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.

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.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves [120]-134).

The addition of Mn(II) to an early stationary-phase Deinococcus radiodurans RI culture could induce a new round of cell division (MnCD effect). The addition of Mn(II) could also stimulate the utilization of glucose and fructose in this bacterium. Class II fructose-1,6-bisphosphate aldolase (FBA) is an Mn-dependent key enzyme in pentose phosphate pathway. Therefore, in this research, we focused on the studies of the fba gene. Base on the gene sequence, FBA protein was composed of 306 amino acids, (M.W., 32.4 kDa¡F pI, 5.4). The expected PCR product size of the fba gene is 9.3 kbp. We had amplified the fba gene by using both Taq DNA polymerase and pfu turbo DNA polymerase. The sequence of the pfu turbo DNA polymerase products showed a higher homology with the fba gene than those of using Taq DNA polymerase. These amplified fba gene was cloned into three expression vectors, pGEX-4T-2, pQE30, and pET28a, and then further expressed in E. coli BL21(DE3)RIL and JM109. The recombinant GST-FBA protein could be overproduced in pTDA2/BL21(DE3)RIL. However, the expressed insoluble protein accumulated as inclusion bodies in the cells and exhibited no enzyme activity. After partial purification, and processing by thrombin protease cleavage, urea treatment, and the addition of Mn(II), this enzyme still showed no activity. The recombinant pEDA2/BL21(DE3)RIL strain cells grew in 18¢J and induced by 0.1mM IPTG could produced a soluble form His-Thrombin-T7-FBA protein which performed a 50X higher activities than those cells grew in 30¢J. This result indicated that decreasing the indicatioin temperature could improve the protein solubility and activity.