| Scopolamine (3) is a tropane alkaloid that has a long history of use as a medicinal agent. The last two steps of scopolamine biosynthesis are accomplished by a bifunctional dioxygenase hyoscyamine 6beta-hydroxylase (H6H, EC 1.14.11.11). The mechanism of H6H catalysis has not been elucidated. The first part of this dissertation will describe mechanistic studies of H6H from Atropa belladonna (AbH6H).;An ongoing goal of this project is to obtain the crystal structure of AbH6H to elucidate its catalytic mechanism. In order to facilitate crystallization, cleavage of the His6-tag was attempted by introducing a tobacco etch virus (TEV) protease cleavage site at the N-terminus of the protein. The AbH6H gene with an N-terminal TEV tag was cloned into pET28a(+), and was transformed into E. coli BL21(DE3) for protein overexpression. After obtaining the TEV tagged AbH6H, TEV protease treatment was applied. Mass spectrometry analysis suggested that the His6-tag was successfully removed.;The second part of this dissertation describes efforts and results of isolation and characterization of antimicrobial compounds from three sources: a strain of Bacillus licheniformis, LeucidalRTM Liquid and from Streptomyces sp. SPS030.;An anticlostridial peptide was isolated from Bacillus licheniformis Ce-7. Based on MS and NMR analysis, the peptide was found to be bacitracin A, a well known antibiotic active against Gram-positive bacteria. Salicylic acid and a didecyl dimethyl ammonium species were isolated from LeucidalRTM Liquid, which are responsible for the activity of LeucidalRTM Liquid against Gram-negative and Gram-positive bacteria, respectively. The anti-microbial compound from Streptomyces sp. SPS030 is still under investigation.;In order to obtain AbH6H, the gene sequence of H6H from Atropa belladonna was cloned into the vector pQE60 with a C-terminal His6-tag. The recombinant H6H was expressed in Escherichia coli JM109 and purified by nickel affinity chromatography. An HPLC method was developed to detect the formation of products from the recombinant AbH6H, allowing for the investigation of the enzymatic activity. Analogues of hyoscyamine (1), the substrate of AbH6H, were synthesized and tested for conversion and enzyme inhibition. Additionally, an 18O-labeled 6beta-hydroxyhyoscyamine (2a) was fed to purified AbH6H to probe the catalytic mechanism of the last step in the biosynthesis of scopolamine. |
