Structure/function analysis of heptosyltransferase I (WaaC)

Heptosyltransferase I (WaaC) is a bacterial enzyme, which transfers heptose (donor), a unique carbohydrate, onto the inner core of the bacterial cell wall (acceptor) of gram negative bacteria. Due to its high conservation among enterobacteriaceae in regard of distribution and function, WaaC constitutes a highly interesting target for the development of new antibiotics and antivirulence based therapies. While the specificity of WaaC for its donor has been studied in some detail, little is known about the WaaC -acceptor specificity. This is largely due to the highly complex amphiphilic nature of lipid A-Kdo2 , the natural acceptor molecule. Lipid A consists of a carbohydrate and fatty acid residue of which the carbohydrate-enzyme interaction is crucial for binding to WaaC. Due to the fatty acid portion the spectroscopic accessibility by NMR and X-ray was very limited until recently it was shown that WaaC does also accept deacetylated lipid A, which makes the acceptor structure now available for detailed studies of the enzyme-carbohydrate interaction. For detailed spectroscopic investigation well defined part structures of the carbohydrate portion are necessary. As carbohydrate fractions from bacterial lipopolysaccharide (LPS) may only be isolated in small amounts and insufficient purity, chemical synthesis of defined subunits is mandatory. Unique sub structures of the WaaC acceptor region were covered and collected by previous projects and can therefore be provided. In this project I shall carry out a comprehensive interdisciplinary investigation of the WaaC - acceptor interaction by use of three different methodological approaches. Organic synthesis will be used to prepare part structures of the natural acceptor molecule that are not covered by our unique substance database and molecular biology will help us to express and isolate the target enzyme WaaC. The interactions of the part structures and the enzyme will then be studied by NMR spectroscopy (STD), crystallography and molecular modelling for structure refinement. The molecular biological, spectroscopic and necessary organic synthetic work will be carried out in cooperation with an interdisciplinary, established and highly acclaimed international group from Canada. The knowledge acquired during the stay abroad will be implemented at the home institution and be used in future projects. The outcome of the project should contribute to a better understanding of bacterial heptosyltransferase-acceptor interaction and translate into novel approaches towards the development of antimicrobial agents to be used in antibiotic therapies.

Within this project the so far unknown interaction of Heptosyltransferase I (WaaC), a bacterial enzyme, with its main substrate could be mapped and therefore studied in great detail. The findings of this project could help to pave the way for new antibiotic substances and antivirulence based therapies. WaaC is a highly conserved bacterial enzyme, which transfers heptose (donor), a unique carbohydrate, onto the inner core of the bacterial cell wall (acceptor) of gram negative bacteria. Due to its high conservation among enterobacteriaceae in regard of distribution and function, WaaC constitutes a highly interesting target for the development of new antibiotics and antivirulence based therapies. While the specificity of WaaC for its donor has been studied in some detail before, little was known about the WaaC -acceptor specificity. This was largely due to the high complexity of lipid A-Kdo2, the natural acceptor molecule which makes it almost insoluble in solvents which would allow pectroscopic determinations. Isolation from bacterial sources and further chemical modification of this molecule allowed this problem to be overcome and detailed analysis became accessible. By the use of protein crystallography it was revealed, that the phosphate and carboxylic groups of the acceptor are crucial for the binding process and also initiate a slight conformational change. The outcome of the project contributes to a better understanding of bacterial heptosyltransferase-acceptor interaction and will translate into novel approaches towards the development of antimicrobial agents to be used in antibiotic therapies.