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Thesis advisor: Mary F. Roberts / Thesis advisor: Jianmin Gao / The research reported in this thesis focused on three proteinaceous virulence factors of the intracellular bacterial pathogen Listeria monocytogenes: listeriolysin O (LLO), broad-range phospholipase C (PC-PLC), and phosphatidylinositol-specific phospholipase C (PI-PLC). Based on sequence homology of LLO with other cholesterol-dependent cytolysins (CDC), the protein has four domains of which domain 4 is thought to anchor the protein to cholesterol-containing surfaces while domain 3 mediates protein-protein binding on the membrane and contributes α-helices that convert to two β-strands that form the large β-barrel pore. It was previously assumed that the sequential and cooperative behaviors of domain 3 in each LLO monomer required D4 to bind to cholesterol-enriched membranes. By cloning and expressing a separate protein containing domains 1, 2, and 3 (D123) and the isolated domain 4 (D4) of LLO, I could uncouple some of the events in its membrane binding and pore-formation. Flow cytometry, used to investigate protein binding to vesicles and to red blood cells, showed that D123 had no membrane affinity on its own, but became membrane-bound when sub-lytic amounts of LLO were added. D123, not membrane-lytic by itself, became hemolytic when trace amounts of LLO were present to provide a membrane anchor for D123 proteins. FRET and fluorescence correlation spectroscopy were used to show that D123 and LLO formed oligomers at nanomolar concentration and could also associate with one another in the solution. These results suggest that D4 provides an initial membrane attachment but need not be present on all monomers to trigger the cooperative conformational change that leads to membrane insertion and pore formation. The gene for L. monocytogenes PC-PLC was obtained, expressed in E. coli and the product protein purified and characterized. The zinc content of this metalloenzyme was analyzed with ICP-MS. The dissociation constants of the three zinc ions proposed as necessary for PC-PLC activity ranged from 0.05 to 60 μM. Enzymatic activities of PC-PLC were analyzed for various substrates, include long-chain phospholipid in vesicles (LUVs, SUVs) and micelles (Triton X-100), and short-chain lipids (diC4PC, diC6PC, diC7PC) mono-dispersed in solutions. Key results include the following: (1) the L. monocytogenes PC-PLC has an acidic pH optimum (in contrast to other bacterial PC-PLC enzymes) consistent with its role in vacuole lysis upon acidification; (2) the preference of PC-PLC for longer chain monomeric substrates is not because of a higher kcat but a reduced Km suggesting some amount of hydrophobicity is important for substrate binding in the active site; (3) the apparent Kd of PC-PLC for Zn2+ derived from kinetics at pH 6.0 (1.94 ± 0.22 μM) is lower that that from ICP-MC; and (4) PC-PLC enzymatic activity is not enhanced by added LLO that generates pores in vesicles (likewise, PC-PLC does not affect the membrane lytic activity of LLO) indicating no synergism between the two virulence factors. These results should aid in understanding the function of PC-PLC in L. monocytogenes pathogenicity. The L. monocytogenes PI-PLC and a variant with reduced catalytic activity were expressed and are currently used in a collaborative project with the Portnoy laboratory at the University of California at Berkeley. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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The principal methylglyoxal (MG)-detoxifying system in most living organisms is the two metalloenzyme Glyoxalase system. Glyoxalase I (GlxI) initially converts the non-enzymatically formed MG-GSH hemithioacetal to the thioester S,D-lactoylglutathione. The hydrolase, Glyoxalase II(GlxII) regenerates GSH and liberates the product D-lactate. Ni2+/Co2+- and Zn2+-activated GlxI enzymes exist in nature. The Ni2+/Co2+-activated GlxI are not active as Zn2+-holoenzymes in spite of the structural similarities to the Zn2+-dependent enzymes. The Zn2+-GlxI enzymes have been investigated heavily relative to the Ni2+/Co2+-activated enzymes, which have been isolated more recently. As part of this study the three GlxI homologs isolated from Pseudomonas aeruginosa were characterized. The homologous genes encode GlxI enzymes of both metal activation type. The Zn2+-activated P. aeruginosa GlxI is difficult to de-metallate compared to the Ni2+/Co2+-activated enzymesreflecting a difference in metal-binding/insertion between the two types of GlxI. The E. coli GlxII was isolated and characterized to determine whether Ni2+/Co2+-activation is a characteristic of the Glx system as a whole in this organism. Inductively coupled plasma mass spectrometry on purified E. coli GlxII confirms that the active protein is a binuclear Zn2+-metalloenzyme. The results to date indicate a detectable isotope effect for the Cd2+-holoenzyme but not the Ni2+-reconstituted enzyme. Chemical crosslinking experiments indicate that the SlyD Ni2+ metallochaperone does not form a complex with E.coli GlxI. This indicates that the E. coli active site is not metallated in vivo by this accessory protein. The principal biophysical experiment in this project was determining of Ni2+-binding stoichiometry for E. coli GlxI by 1H-15N heteronuclear single quantum coherence (HSQC) NMR. The GlxI dimer reorganization ceases when the metal:dimer stoichiometry reaches 0.5 during apoenzyme titration. This finding supports previous studies that indicate half-of-the-sites metal binding in this enzyme.

It has been suggested for many decades that the essential and ubiquitous enzyme fructose 1,6-bisphosphate aldolase (FBA) could be a good drug target against bacteria and fungi, since lower organisms possess a metal-dependent (Class II) FBA, as opposed to higher organisms which possess a Schiff-base forming, metal-independent (Class I) FBA. The purpose of this doctoral project was to purify and study the inhibition of Class II FBA from pathogenic organisms. The capacity of various thiol compounds, as well as various derivatives of the metal-chelating compound dipicolinic acid, to inhibit the purified Class II FBAs from Mycobacterium tuberculosis, Pseudomonas aeruginosa, Bacillus cereus, and Magnaporthe grisea, was compared. The genes were subcloned in the Escherichia coli vector pT7-7 and the enzymes purified to near homogeneity, and characterized using a coupled assay. A small fed-batch fermentor was used to express the enzymes in E. coli, and yields of up to 2 grams of purified protein per liter of bacterial culture were obtained. The commercially available compound 2,3-dimercaptopropane sulfonate was found to be the most effective inhibitor against the aldolase from M. tuberculosis, with a second order binding rate constant of 500 +/- 4 M-1 s-1, which is three times and twenty times higher than the constants obtained with dipicolinic acid and EDTA, respectively. In an attempt to detect the enzyme dynamics during catalysis or inhibition, tryptophan residues were used as reporter groups and introduced by site-directed mutagenesis into the catalytic mobile loops and near the active site of the aldolases from M. tuberculosis, P. aeruginosa and B. cereus. The kinetic characterization of the mutants is described; as well as the effect of substrate binding on the steady-state and time-resolved fluorescence signals. Finally, the possibility of using the recombinant Class II FBP aldolases for industrial chemical synthesis was explored by measuring the enzymatic stability in organic solvents, at high temperatures and at different pH conditions. Surprisingly, the commercial Class I enzyme from rabbit muscle was more stable than the metalloenzymes in most conditions tested. The results presented in this thesis will be useful for the future design of Class II FBP aldolase inhibitors.

The principal methylglyoxal (MG)-detoxifying system in most living organisms is the two metalloenzyme Glyoxalase system. Glyoxalase I (GlxI) initially converts the non-enzymatically formed MG-GSH hemithioacetal to the thioester S,D-lactoylglutathione. The hydrolase, Glyoxalase II(GlxII) regenerates GSH and liberates the product D-lactate. Ni2+/Co2+- and Zn2+-activated GlxI enzymes exist in nature. The Ni2+/Co2+-activated GlxI are not active as Zn2+-holoenzymes in spite of the structural similarities to the Zn2+-dependent enzymes. The Zn2+-GlxI enzymes have been investigated heavily relative to the Ni2+/Co2+-activated enzymes, which have been isolated more recently. As part of this study the three GlxI homologs isolated from Pseudomonas aeruginosa were characterized. The homologous genes encode GlxI enzymes of both metal activation type. The Zn2+-activated P. aeruginosa GlxI is difficult to de-metallate compared to the Ni2+/Co2+-activated enzymesreflecting a difference in metal-binding/insertion between the two types of GlxI. The E. coli GlxII was isolated and characterized to determine whether Ni2+/Co2+-activation is a characteristic of the Glx system as a whole in this organism. Inductively coupled plasma mass spectrometry on purified E. coli GlxII confirms that the active protein is a binuclear Zn2+-metalloenzyme. The results to date indicate a detectable isotope effect for the Cd2+-holoenzyme but not the Ni2+-reconstituted enzyme. Chemical crosslinking experiments indicate that the SlyD Ni2+ metallochaperone does not form a complex with E.coli GlxI. This indicates that the E. coli active site is not metallated in vivo by this accessory protein. The principal biophysical experiment in this project was determining of Ni2+-binding stoichiometry for E. coli GlxI by 1H-15N heteronuclear single quantum coherence (HSQC) NMR. The GlxI dimer reorganization ceases when the metal:dimer stoichiometry reaches 0.5 during apoenzyme titration. This finding supports previous studies that indicate half-of-the-sites metal binding in this enzyme.

It has been suggested for many decades that the essential and ubiquitous enzyme fructose 1,6-bisphosphate aldolase (FBA) could be a good drug target against bacteria and fungi, since lower organisms possess a metal-dependent (Class II) FBA, as opposed to higher organisms which possess a Schiff-base forming, metal-independent (Class I) FBA. The purpose of this doctoral project was to purify and study the inhibition of Class II FBA from pathogenic organisms. The capacity of various thiol compounds, as well as various derivatives of the metal-chelating compound dipicolinic acid, to inhibit the purified Class II FBAs from Mycobacterium tuberculosis, Pseudomonas aeruginosa, Bacillus cereus, and Magnaporthe grisea, was compared. The genes were subcloned in the Escherichia coli vector pT7-7 and the enzymes purified to near homogeneity, and characterized using a coupled assay. A small fed-batch fermentor was used to express the enzymes in E. coli, and yields of up to 2 grams of purified protein per liter of bacterial culture were obtained. The commercially available compound 2,3-dimercaptopropane sulfonate was found to be the most effective inhibitor against the aldolase from M. tuberculosis, with a second order binding rate constant of 500 +/- 4 M-1 s-1, which is three times and twenty times higher than the constants obtained with dipicolinic acid and EDTA, respectively. In an attempt to detect the enzyme dynamics during catalysis or inhibition, tryptophan residues were used as reporter groups and introduced by site-directed mutagenesis into the catalytic mobile loops and near the active site of the aldolases from M. tuberculosis, P. aeruginosa and B. cereus. The kinetic characterization of the mutants is described; as well as the effect of substrate binding on the steady-state and time-resolved fluorescence signals. Finally, the possibility of using the recombinant Class II FBP aldolases for industrial chemical synthesis was explored by measuring the enzymatic stability in organic solvents, at high temperatures and at different pH conditions. Surprisingly, the commercial Class I enzyme from rabbit muscle was more stable than the metalloenzymes in most conditions tested. The results presented in this thesis will be useful for the future design of Class II FBP aldolase inhibitors.

The Glyoxalase system is composed of two metalloenzymes, Glyoxalase I and Glyoxalase II, that catalyze the conversion of toxic, metabolically produced alpha-ketoaldehydes, such as methyglyoxal, in the presence of a thiol cofactor, such as glutathione, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the Glyoxalase system, Glyoxalase I (GlxI), in yeast, protozoa, animals, human, plants and Gram-negative bacteria suggest two metal activation classes, zinc-activation or non-zinc-activation (but exhibiting selective nickel/cobalt-activation). This thesis provides the key discoveries of the Glyoxalase system from Gram-positive microorganisms using the major thiol cofactor/cosubstrate that produced within that particular organisms as well as the relatedness of the proteins in the same beta-alpha-beta-beta-beta protein superfamily.

The Glyoxalase system is composed of two metalloenzymes, Glyoxalase I and Glyoxalase II, that catalyze the conversion of toxic, metabolically produced alpha-ketoaldehydes, such as methyglyoxal, in the presence of a thiol cofactor, such as glutathione, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the Glyoxalase system, Glyoxalase I (GlxI), in yeast, protozoa, animals, human, plants and Gram-negative bacteria suggest two metal activation classes, zinc-activation or non-zinc-activation (but exhibiting selective nickel/cobalt-activation). This thesis provides the key discoveries of the Glyoxalase system from Gram-positive microorganisms using the major thiol cofactor/cosubstrate that produced within that particular organisms as well as the relatedness of the proteins in the same beta-alpha-beta-beta-beta protein superfamily.