This research opens up a new direction of β-lactams design: inhibiting enzymes in the ATP-dependent carboxylate-amine ligase superfamily, which has broad therapeutic potential in many disease areas.

Mechanistic basis for ATP-dependent inhibition of glutamine synthetase by Tabtoxinine-β-lactam

256th National Meeting (2018)

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