MshA is a glycosyltransferase that synthesizes the precursor of mycothiol, a low-molecular-weight thiol found exclusively in Actinomycetes, including the virulent pathogen Mycobacterium tuberculosis (Mtb). The structure of MshA from Mtb (herein coined as TbMshA) and its complex with uridine diphosphate N-acetyl-glucosamine (UDP-GlcNAc) have been solved to resolutions of 2.32 A and 2.89 A respectively. Both structures form two monomers in the asymmetric unit cell and exhibit typical beta/alpha/beta Rossmann folds. Upon binding of UDP-GlcNAc, the C-terminal domain of TbMshA undergoes conformational changes in order to interact with UDP-GlcNAc at the binding site. In addition, ligand-bound TbMshA structure enables the identification of critical residues for enzymatic interactions, especially the residue Glu-353 (E353) at the active site that is believed to serve as a nucleophile in the sugar transfer of TbMshA. In order to verify this, a mutant of TbMshA with a single amino acid mutation from glutamate to glutamine at residue 353 is generated. The mutant (E353Q) has shown reduced enzyme activity by more than four-fold compared to the wild-type TbMshA (Vmax for wild-type is 0.17 plus/minus 0.02 microM sec^-1, whereas Vmax for E353Q is 0.04 plus/minus 0.01 microM sec-1). The kcat/Km for wild-type TbMshA (3.5 plus/minus 1.1 * 10^3 M^-1 sec^-1) is an order of magnitude higher than that of the mutant (0.3 plus/minus 0.1 * 10^3 M^-1 sec^-1), indicating the catalytic efficiency is greatly suppressed by the mutation. Mass spectrometry data also reveals that E353Q is unable to form the product of the reaction catalyzed by the wild-type TbMshA. These findings suggest the important role of Glu-353 in the structure and activity of TbMshA.

A vast literature acknowledges the corruptibility of regulators; however, empirical tests on the matter have been limited to two-agent models examining the rulemaking process and price regulation of natural monopolies. It remains an open question whether political contributions, by driving legislative pressure, can entice laxity from regulators in their application of the rules. To remedy this issue, I observe the highly-regulated coal mining industry for which there exists several points for inspector discretion. By comparing the outcomes with Congressional coal mining contribution levels, I am able to ascertain capture across several dimensions. Specifically, I find that contributions are associated with agency inspectors using their discretion to preempt violations requiring follow-up inspection, grant more inspections to waive safety requirements, conduct shorter inspections, and grant lower penalties. It is troubling that these findings occur at relatively low levels of contributions, suggesting a high level of corruptibility on the part of regulators.

This research shows that worst-case methane-air detonation loading on coal mine seals could be more severe than the design loads required by federal regulations, and therefore mine seals should be designed with sufficient ductility beyond the elastic regime. For this study, reinforced concrete mine seals were designed according to traditional protective structural design methods to meet the federal regulation requirements, and then the response to worst-case loads was analyzed in a single-degree-ofreedom model. Coal mine seals designed to resist the regulation loads elastically experienced support rotations up to 4.27 deg when analyzed with the worst-case loads. The analysis showed that coal mine seals designed to satisfy the federal regulations can survive worst-case methane-air detonations if they have sufficient ductility, but will undergo permanent, inelastic deformation.