Structural and Biochemical Investigation of Substrate Recognition and Shuttling in Natural Product Biosynthesis.

Polyketide natural products are chemically complex, bioactive small molecules with multiple therapeutic applications. Modular polyketide synthases (PKSs), which biosynthesize polyketides, are organized into assembly lines with modules that successively extend and in some cases modify specific pathway intermediates. Throughout biosynthesis an acyl carrier domain (ACP) tethers and transports intermediates to each catalytic domain within its module then transfers the fully processed intermediate to the next module. Employing x-ray crystallography, electron cryo-microscopy (cryo EM), and biochemical experiments, this thesis investigates the substrate and ACP specificity of PKS catalytic domains, the architecture of a module, dynamics of the catalytic domains and the ACP during a catalytic cycle, and the mechanisms of substrate transfer between modules. A PKS β-module is comprised of a ketosynthase (KS), an acyltransferase (AT), and a ketoreductase (KR), which elongate (KS and AT) and modify (KR) the polyketide intermediate. Crystal structures of a KS-AT di-domain and a KR domain along with structural comparison to homologs provided insights into the molecular basis of catalysis and substrate specificity. Cryo EM provided the first structure of an entire PKS β-module. The architecture of the PKS β-module forms a single chamber, in which the intramodular ACP can access all active sites. Incubation with different combinations of the natural substrates allowed visualization of the ACP at distinct positions during the catalytic cycle. The cryo-EM reconstructions and crystal structures revealed the ACP docking site on each catalytic domain and important protein-protein interactions that were validated by biochemical experiments. Docking domains promote modular association and intermediate transfer between modules. Structural and biochemical analysis identified a Class 2 docking domain from cyanobacterial PKSs with a novel docking strategy for promoting intermediate chain transfer. Through structural and biochemical analysis we also identified a thioesterase domain, which appears to function in intermediate transfer by releasing a pathway intermediate from an ACP for transfer to the next module in the pathway. The information presented in this thesis provides an important advance in our understanding of the PKS biosynthetic machinery and new engineering tools that will facilitate bioengineering efforts to produce novel small molecules with desired therapeutic activity.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/102351/1/jwhicher_1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102351/2/jwhicher_2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102351/3/jwhicher_3.pd

The yeast 14-3-3 proteins BMH1 and BMH2 differentially regulate rapamycin-mediated transcription

Synopsis 14-3-3 proteins are highly conserved and have been found in all eukaryotic organisms investigated. They are involved in many varied cellular processes, and interact with hundreds of other proteins. Among many other roles in cells, yeast 14-3-3 proteins have been implicated in rapamycin-mediated cell signalling. We determined the transcription profiles of bmh1 and bmh2 yeast after treatment with rapamycin. We found that, under these conditions, BMH1 and BMH2 are required for rapamycin-induced regulation of distinct, but overlapping sets of genes. Both Bmh1 and Bmh2 associate with the promoters of at least some of these genes. BMH2, but not BMH1, attenuates the repression of genes involved in some functions required for ribosome biogenesis. BMH2 also attenuates the activation of genes sensitive to nitrogen catabolite repression

Structure and function of the RedJ protein, a thioesterase from the prodiginine biosynthetic pathway in streptomyces coelicolor

Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a beta-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 angstrom resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole