Studies on recently identified activities of the Jumonji-C oxygenase JMJD4 and the bacterial class D nucleophilic serine β-lactamases

The Jumonji-C oxygenase JMJD4 and the bacterial class D nucleophilic serine β-lactamases (SBLs) have been recently found to catalyse unprecedented cyclisation reactions. Biochemical, biophysical, and inhibition studies were carried out to investigate the formation of β-lactones from carbapenems by class D SBLs, and the JMJD4 catalysed γ-lactam ring formation of eukaryotic release factor 1 (eRF1). Chapter 1 gives an introduction to the β-lactamase and oxygenase enzyme families to which the class D SBLs and JMJD4 belong, respectively.
The mechanism of β-lactone formation by class D SBLs was investigated through biochemical studies with variants of wild-type OXA-48 and OXA-23 in which active site residues not directly involved in catalysis were substituted (Chapter 2). The results reveal that active site residues that interact with the carbapenem-derived C-6 hydroxyethyl side chain impact both activity and product profile. OXA-23 V128L and the clinically observed variant OXA-48 V120L were observed to extend the β-lactone formation activity of class D SBLs to 1β-hydrogen substituted carbapenems.
β-Lactones are capable of re-acylating the nucleophilic serine residues in the active sites of some SBLs and could act as inhibitors. Inhibition studies revealed the Δ1 imine tautomers of β-lactones derived from meropenem were less potent than their parent carbapenem meropenem (Chapter 3). Analyses employing NMR, and mass spectrometry indicated that the complex derived from class D SBLs and β-lactones can (re-)form β-lactones to a greater degree than observed with the corresponding complex derived from carbapenems.
Carbapenems bearing electron-donating and electron-withdrawing C-2 thiophenol side chains were used to investigate the mechanism of class D SBL carbapenemase activity (Chapter 4). It was proposed that the electronic properties of this C-2 side chain might influence kinetics and product distribution. The NMR studies showed differences in the turnover rate and the extent of β-lactone formation with carbapenem analogues.
Studies on the human JMJD4-catalysed oxidative γ-lactam ring formation with eRF1 are described in Chapter 5. eRF1 is responsible for translation termination and functional studies have shown that hydroxylation of eRF1 improve translation termination efficiency. Although the initial (4R)-hydroxylation of eRF1 by JMJD4 is established, less is known about the process by which Lys63 is modified to form a γ-lactam ring. Biochemical studies confirmed that γ-lactam ring formation depends on catalytic activity of JMJD4, and that Fe(II) and 2-oxoglutarate are required for and that L-ascorbate promotes its formation. Functional analysis of the mouse JMJD4 orthologue demonstrated that the oxidative γ-lactam ring formation is conserved; however, the yeast orthologue showed only hydroxylation activity.
Overall, the research described in this thesis provides new insights into the molecular mechanisms of β-lactone formation catalysed by class D SBLs and γ-lactam ring formation catalysed by JMJD4.

Keywords: post-translational modification
mutagenesis, site-directed
kinetics
enzymology
Advisor: Schofield, C