Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.

Knowledge management capabilities and organizational performance: an investigation into the effects of knowledge infrastructure and processes on organizational performance

Knowledge is one of the most important assets for surviving in the modern business environment. The effective management of that asset mandates continuous adaptation by organizations, and requires employees to strive to improve the company's work processes. Organizations attempt to coordinate their unique knowledge with traditional means as well as in new and distinct ways, and to transform them into innovative resources better than those of their competitors. As a result, how to manage the knowledge asset has become a critical issue for modern organizations, and knowledge management is considered the most feasible solution. Knowledge management is a multidimensional process that identifies, acquires, develops, distributes, utilizes, and stores knowledge. However, many related studies focus only on fragmented or limited knowledge-management perspectives. In order to make knowledge management more effective, it is important to identify the qualitative and quantitative issues that are the foundation of the challenge of effective knowledge management in organizations. The main purpose of this study was to integrate the fragmented knowledge management perspectives into the holistic framework, which includes knowledge infrastructure capability (technology, structure, and culture) and knowledge process capability (acquisition, conversion, application, and protection), based on Gold's (2001) study. Additionally, because the effect of incentives ̶̶ which is widely acknowledged as a prime motivator in facilitating the knowledge management process ̶̶ was missing in the original framework, this study included the importance of incentives in the knowledge management framework. This study also identified the relationship of organizational performance from the standpoint of the Balanced Scorecard, which includes the customer-related, internal business process, learning & growth, and perceptual financial aspects of organizational performance in the Korean business context. Moreover, this study identified the relationship with the objective financial performance by calculating the Tobin's q ratio. Lastly, this study compared the group differences between larger and smaller organizations, and manufacturing and nonmanufacturing firms in the study of knowledge management. Since this study was conducted in Korea, the original instrument was translated into Korean through the back translation technique. A confirmatory factor analysis (CFA) was used to examine the validity and reliability of the instrument. To identify the relationship between knowledge management capabilities and organizational performance, structural equation modeling (SEM) and multiple regression analysis were conducted. A Student's t test was conducted to examine the mean differences. The results of this study indicated that there is a positive relationship between effective knowledge management and organizational performance. However, no empirical evidence was found to suggest that knowledge management capabilities are linked to the objective financial performance, which remains a topic for future review. Additionally, findings showed that knowledge management is affected by organization's size, but not by type of organization. The results of this study are valuable in establishing a valid and reliable survey instrument, as well as in providing strong evidence that knowledge management capabilities are essential to improving organizational performance currently and making important recommendations for future research.