| Acrylamide is a neurotoxin and suspected carcinogen that is produced by industrial processes and during the cooking of certain foods. In this first portion of this study, the microbial diversity of acrylamide metabolism has been expanded through the isolation and characterization of a new strain of Rhodopseudomonas palustris capable of growth with acrylamide under photoheterotrophic conditions. The newly isolated strain grew rapidly with acrylamide under photoheterotrophic conditions by rapid deamidation to acrylate, and subsequent degradation of acrylate (the rate-limiting reaction). 13C NMR studies of [1,2,3] 13C acrylamide confirmed the rapid conversion of acrylamide to acrylate, and by using concentrated cell suspensions 13C acrylate consumption occurred with the production and then degradation of 13C propionate. While R. palustris strain Ac1 grew well and with comparable doubling times for each of acrylamide, acrylate, and propionate, R. palustris strain CGA009 was incapable of significant acrylamide- or acrylate-dependent growth over the same time course, but grew comparably with propionate. These results provide the first demonstration of anaerobic photoheterotrophic bacterial acrylamide catabolism and provide evidence for a new pathway for acrylate catabolism involving propionate as an intermediate.; The second part of this dissertation investigates the mechanism in which NADPH:2-Ketopropyl-Coenzyme M oxidoreductase/carboxylase (2-KPCC) acts on its respective substrates. To facilitate these studies, an expression system was developed to overexpress the enzyme in E. coli. Subsequently site-directed mutagenesis was utilized to alter specific residues that were identified in the crystal structure as important for either catalysis or substrate binding. Specifically, the cysteines involved in the redox active disulfide were targeted for their role in shifting from an internal disulfide to form a mixed disulfide between interchange cysteine and the CoM moiety of the substrate, followed by release of the CoM and the reformation of the internal disulfide. Substitution of either of these two residues with alanine results in a redox inactive enzyme, although the enzyme was still able to catalyze the non-reductive decarboxylation of acetoacetate. The spectroscopic features of this mutated flavoenzyme were used to capture the thiol-flavin charge transfer, the NADP + flavin charge transfer, and the flavin C4a-C87 adduct. |
