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 | Research Achievements |  |
| 1985 - 2007
| Protein binding |
| | The binuclear metallohydrolase 3'-5' exonuclease is part of the DNA replication process and catalyses the hydrolysis of 3'-terminal nucleotides from single-stranded DNA. Its primary role is in proofreading during replication, thus ensuring genome integrity. 3'-5' exonucleases from different sources have a strict requirement for divalent metal ions, in particular Mn(II), Zn(II) and Mg(II), but it is unknown which metal ion is preferred in vivo. Here, the formation of catalytically active binuclear Mn(II)-Mn(II) centres in the 3'-5' exonuclease subunit of Escherichia coli DNA polymerase III was monitored by room temperature electron paramagnetic resonance. In the absence of the substrate analogue thymidine-5'-monophosphate only one Mn(II) ion binds with a Kd1 of ~94 ìM. Upon addition of the substrate analogue two Mn(II) ions bind to the active site with Kd1 ~13 ìM and Kd2 ~420 ìM. Thus, binding of the substrate analogue does not only increase the metal ion affinity of site 1 (Kd1) but it facilitates the binding of a second Mn(II) (Kd2), leading to the formation of a binuclear centre. The exclusive observation of multi-line features between 260 mT and 300 mT in the low-temperature spectrum of 3'-5' exonuclease in the presence of Mn(II) and thymidine-5'-monophosphate indicates an exchange coupling interaction between the bound Mn(II) ions. In the absence of the substrate analogue low-temperature EPR spectra consistent with mononuclear Mn(II) centres were recorded. Thus, it is envisioned that the substrate-dependent formation of catalytically active binuclear metal centres in 3'-5' exonucleases is a mechanism to regulate enzyme activity. | | Keywords: | substrate analogue, nucleotides, DNA, divalent metal ions, binuclear metallohydrolase 3'-5' exonuclease |
| | 1985 - 2007
| Mycophenolic acid (MPA) |
| | Mycophenolic acid (MPA) is a drug that has found widespread use as an immunosuppressive agent which limits rejection of transplanted organs. Optimal use of this drug is hampered by gastrointestinal side-effects which can range in severity. One mechanism by which MPA causes gastropathy may involve a direct interaction between the drug and gastric phospholipids. In order to combat this interaction we have investigated the potential of MPA to coordinate Cu(II), a metal which has been used to inhibit gastropathy associated with use of the NSAID indomethacin. Using a range of spectroscopic techniques we show that Cu(II) is coordinated to two MPA molecules via carboxylates and, at low pH, water ligands. The copper complex formed is stable in solution as assessed by mass spectrometry and 1H NMR diffusion experiments. Competition studies with glycine and albumin indicate that the copper-MPA complex will release Cu(II) to amino acids and proteins thereby allowing free MPA to be transported to its site of action. Transfer to serum albumin proceeds via a Cu(MPA)(albumin) ternary complex. These results raise the possibility that copper complexes of MPA may be useful in a therapeutic situation. | | Keywords: | MPA, gastric phospholipids, Cu(II), Mycophenolic acid |
| | 1985 - 2007
| Multifield saturation magnetization |
| | The purple acid phosphatase from sweet potato is the first reported example of an enzyme containing binuclear Fe-Mn centres. Multifield saturation magnetization data over a temperature range from 2 to 200 K indicates that these centres are strongly antiferromagnetically coupled. Metal ion analysis shows an excess of Fe over Mn. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centres (Fe(III)-apo and Fe(III)-Zn(II)) and Cu(II). There were no resonances from either Mn(II) or binuclear Fe-Mn centres. Oxidation and reduction of the enzyme indicated that homobinuclear metal centres (Fe(III)-Fe(III) , Mn(III)-Mn(III) and Mn(II)-Mn(II)) were not present in the enzyme. Together with a comparison of spectral properties and sequence homologies between known purple acid phosphatases the spectroscopic data strongly indicate the presence of Fe(III)-Mn(II) centres in the active site of the sweet potato enzyme. Due to the strong antiferromagnetism it is likely that the metal ions in the sweet potato enzyme are linked via a µ-oxo bridge, in contrast to other known purple acid phosphatases where a µ-hydroxo bridge is present. Differences in metal ion composition and bridging may affect substrate specificities and hence the biological function of different purple acid phosphatases. | | Keywords: | purple acid phosphatase, sweet potato, metal ion, EPR |
| | 1985 - 2007
| CW EPR spectra |
| | Analysis of the results of continuous wave and pulsed electron paramagnetic resonance spectroscopy and EPR potentiometric titration experiments have shown that the Mo(V) High-g Unsplit Type-2 species is the intermediate species formed during the catalytic reduction of DMSO reductase from Rhodobacter capsulatus. The spin Hamiltonian parameters for the Mo(V) High-g Unsplit Type-2 species obtained from naturally abundandant and 95Mo enriched DMSO reductase reveal that the unpaired electron is present in a |dz2> ground state molecular orbital and that the geometry of the active site Mo centre is trigonal prismatic. Since the tigonal prismatic geometry of the Mo center is retained upon reduction of the resting (Mo(VI)), to Mo(V) and Mo(IV) the active site within DMSO reductase is an example of an entatic state The Mo(V) High-g Split species, previously proposed to be catalytically relevant in the reductive direction, has been shown to be involved in the oxidative half reaction.
Observation of a continuous wave (CW) EPR spectrum attributable to the Low-g Type-I Mo(V) species and a sulfur centered radical generated upon dithionite reduction of dimethylsulfoxide reductase from the photosynthetic bacterium Rhodobacter capsulatus. Computer simulation of the naturally abundant and 95Mo enriched Low-g Type-I Mo(V) CW EPR spectrum reveals that while the magnitudes of the principal components of the g and A matrices resemble the Slow Mo(V) centre found in desulfo xanthine oxidase, their orientation is quite different and the largest 95Mo hyperfine component is associated with the smallest g value rather than the largest g value. The coordination sphere of the Low-g Type-I Mo(V) species consists of an an ene-dithiolene (P-MGD), Ser-147 and a protonated oxo group, which form the base of the trigonal prism. An oxo group and a deprotonated hydroxyethane moiety from Bicine complete the corrdination sphere by capping the trigonal prism. The g and triclinic A(95Mo) matrices are consistent with the unpaired electron located in the ground state molecular orbital in which the x and y axes are located between Mo-ligand bonds and a1 > b1, c1 with b1 and c1 greater than zero. In addition to the Low-g Type-I Mo(V) species the CW EPR spectrum exhibits an orthorhombic g matrix (gz = 2.0545, gy = 2.0182, gx = 1.999) with small 95Mo (A2 = 5.0x10-4 cm-1) hyperfine coupling on the gy resonance. Both 3-pulse ESEEM and HYSCORE spectra revealed the presence of one or more weakly coupled protons and isotropic hyperfine coupling (Aiso(14N) = 6.7 MHz) to a single nitrogen nucleus. The CW- and pulsed-EPR results are consistent with an unpaired electron centered on sulfur atom (S1) of Q-MGD which is delocalized onto the pyranopterin ring system. These results implicate sulfur centered radicals in the stabilization of the charge on the molybdenum ion in DMSO reductase and/or electron transfer between the native electron donor DorC and the Mo center via the Q-MGD. | | Keywords: | epr (cw and pulsed), endor, eldor bioinorganic chemistry, metalloproteins, XSophe, Molecular Sophe, Resonanz, high performance computing |
| | 1985 - 2007
| EPR Facility |
| | I am committed to maintaining a world class EPR facility and providing expertise in EPR spectroscopy to users within and outside the University of Queensland. Towards this end my group have:
• during the last two years, undertaken the development of an integrated approach for the computer simulation of continuous wave and pulsed EPR and ENDOR spectra, energy level diagrams, transition roadmaps and transition surfaces. This approach, based on molecular structure, will revolutionise the 3-dimensional molecular characterisation of paramagnetic materials using EPR spectroscopy as until now the analysis of complex CW and pulsed EPR spectra has been based on a spin system rather than molecular structure. The approach employing object oriented programming has involved the development of a:
• completely new X-windows interface (Molecular Sophe) written in C++ and employing the GUI builder BxPro,
• general C++ parser allowing the input/output of spectral, spin Hamiltonian and structural parameters and enabling the expansion of experiments (pulse sequences) to be easily integrated in the future,
• C++ version of Sophe for the analysis of CW and pulsed EPR and ENDOR spectra. This software has been based around the SOPHE grid (patented) and has employed the mosaic misorientation linewidth model, frequency domain pulsed simulations, Floquet theory and distributions of spin Hamiltonian and structural (internuclear distances and orientations) parameters.
A working version of Molecular Sophe v2.0.63 is available
| | Links: | EPR |
| Keywords: | epr (cw and pulsed), endor, eldor bioinorganic chemistry, metalloproteins, XSophe, Molecular Sophe, Resonanz, high performance computing |
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