Graduation date: 1993

Graduation date: 1991

Graduation date: 1993

Thesis (Ph. D.)--Washington State University, December 2009. / Title from PDF title page (viewed on Dec. 15, 2009). "Department of Horticulture and Landscape Architecture." Includes bibliographical references.

Thesis--Ph. D., University of Hong Kong. / Typewritten.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2007. / Committee Chair: Sheldon W. May ; Committee Members: Donald F. Doyle, Leslie T. Gelbaum, Stanley H. Pollock, and James Powers. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Polyketides are a class of small molecules synthesized by a broad spectrum of bacteria, plants, and fungi, and many exhibit powerful bioactive properties. The number of clinically-relevant compounds adapted from polyketide scaffolds is growing, eliciting attempts from synthetic organic chemists to construct polyketide-related compounds in the laboratory from simple chemical building blocks. Unfortunately, the current efficiency by which a skilled artisan can synthesize even small quantities of a polyketide is severely limited by the functional and stereochemical complexity of these compounds. Conceptually, it would be much simpler to genetically reprogram the enzymes responsible for polyketide biosynthesis to produce designer molecules; however, the massive size of polyketide synthase enzymes has hindered efforts towards understanding critical features of their structures and mechanisms. Only very recently has structural information become available for enzymes involved in polyketide biosynthesis, providing an initial glimpse into the inner workings of these subcellular pharmaceutical factories. It will not be possible for mankind to fully realize the potential of engineered polyketide synthases without understanding how their architectures govern the molecules they have evolved to produce. In this work, the structure and mechanism of several enzymes involved in polyketide biosynthesis is investigated. An unprecedented architecture for the ketoreductase-enoylreductase didomain from the second module of the spinosyn polyketide synthase reveals structural divergence from the related mammalian fatty acid synthase, and reconstituted in vitro activity of the enoylreductase domain indicates the isolated enzyme retains activity apart from its parent polyketide synthase module. The dehydratase domain isolated from the tenth module of the rifamycin polyketide synthase, previously hypothesized to only form double bonds with (Z) geometry, was found to have altered stereoselectivity dependent on the carrier handle bound to the substrate. The enoyl-isomerase domain, isolated from the fourteenth module of the bacillaene polyketide synthase, utilizes a catalytic mechanism that relies only on a single active site histidine. A series of ketosynthase domains from trans-acyltransferase polyketide synthases reveal how polyketides bind covalently to the active site of the ketosynthase, and how the flanking subdomain of the ketosynthase is used as an anchor point for the assembly of the polyketide synthase megacomplex.

A network of three classes of proteins consisting of bHLH and MYB transcription factors and a WD40 repeat protein - TRANSPARENT TESTA GLABRA1 (TTG1) act in concert to activate trichome initiation and patterning in Arabidopsis. These proteins also regulate the flavonoid-based pigment biosynthetic pathway in almost all higher plants including Arabidopsis. Using TTG1-YFP translational fusions, I show that TTG1 is expressed ubiquitously in Arabidopsis leaves and is preferentially localized in the nuclei of trichomes at all developmental stages. Using conditional transgenic alleles I demonstrate that TTG1 directly regulates the same genes as GL3. In vivo binding of GL3, GL1 and TTG1 to the promoters of GL2, TTG2, CPC and ETC1 establishes that these genes are major transcriptional targets for the TTG1-bHLH-MYB regulatory complex. By co-precipitation, I confirm that TTG1 interacts with the GL3 (bHLH) and GL1 (Myb) proteins in vivo, forming a complex. The loss of members of the TTG1 complex through mutation, affects the subcellular distribution of other complex members. Using particle bombardment, I show that TTG1, GL3, GL1 and GL2 do not move between adjacent epidermal cells while CPC does move to neighboring cells. These data support a model for the TTG1 complex directly regulating activators and repressors and the movement of repressors to affect trichome patterning on the Arabidopsis leaf. In addition, I also show that GL3 is recruited to its own promoter in a GL1-independent manner, which results in decreased GL3 expression, suggesting the presence of a GL3 negative auto-regulatory loop. Expression studies using GL3-GR (GL3-glucocorticoid receptor) and TTG1-GR fusions reveal direct regulation of the late anthocyanin biosynthetic genes, but not of early biosynthetic genes. Taken together, our results provide insights on the molecular mechanisms by which the combinatorial TTG1-bHLH-MYB regulatory complexes activate and repress both developmental and biosynthetic pathways in Arabidopsis.

published_or_final_version / abstract / toc / Botany / Doctoral / Doctor of Philosophy

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