Metallocofactors are ubiquitous in nature, serving multiple purposes in proteins. These metallocofactors typically act as the site of catalysis or as an electron relay to move electrons within the protein, or within the cell, and are very energetically costly to manufacture. Yet, in nature it can appear that supernumerary, or ‘auxiliary’ cofactors are apparent, with no clear function. In this thesis, I address the question of what roles additional cofactors play, and why they are retained. The radical S-adenosylmethionine (AdoMet) enzyme superfamily has displayed great diversity in the cofactor requirements for its members. Some members of this family contain only the canonical [4Fe-4S] cluster, which reductively cleaves AdoMet to initiate chemistry, while others have additional [2Fe-2S] or [4Fe-4S] clusters. Even greater cofactor complexity is seen with the B12-dependent subclass, featuring a cobalamin-binding domain in addition to the canonical FeS cluster. The majority of this thesis has focused on using the technique of protein film electrochemistry (PFE) to study members of various subclasses of this superfamily: a dehydrogenase: BtrN, two methylthiotransferases: MiaB and RimO, as well as OxsB and TsrM, two B12-dependent enzymes. By evaluating the redox properties of members of different subclasses, we have been able to shed light on the redox properties of this superfamily, in general, and observed that the redox properties of auxiliary clusters can differ widely between subclasses (e.g. BtrN versus MiaB). PFE has also been used to evaluate five ferredoxins that are possible electron donors for MiaB from Thermotoga maritima. Additionally, bacterial cytochrome c peroxidases (bCCPs) are diheme enzymes catalyzing the detoxification of hydrogen peroxide; however, a novel subclass of bCCPs containing a third heme-binding motif has been identified in enteric pathogens. Protein film electrochemistry has been used to study the redox properties of Escherichia coli YhjA, a member of this subgroup. Further characterization of this novel bCCP was achieved with electron paramagnetic resonance, optical spectroscopy, and steady-state kinetics. Through characterizing YhjA and members of the AdoMet radical enzyme superfamily, we have shed light on the role these additional cofactors play in the mechanism and how these enzymes are tuned for their specific chemistries. / 2018-08-11T00:00:00Z

For many enzymes to function correctly they must have the freedom to display a level of dynamics or communication during their catalytic cycle. The effects that protein dynamics and communication can have are wide ranging, from changes in substrate specificity or product profiles, to speed of reaction or switching activity on or off. This project investigates the protein dynamics and communication in two separate systems, a non-ribosomal peptide synthetase (NRPS), and a protein arginine methyltransferase (PRMT). PRMT1, the enzyme responsible for 80% of arginine methylation in humans, has been implicated in a variety of disease states when functioning incorrectly. For this reason, much focus has been placed on better understanding how PRMT1 determines which products it creates and at what times. This project aims to shed light on how dynamics and communication within PRMT1 dictate its activity. We have to this point developed a protocol for creating and purifying a linked PRMT1 construct which will enable us to conduct the necessary experiments capable of answering our larger questions about the PRMT1 catalytic mechanism. Our collaborators in the Zhan lab discovered the presence of a methyltransferase (Mt) in the two NRPS systems they study, which produce two different and medically relevant compounds, bassianolide and beauvericin. The Hevel lab is well suited to study methyltransferases and so were asked to help evaluate the role of these Mt domains and how they affect the production of the relevant natural products. Achieving a more complete understanding of these systems will move us closer toward the “holy grail” of being able to manipulate and harness NRPS systems for the engineering of novel medically relevant compounds. This project has found that the Mt domain substrate specificity is affected by the surrounding protein domains, or even small portions of them.

Many higher plants can synthesize methyl chloride gas via a common metabolic route, also known as the biological chloride methylation. The reaction is catalyzed by an S-adenosyl-L- methionine (AdoMet) dependent halide/thiol methyltransferase (H/TMT). It is speculated that plants use chloride methylation to remove excess chloride via volatilization and hence maintain homeostatic levels of cytoplasmic chloride ion, suggesting a role of H/TMT in salt tolerance. In this project, the effect of engineering a Brassica oleracea thiol methyltransferase (BoTMT) into tomato was studied to determine the physiological relevance of this enzyme in conferring salt tolerance. Transgenic tomato plants acquired the ability to release methyl chloride in response to NaCl treatment, but exhibited no greater tolerance to NaCl, based on several morphological and physiological measurements, as compared to the wild-type plants. The results indicate that AdoMet dependent chloride methylation is unlikely to contribute to an increase in salt tolerance in higher plants.

Many higher plants can synthesize methyl chloride gas via a common metabolic route, also known as the biological chloride methylation. The reaction is catalyzed by an S-adenosyl-L- methionine (AdoMet) dependent halide/thiol methyltransferase (H/TMT). It is speculated that plants use chloride methylation to remove excess chloride via volatilization and hence maintain homeostatic levels of cytoplasmic chloride ion, suggesting a role of H/TMT in salt tolerance. In this project, the effect of engineering a Brassica oleracea thiol methyltransferase (BoTMT) into tomato was studied to determine the physiological relevance of this enzyme in conferring salt tolerance. Transgenic tomato plants acquired the ability to release methyl chloride in response to NaCl treatment, but exhibited no greater tolerance to NaCl, based on several morphological and physiological measurements, as compared to the wild-type plants. The results indicate that AdoMet dependent chloride methylation is unlikely to contribute to an increase in salt tolerance in higher plants.

No description available.

The persistence and propagation of bacterial antibiotic resistance presents significant challenges to the treatment of drug resistant bacteria with current antimicrobial chemotherapies, while a dearth in replacements for these drugs persists. The thiopeptide family of antibiotics may represent a potential source for new drugs and thiostrepton, the prototypical member of this antibiotic class, is the primary subject under study in this thesis. Using a facile semi-synthetic approach novel, regioselectively-modified thiostrepton derivatives with improved aqueous solubility were prepared. In vivo assessments found these derivatives to retain significant antibacterial ability which was determined by cell free assays to be due to the inhibition of protein synthesis. Moreover, structure-function studies for these derivatives highlighted structural elements of the thiostrepton molecule that are important for antibacterial activity. Organisms that produce thiostrepton become insensitive to the antibiotic by producing a resistance enzyme that transfers a methyl group from the co-factor S-adenosyl-L-methionine (AdoMet) to an adenosine residue at the thiostrepton binding site on 23S rRNA, thus preventing binding of the antibiotic. Extensive site-directed mutagenesis was performed on this enzyme to generate point mutations at key active site residues. Ensuing biochemical assays and co-factor binding studies on these variants identified amino acid residues in the active site that are essential to the formation of the AdoMet binding pocket and provided direct evidence for the involvement of an active site arginine in the catalytic mechanism of the enzyme. Certain bacteria that produce neither thiostrepton nor the resistance methyltransferase express the thiostrepton binding proteins TIP-AL and TIP-AS, that irreversibly bind to the antibiotic, thereby conferring resistance by sequestration. Here, it was found that the point mutation of the previously identified reactive amino acid in TIP-AS did not affect covalent binding to the antibiotic, which was immediately suggestive of a specific, high affinity non-covalent interaction. This was confirmed in binding studies using chemically synthesized thiostrepton derivatives. These studies further revealed structural features from thiostrepton important in this non-covalent interaction. Together, these results indicate that thiostrepton binding by TIP-AS begins with a specific non-covalent interaction, which is necessary to properly orient the thiostrepton molecule for covalent binding to the protein. Finally, the synthesis of a novel AdoMet analogue is reported. The methyl group of AdoMet was successfully replaced with a trifluoromethyl ketone moiety, however, the hydrated form (germinal diol) of this compound was found to predominate in solution. Nevertheless, the transfer of this trifluoroketone/ trifluoropropane diol group was demonstrated with the thiopurine methyltransferase.

Thiostrepton (TS: TS; C72H85N19O18S5) is a thiazoline antibiotic that is effective against Gram-positive bacteria and the malarial parasite, Plasmodium falciparum. Tight binding of TS to the bacterial L11-23S ribosomal RNA (rRNA) complex of the large 50S ribosomal unit inhibits protein biosynthesis. The TS producing organism, Streptomyces azureus, biosynthesizes thiostrepton-resistance methyltransferase (TSR), an enzyme that uses S-adenosyl-L-methionine (AdoMet) as a methyl donor, to modify the TS target site. Methylation of A1067 (Escherichia coli ribosome numbering) by TSR circumvents TS binding. The S. azureus tsr gene was overexpressed in E. coli and the protein purified for biochemical characterization. Although the recombinant protein was produced in a soluble form, its tendency to aggregate made handling a challenge during the initial stages of establishing a purification protocol. Different purification conditions were screened to generate an isolation protocol that yields milligram quantities of protein with little aggregation and sufficient purity for crystallographic studies. Enzymological characterization of TSR was carried out using an assay to monitor AdoMet-dependent ([methyl-3H]-AdoMet) methylation of the rRNA substrate by liquid scintillation counting. During the optimization of assay, it was found that, although this method is frequently employed, it is very time and labour intensive. A scintillation proximity assay was investigated to evaluate whether it could be a method for collecting kinetic data, and was found that further optimization is required. Comparative sequence analysis of TSR has shown it to be a member of the novel Class IV SpoUT family of AdoMet-dependent MTases. Members of this class possess a non-canonical AdoMet binding site containing a deep trefoil knot. Selected SpoUT family proteins were used as templates to develop a TSR homology model for monomeric and dimeric forms. Validation of the homology models was performed with structural validation servers and the model was then used as the basis of ongoing mutagenesis experiments. The X-ray crystal structure of TSR bound with AdoMet (2.45 Å) was elucidated by our collaborators, Drs. Mark Dunstan and Graeme Conn (University of Manchester). This structure confirms TSR MTase’s membership in the SpoUT MTase family with a deep trefoil knot in the catalytic domain. The AdoMet bound in the crystal structure is in an extended conformation not previously observed in SpoUT MTases. RNA docking simulations revealed some features that may be relevant to binding and recognition of TSR to the L11 binding domain of the RNA substrate. Two structure-activity studies were conducted to investigate the TS-rRNA interaction and TS solubility. Computational analyses of TS conformations, molecular orbitals and dynamics provided insight into the possible modes of TS binding to rRNA. Single-site modification of TS was attempted, targeting the dehydroalanine and dehydrobutyrine residues of the antibiotic. These moieties were modified using the polar thiol, 2-mercaptoethanesulfonic acid (2-MESNA). Similar modifications had been previously used to improve solubility and bioavailability of antibiotics. The resulting analogue was structurally characterized (NMR and mass spectrometry) and showed antimicrobial activity against Bacillus subtilis and Staphylococcus aureus.

Thiostrepton (TS: TS; C72H85N19O18S5) is a thiazoline antibiotic that is effective against Gram-positive bacteria and the malarial parasite, Plasmodium falciparum. Tight binding of TS to the bacterial L11-23S ribosomal RNA (rRNA) complex of the large 50S ribosomal unit inhibits protein biosynthesis. The TS producing organism, Streptomyces azureus, biosynthesizes thiostrepton-resistance methyltransferase (TSR), an enzyme that uses S-adenosyl-L-methionine (AdoMet) as a methyl donor, to modify the TS target site. Methylation of A1067 (Escherichia coli ribosome numbering) by TSR circumvents TS binding. The S. azureus tsr gene was overexpressed in E. coli and the protein purified for biochemical characterization. Although the recombinant protein was produced in a soluble form, its tendency to aggregate made handling a challenge during the initial stages of establishing a purification protocol. Different purification conditions were screened to generate an isolation protocol that yields milligram quantities of protein with little aggregation and sufficient purity for crystallographic studies. Enzymological characterization of TSR was carried out using an assay to monitor AdoMet-dependent ([methyl-3H]-AdoMet) methylation of the rRNA substrate by liquid scintillation counting. During the optimization of assay, it was found that, although this method is frequently employed, it is very time and labour intensive. A scintillation proximity assay was investigated to evaluate whether it could be a method for collecting kinetic data, and was found that further optimization is required. Comparative sequence analysis of TSR has shown it to be a member of the novel Class IV SpoUT family of AdoMet-dependent MTases. Members of this class possess a non-canonical AdoMet binding site containing a deep trefoil knot. Selected SpoUT family proteins were used as templates to develop a TSR homology model for monomeric and dimeric forms. Validation of the homology models was performed with structural validation servers and the model was then used as the basis of ongoing mutagenesis experiments. The X-ray crystal structure of TSR bound with AdoMet (2.45 Å) was elucidated by our collaborators, Drs. Mark Dunstan and Graeme Conn (University of Manchester). This structure confirms TSR MTase’s membership in the SpoUT MTase family with a deep trefoil knot in the catalytic domain. The AdoMet bound in the crystal structure is in an extended conformation not previously observed in SpoUT MTases. RNA docking simulations revealed some features that may be relevant to binding and recognition of TSR to the L11 binding domain of the RNA substrate. Two structure-activity studies were conducted to investigate the TS-rRNA interaction and TS solubility. Computational analyses of TS conformations, molecular orbitals and dynamics provided insight into the possible modes of TS binding to rRNA. Single-site modification of TS was attempted, targeting the dehydroalanine and dehydrobutyrine residues of the antibiotic. These moieties were modified using the polar thiol, 2-mercaptoethanesulfonic acid (2-MESNA). Similar modifications had been previously used to improve solubility and bioavailability of antibiotics. The resulting analogue was structurally characterized (NMR and mass spectrometry) and showed antimicrobial activity against Bacillus subtilis and Staphylococcus aureus.

The persistence and propagation of bacterial antibiotic resistance presents significant challenges to the treatment of drug resistant bacteria with current antimicrobial chemotherapies, while a dearth in replacements for these drugs persists. The thiopeptide family of antibiotics may represent a potential source for new drugs and thiostrepton, the prototypical member of this antibiotic class, is the primary subject under study in this thesis. Using a facile semi-synthetic approach novel, regioselectively-modified thiostrepton derivatives with improved aqueous solubility were prepared. In vivo assessments found these derivatives to retain significant antibacterial ability which was determined by cell free assays to be due to the inhibition of protein synthesis. Moreover, structure-function studies for these derivatives highlighted structural elements of the thiostrepton molecule that are important for antibacterial activity. Organisms that produce thiostrepton become insensitive to the antibiotic by producing a resistance enzyme that transfers a methyl group from the co-factor S-adenosyl-L-methionine (AdoMet) to an adenosine residue at the thiostrepton binding site on 23S rRNA, thus preventing binding of the antibiotic. Extensive site-directed mutagenesis was performed on this enzyme to generate point mutations at key active site residues. Ensuing biochemical assays and co-factor binding studies on these variants identified amino acid residues in the active site that are essential to the formation of the AdoMet binding pocket and provided direct evidence for the involvement of an active site arginine in the catalytic mechanism of the enzyme. Certain bacteria that produce neither thiostrepton nor the resistance methyltransferase express the thiostrepton binding proteins TIP-AL and TIP-AS, that irreversibly bind to the antibiotic, thereby conferring resistance by sequestration. Here, it was found that the point mutation of the previously identified reactive amino acid in TIP-AS did not affect covalent binding to the antibiotic, which was immediately suggestive of a specific, high affinity non-covalent interaction. This was confirmed in binding studies using chemically synthesized thiostrepton derivatives. These studies further revealed structural features from thiostrepton important in this non-covalent interaction. Together, these results indicate that thiostrepton binding by TIP-AS begins with a specific non-covalent interaction, which is necessary to properly orient the thiostrepton molecule for covalent binding to the protein. Finally, the synthesis of a novel AdoMet analogue is reported. The methyl group of AdoMet was successfully replaced with a trifluoromethyl ketone moiety, however, the hydrated form (germinal diol) of this compound was found to predominate in solution. Nevertheless, the transfer of this trifluoroketone/ trifluoropropane diol group was demonstrated with the thiopurine methyltransferase.

No description available.