Turku Centre for Biotechnology
Supervisor: Patrik Jones
The objective is to understand the mechanism for regulation of NADP(H)-homeostasis in prokaryotes and to identify and understand the role of novel components that participate in this process. NADP(H) is a central electron acceptor/donor in most organisms. Unlike the electron acceptor/donor of glycolytic catabolism, NAD(H), the primary metabolic role of NADP(H) is to donate electrons in anabolic reactions required for cellular growth and superoxide defence systems (Pollak, N., Biochem J 402, 205-218 (2007)). A basic hypothesis for the study is that the relative ratio of reduced to oxidized pyridine nucleotide cofactors is maintained under "normal" conditions (metabolic homeostasis), although it may change drastically in response to changes in environmental conditions. This ratio influences more than 100 metabolic reactions including several medically relevant processes (Sauer, U. 2004. J. Biol Chem 279, 6613-6619). Although the control of this ratio has important thermodynamic implications for biotechnological applications (Veit, A., 2008. Microbial Biotechnol. 1, 382-39; Walton, A., and Stewart, J. 2004. Biotechnol Prog 20, 403-411), whole cell metabolism (Henry, C. 2007. Biophys J 92, 1792-1805), and defence against Reactive Oxygen Species (Ralser, M., 2007. J Biol 6, 10), there is currently no understanding of the mechanism that regulates this ratio in prokaryotes.
The aim of this project is to address this medically and biotechnologically important question with a combined computational and experimental systems approach. Targeted network data will be generated by whole-systems analysis using selected mutants and environmental conditions relevant for NADPH-metabolism, followed by explorative bioinformatics analysis and experimental verification of predicted cause-effect relationships.
The initial candidate genes for modification of NADPH-metabolism include those gene products which are directly involved in the reduction or oxidation of NADP(H); pntAB, zwf and sthA. In previous work in our laboratory it was found that deletion or over-expression of PntAB resulted in strong changes in metabolic flux through the pentose phosphate pathway (unpublished). Transcriptional regulation of genes encoding key enzymes of glycolysis (Pgi) and the PP pathway (Zwf) has also been demonstrated to vary in response to changes in NADP(H)-metabolism (Sauer, 2004), whilst post-transcriptional regulatory mechanisms such as dimerization also cannot be ruled out . Despite these insights, the relative importance of protein- vs. metabolite-level regulation and the mechanism for sensing and regulation of NADP(H)-homeostasis in prokaryotes remains to be answered.
The project is expected to significantly expand our understanding of overall NADP(H)- metabolism and to generate novel insight into mechanisms for homeostatic regulation of oxidation-reduction metabolism, a topic that is of central importance for microbial metabolism.