This thesis is focussed on structural and functional studies of a novel glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. GpdQ is highly promiscuous and is the first known phosphatase which is capable of degrading all three classes of phosphate esters (mono-, di- and triesters). Remarkably, GpdQ is also able to hydrolyse stable aliphatic phosphate esters and has been shown to degrade the hydrolysis product of the nerve agent VX. For these reasons, GpdQ has been realised to have potential as a powerful bioremediator for the removal of organophosphate pesticides and nerve agents. GpdQ is a binuclear metallohydrolase in which one of the metal ions is very weakly bound. Chapter 1 introduces the catalytic mechanisms of binuclear metallohydrolases by examining two related phosphate ester-degrading enzymes. Since one of the main features of catalysis addressed in this thesis are the differential metal binding affinities of GpdQ, Chapter 1 also canvasses a range of other binuclear metallohydrolases with similar behaviour. Chapter 2 examines the structural and evolutionary relationship between GpdQ and a number of other related enzymes. Using genome database searches, the two most closely related enzymes are identified. In performing these searches, a novel, putative binuclear metallohydrolase from Homo sapiens is also discovered. This enzyme, Hsa_aTRACP, is most closely related to PAPs, however construction of a homology model indicates that the active site tyrosine residue of PAP is replaced by histidine. In this respect, it may represent an evolutionary link to Ser/Thr protein phosphatases and GpdQ. The biology and chemistry of this putative enzyme is discussed. PAPs are the only binuclear enzymes with an established heterovalent active site of the type Fe(III)-M(II) (where M=Fe, Zn or Mn) whereas the majority of enzymes in this family have homovalent metal centres, including GpdQ and Ser/Thr protein. This is brought about due to the nature of the coordination sphere imposed by the enzyme. The activity of GpdQ can be reconstituted in the presence of Co(II), Zn(II), Mn(II) and Cd(II). Chapter 3 examines the kinetic properties of a binuclear homovalent system by studying the kinetic properties of Cd(II)-substituted GpdQ and a corresponding model complex. This comparative study leads to the identification of a terminal hydroxide molecule as the likely reaction-initiating nucleophile in Cd(II)-GpdQ with a pKa of 9.4. In Chapter 4, a detailed study of the structural, kinetic and spectroscopic behaviour of Co(II)-substituted GpdQ is presented. This chapter specifically probes the formation of the binuclear active site, the role of the metal ions in catalysis, the identity of the nucleophile and the potential role of any first or second coordination sphere residues in the regulation of enzyme activity, proton donation and metal ion coordination. Based on these findings, a detailed reaction mechanism is proposed in which the substrate itself promotes the formation of the catalytically competent binuclear centre and phosphorolysis occurs following nucleophilic attack by a terminal hydroxide molecule. A potential role of Asn80 (a ligand of one of the metal ions) in regulating both substrate and metal binding, and the role of the bridging hydroxide molecule in the activation of the terminal nucleophile is proposed. Chapter 5 employs a combination of kinetic and spectroscopic techniques to probe the proposed catalytic mechanism of GpdQ in depth. The formation of the catalytically competent binuclear centre is observed in pre-steady state studies, an integral first step in the catalytic mechanism. The dissociation and rate constants associated with formation of the binuclear centre are quantified. The rate of substrate turnover in GpdQ is relatively modest but is enhanced by a structural rearrangement involving the flexible Asn80 ligand. This structural change fine-tunes the reaction mechanism, leading to optimal reactivity. The steady-state kinetic properties of a series of metal ion derivatives (Co(II), Cd(II) and Mn(II)) of GpdQ and their reactivity towards a number of substrates are also compared. These findings lead to the conclusion that the reaction mechanism of GpdQ is modulated by both substrate and metal ion. In this respect, GpdQ is adaptive to the environmental conditions to which it is exposed by employing a flexible mechanistic strategy to achieve catalysis. Chapter 6 correlates the electronic and geometric structure of the binuclear centre in GpdQ as a means to probe specific aspects of the mechanism. This study uses the wild type enzyme and a site-directed mutant (Asn80Asp) to examine the structure of the metal ions at two stages of catalysis. The role of the bridging hydroxide molecule in nucleophilic activation is specifically addressed by monitoring changes in the electronic exchange interaction and other structural parameters as a result of phosphate binding. Also, the coordination environment of the metal ions in both the free enzyme and the phosphate-bound enzyme of wild type and Asn80Asp GpdQ were assessed against the currently proposed structures. The findings in this chapter corroborate the proposed catalytic mechanism of GpdQ. In summary, this project led to a detailed understanding of the mechanism of GpdQ, and provided insight into how both the metal ion composition and the identity of the substrate may modulate this mechanism. The knowledge gained may lead to the design of catalytically more efficient derivatives (mutants) of GpdQ for application in bioremediation.

This thesis is focussed on structural and functional studies of a novel glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. GpdQ is highly promiscuous and is the first known phosphatase which is capable of degrading all three classes of phosphate esters (mono-, di- and triesters). Remarkably, GpdQ is also able to hydrolyse stable aliphatic phosphate esters and has been shown to degrade the hydrolysis product of the nerve agent VX. For these reasons, GpdQ has been realised to have potential as a powerful bioremediator for the removal of organophosphate pesticides and nerve agents. GpdQ is a binuclear metallohydrolase in which one of the metal ions is very weakly bound. Chapter 1 introduces the catalytic mechanisms of binuclear metallohydrolases by examining two related phosphate ester-degrading enzymes. Since one of the main features of catalysis addressed in this thesis are the differential metal binding affinities of GpdQ, Chapter 1 also canvasses a range of other binuclear metallohydrolases with similar behaviour. Chapter 2 examines the structural and evolutionary relationship between GpdQ and a number of other related enzymes. Using genome database searches, the two most closely related enzymes are identified. In performing these searches, a novel, putative binuclear metallohydrolase from Homo sapiens is also discovered. This enzyme, Hsa_aTRACP, is most closely related to PAPs, however construction of a homology model indicates that the active site tyrosine residue of PAP is replaced by histidine. In this respect, it may represent an evolutionary link to Ser/Thr protein phosphatases and GpdQ. The biology and chemistry of this putative enzyme is discussed. PAPs are the only binuclear enzymes with an established heterovalent active site of the type Fe(III)-M(II) (where M=Fe, Zn or Mn) whereas the majority of enzymes in this family have homovalent metal centres, including GpdQ and Ser/Thr protein. This is brought about due to the nature of the coordination sphere imposed by the enzyme. The activity of GpdQ can be reconstituted in the presence of Co(II), Zn(II), Mn(II) and Cd(II). Chapter 3 examines the kinetic properties of a binuclear homovalent system by studying the kinetic properties of Cd(II)-substituted GpdQ and a corresponding model complex. This comparative study leads to the identification of a terminal hydroxide molecule as the likely reaction-initiating nucleophile in Cd(II)-GpdQ with a pKa of 9.4. In Chapter 4, a detailed study of the structural, kinetic and spectroscopic behaviour of Co(II)-substituted GpdQ is presented. This chapter specifically probes the formation of the binuclear active site, the role of the metal ions in catalysis, the identity of the nucleophile and the potential role of any first or second coordination sphere residues in the regulation of enzyme activity, proton donation and metal ion coordination. Based on these findings, a detailed reaction mechanism is proposed in which the substrate itself promotes the formation of the catalytically competent binuclear centre and phosphorolysis occurs following nucleophilic attack by a terminal hydroxide molecule. A potential role of Asn80 (a ligand of one of the metal ions) in regulating both substrate and metal binding, and the role of the bridging hydroxide molecule in the activation of the terminal nucleophile is proposed. Chapter 5 employs a combination of kinetic and spectroscopic techniques to probe the proposed catalytic mechanism of GpdQ in depth. The formation of the catalytically competent binuclear centre is observed in pre-steady state studies, an integral first step in the catalytic mechanism. The dissociation and rate constants associated with formation of the binuclear centre are quantified. The rate of substrate turnover in GpdQ is relatively modest but is enhanced by a structural rearrangement involving the flexible Asn80 ligand. This structural change fine-tunes the reaction mechanism, leading to optimal reactivity. The steady-state kinetic properties of a series of metal ion derivatives (Co(II), Cd(II) and Mn(II)) of GpdQ and their reactivity towards a number of substrates are also compared. These findings lead to the conclusion that the reaction mechanism of GpdQ is modulated by both substrate and metal ion. In this respect, GpdQ is adaptive to the environmental conditions to which it is exposed by employing a flexible mechanistic strategy to achieve catalysis. Chapter 6 correlates the electronic and geometric structure of the binuclear centre in GpdQ as a means to probe specific aspects of the mechanism. This study uses the wild type enzyme and a site-directed mutant (Asn80Asp) to examine the structure of the metal ions at two stages of catalysis. The role of the bridging hydroxide molecule in nucleophilic activation is specifically addressed by monitoring changes in the electronic exchange interaction and other structural parameters as a result of phosphate binding. Also, the coordination environment of the metal ions in both the free enzyme and the phosphate-bound enzyme of wild type and Asn80Asp GpdQ were assessed against the currently proposed structures. The findings in this chapter corroborate the proposed catalytic mechanism of GpdQ. In summary, this project led to a detailed understanding of the mechanism of GpdQ, and provided insight into how both the metal ion composition and the identity of the substrate may modulate this mechanism. The knowledge gained may lead to the design of catalytically more efficient derivatives (mutants) of GpdQ for application in bioremediation.

This thesis is focussed on structural and functional studies of a novel glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. GpdQ is highly promiscuous and is the first known phosphatase which is capable of degrading all three classes of phosphate esters (mono-, di- and triesters). Remarkably, GpdQ is also able to hydrolyse stable aliphatic phosphate esters and has been shown to degrade the hydrolysis product of the nerve agent VX. For these reasons, GpdQ has been realised to have potential as a powerful bioremediator for the removal of organophosphate pesticides and nerve agents. GpdQ is a binuclear metallohydrolase in which one of the metal ions is very weakly bound. Chapter 1 introduces the catalytic mechanisms of binuclear metallohydrolases by examining two related phosphate ester-degrading enzymes. Since one of the main features of catalysis addressed in this thesis are the differential metal binding affinities of GpdQ, Chapter 1 also canvasses a range of other binuclear metallohydrolases with similar behaviour. Chapter 2 examines the structural and evolutionary relationship between GpdQ and a number of other related enzymes. Using genome database searches, the two most closely related enzymes are identified. In performing these searches, a novel, putative binuclear metallohydrolase from Homo sapiens is also discovered. This enzyme, Hsa_aTRACP, is most closely related to PAPs, however construction of a homology model indicates that the active site tyrosine residue of PAP is replaced by histidine. In this respect, it may represent an evolutionary link to Ser/Thr protein phosphatases and GpdQ. The biology and chemistry of this putative enzyme is discussed. PAPs are the only binuclear enzymes with an established heterovalent active site of the type Fe(III)-M(II) (where M=Fe, Zn or Mn) whereas the majority of enzymes in this family have homovalent metal centres, including GpdQ and Ser/Thr protein. This is brought about due to the nature of the coordination sphere imposed by the enzyme. The activity of GpdQ can be reconstituted in the presence of Co(II), Zn(II), Mn(II) and Cd(II). Chapter 3 examines the kinetic properties of a binuclear homovalent system by studying the kinetic properties of Cd(II)-substituted GpdQ and a corresponding model complex. This comparative study leads to the identification of a terminal hydroxide molecule as the likely reaction-initiating nucleophile in Cd(II)-GpdQ with a pKa of 9.4. In Chapter 4, a detailed study of the structural, kinetic and spectroscopic behaviour of Co(II)-substituted GpdQ is presented. This chapter specifically probes the formation of the binuclear active site, the role of the metal ions in catalysis, the identity of the nucleophile and the potential role of any first or second coordination sphere residues in the regulation of enzyme activity, proton donation and metal ion coordination. Based on these findings, a detailed reaction mechanism is proposed in which the substrate itself promotes the formation of the catalytically competent binuclear centre and phosphorolysis occurs following nucleophilic attack by a terminal hydroxide molecule. A potential role of Asn80 (a ligand of one of the metal ions) in regulating both substrate and metal binding, and the role of the bridging hydroxide molecule in the activation of the terminal nucleophile is proposed. Chapter 5 employs a combination of kinetic and spectroscopic techniques to probe the proposed catalytic mechanism of GpdQ in depth. The formation of the catalytically competent binuclear centre is observed in pre-steady state studies, an integral first step in the catalytic mechanism. The dissociation and rate constants associated with formation of the binuclear centre are quantified. The rate of substrate turnover in GpdQ is relatively modest but is enhanced by a structural rearrangement involving the flexible Asn80 ligand. This structural change fine-tunes the reaction mechanism, leading to optimal reactivity. The steady-state kinetic properties of a series of metal ion derivatives (Co(II), Cd(II) and Mn(II)) of GpdQ and their reactivity towards a number of substrates are also compared. These findings lead to the conclusion that the reaction mechanism of GpdQ is modulated by both substrate and metal ion. In this respect, GpdQ is adaptive to the environmental conditions to which it is exposed by employing a flexible mechanistic strategy to achieve catalysis. Chapter 6 correlates the electronic and geometric structure of the binuclear centre in GpdQ as a means to probe specific aspects of the mechanism. This study uses the wild type enzyme and a site-directed mutant (Asn80Asp) to examine the structure of the metal ions at two stages of catalysis. The role of the bridging hydroxide molecule in nucleophilic activation is specifically addressed by monitoring changes in the electronic exchange interaction and other structural parameters as a result of phosphate binding. Also, the coordination environment of the metal ions in both the free enzyme and the phosphate-bound enzyme of wild type and Asn80Asp GpdQ were assessed against the currently proposed structures. The findings in this chapter corroborate the proposed catalytic mechanism of GpdQ. In summary, this project led to a detailed understanding of the mechanism of GpdQ, and provided insight into how both the metal ion composition and the identity of the substrate may modulate this mechanism. The knowledge gained may lead to the design of catalytically more efficient derivatives (mutants) of GpdQ for application in bioremediation.

Mycobacterium species, which contain the causative agent for human tuberculosis (TB), produce inositol derivatives including mycothiol (MSH).  MSH is a unique and dominant cytosolic thiol that protects mycobacterial pathogens against the damaging effects of reactive oxygen species and is involved in antibiotic detoxification.  Therefore, MSH is considered a potential drug target.  The deacetylase MshB catalyzes the committed step in MSH biosynthesis by converting N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins) to 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins).  In this dissertation, we present detailed functional analysis of MshB.  Our work has shown that MshB is activated by divalent metal ions that can switch between Zn2+ and Fe2+ depending on environmental conditions, including  metal ion availability and oxidative conditions.  MshB employs a general acid-base catalyst mechanism wherein the Asp15 functions as a general base to activate the metal-bound water nucleophile for attack of the carbonyl carbon on substrate.  Proton-transfer from a general acid catalyst facilitates breakdown of the tetrahedral intermediate and release of products.  A dynamic tyrosine was identified that regulates access to the active site and participates in catalysis by stabilizing the oxyanion intermediate.  Molecular docking simulations suggest that the GlcNAc moiety on GlcNAc-Ins is stabilized by hydrogen bonding interactions with active site residues, while a hydrophobic stacking interaction between the inositol ring and Met98 also appears to contribute to substrate affinity for MshB.  Additional binding interactions with side chains in a hydrophobic cavity adjacent to the active site were suggested when the docking experiments were carried out with large amidase substrates.  Together the results from this study provide groundwork for the rational design of specific inhibitors against MshB, which may circumvent current challenges with TB treatment. / Ph. D.