This thesis describes structural and functional studies of the enzyme Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes and examines the properties of small mimics of this enzyme and related binuclear metallohydrolases such as the metallo-ß-lactamases. Chapter 1 will introduce the research to date regarding mechanism and structure of GpdQ, metallo-ß-lactamases and other dinuclear metallohydrolases. An overview of the different approaches in ligand design for biomimetic complexes will be given. The materials and experimental methods will be described in Chapter 2.
The focus of Chapter 3 will be the close examination of the enzyme GpdQ and its practicability for applications in bioremediation. Implications of the hexameric structure on the enzyme activity will be studied using a mutant obtained from directed evolution. The binding of Fe(II), Co(II), Zn(II), Mn(II) and Cd(II) to the enzyme active site and the role of the metals ions in phosphoester hydrolysis will be investigated using kinetic assays, atomic absorption spectroscopy and magnetic circular dichroism. Furthermore, the role of the hydrogen bond network surrounding the active site will be investigated using two site directed mutants (Ser127Ala and Tyr19Phe).
The syntheses of four dinuclear Zn(II) complexes will be reported in Chapter 4 along with their crystal structures, mass spectrometry and NMR experiments. Mechanistic investigation of the complexes, using an 18O-labeling experiment and 31P-NMR spectroscopy, showed that the nucleophile in phosphoester hydrolysis is a terminal water molecule. Moreover, plots of the catalytic activity towards the activated substrate bis(2,4-dinitrophenyl)phosphate (BDNPP) versus the Hammett parameters of the residues in the para-position of the phenolic oxygen demonstrated that the activity of the complexes can be tuned: the less electron withdrawing the functional group (CH3 > Br > NO2) the higher the activity towards BDNPP.
In Chapter 5 a detailed study of two Cd(II) complexes, which serve as competent phosphoesterase models, will be reported. One of the complexes also exhibits metallo-ß-lactamase activity employing one of the ligand alcohol arm donors as a nucleophile. A mechanism was proposed after in-solution IR, mass spectrometry, 13C-NMR and UV-Vis studies using penicillin G and nitrocefin. Under non-aqueous conditions a blue intermediate, previously reported in a metallo-ß-lactamase, was observed.
Chapter 6 employs a detailed spectroscopic, magnetochemical and kinetic analysis of five dinuclear Co(II) complexes. Using magnetic susceptibility and variable field-variable temperature magnetic circular dichroism (VTVH MCD) measurements, as well as density functional theory (DFT) calculations, detailed insight was gained about the magnetic exchange processes in these types of systems. The Co(II) complexes were competent phosphoesterase mimics. Similar to the Cd(II) complex, only the Co(II) complex with alkoxide donors is active towards ß-lactams. The inactive Co(II) complexes were, however, able to bind β-lactams such as penicillin and nitrocefin, as shown by mass spectrometry and MCD measurements.
The syntheses of two ligands which aim to generate more appropriate structural and functional GpdQ biomimetics are reported in Chapter 7. Crystal structure analysis showed that the Zn(II) complexes of these ligands crystallized in a mixed 6,5-coordinate environment. NMR and mass spectral analysis, however, suggested that these complexes exist mainly as complexes with two hexacoordinate Zn(II) ions in solution. This is supported by the findings from MCD analysis of the Co(II) derivatives of the two asymmetric ligands.
Finally, Chapter 8 will highlight the efforts made towards the immobilization of GpdQ and biomimetics on solid supports for bioremediation purposes. PAMAM (poly amido amine) dendrimer modified magnetite nanoparticles and Merrifield resin were employed as insoluble supports. Thorough characterization using transmission election microscopy (TEM), X-ray photoelectron spectroscopy (XPS), microanalysis and IR was conducted. The kinetic analysis showed that both the enzyme and biomimetics can be stored on the solid support without loss of activity.
In summary, this project has led to an enhanced understanding of the metal ion binding and active site structural features of GpdQ and, ultimately, to a mutant (Ser127Ala) with improved ability to hydrolyze organophosphates. Furthermore, a number of Zn(II), Cd(II) and Co(II) complexes were spectroscopically and mechanistically characterized, some of them among the most active biomimetics towards organophosphates reported to date. A first step was made towards developing a practical and recyclable system for bioremediation by immobilizing GpdQ and the biomimetics on solid supports.