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Nonribosomal peptide synthetases (NRPSs) are large, modular proteins responsible for biosynthesis of many medicinally important peptide natural products. Although detailed genetic and biochemical experiments have yielded deep insights into the principles of nonribosomal peptide biosynthesis in recent years, little is known about the in vitro properties of these synthetases that are generally unstable and difficult to reconstitute. In this thesis, three overlooked cellular factors, macromolecular crowding, protein organization, and proofreading enzyme, that influence the activities of these multifunctional enzymes were investigated using enterobactin synthetase as a model system. First, we investigated the macromolecular crowding effect on the activities of the enterobactin NRPS. It was found that macromolecular crowding significantly increase product specificity of the synthetase. Crowding most likely exerts this effect through inducing conformational changes in the multifunctional EntF of the synthetase. Moreover, we investigated protein organization in the enterobactin pathway in vivo and in vitro. All the enzymes involved in this biosynthetic pathway were found to be co-immunoprecipitated from E. coli cells cross-linked by formaldehyde despite the fact that these biosynthetic enzymes were not found to form stable protein complexes in vitro. Furthermore, the influence of type II thioesterase EntH on enterobactin biosynthesis was investigated. We found that EntH evolves its distinctive ability to recognize the aberrant intermediates from the versatile catalytic platform of hotdog proteins and suggests that type II thioesterases are able to actively search for the wrongly incorporated intermediates in nonribosomal peptide synthesis. In summary, the results described in this thesis show that these three factors are critical to correct understanding of biosynthesis of NRPSs and indispensable in their biotechnological utilizations.

Enzymatic formation of supramolecular nanofibers is demonstrated as a novel approach to induce intracellular hydrogelation and control the fate of cells or cellular functions, which can lead to a new paradigm for developing biomaterials to manage cellular artificial nanostructures (CAN), understand cellular functions beyond the molecular level, and create novel therapeutics.

(Figure Presented) Control from within: Supramolecular hydrogelation to form nanostructures intracellularly (see picture) has been developed as a new methodology to control the fate of cells. Enzyme-regulated self-assembly of small molecules inside cells could lead to a new paradigm for managing cellular processes, understanding cellular functions, and developing new therapeutics through supramolecular chemistry. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA.

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel strain of coronavirus. Examination of the immune responses of patients who have recovered from SARS should provide important information for design of a safe and effective vaccine. We determined the continuous viral epitopes targeted by antibodies in plasma samples from convalescent SARS patients through biopanning with a vast M13 phage display dodecapeptide library. These epitopes converged to very short peptide fragments, one on each of the structural proteins spike and nucleocapsid and the nonstructural proteins 3a, 9b, and nsp 3. Immunoassays found that most of the patients who had recovered from SARS developed complementary antibodies to the epitope-rich region on the spike S2 protein, indicating that this is an immunodominant site on the viral envelope comprising the spike, matrix, and small envelope glycoproteins. These S2-targeting antibodies were shown to effectively neutralize the coronavirus, indicating that they provided protective immunity to help the patients recover from the viral infection. These results suggest that the SARS coronavirus might have an antigenic profile distinct from those of other human or animal coronaviruses. Due to the tested safety and protective effects of the convalescent-phase serological antibodies, identification of their complementary antigens may enable the design of an epitope-based vaccine to prevent potential antibody-mediated immunuopathology.