Nitric oxide synthase in Physarum polycephalum: Molecular characterization and biological role

Nitric oxide (NO) synthases are a recently discovered family of enzymes that catalyze the conversion of L-arginine to citrulline and nitric oxide. Biological roles in mammals include control of blood pressure by vasodilatation, transmission of nerve signals and host defense against infections. The spectrum of physiological actions of NO is continuously increased by detection of new roles of this molecule. The latest achievement along this line has been the detection of a role of NO synthase in host defense to pathogens in plants. NO synthases from mammals, insects and molluscs have been cloned. So far, however, no NO synthase from a plant, fungus or bacterium has been characterized at the molecular level. Stimulated by previous reports on the formation of pteridines by Physarum polycephalum, we have purified and characterized an NO synthase from Physarum, which showed biochemical characteristics remarkably similar to the inducible isoform of NO synthase in mammals. Its activity does not depend on calcium(2+) concentrations, it has a molecular weight of 130 kDa, and contains the same cofactors bound, i.e. FAD, FMN, and tetrahydrobiopterin. We then succeeded in cloning a 600 bp polymerase chain reaction product, the deduced reading frame of which showed a striking, highly significant homology to all other NO synthase sequences available, but to no other proteins. This provided the key step to proceed to the submitted project, the molecular and functional characterization of NO synthase. We plan as a first main goal to use classical molecular biology techniques to clone and recombinantly express NO synthase from Physarum, which would be the first NO synthase cloned from a fungus, plant or bacterium. This would not only give exciting new insights into the primary structure-function relationship and the evolution of the enzyme, but provide us with tools essential for our second main goal, the investigation of the biological role of NO in the slime mold Physarum polycephalum. This organism is an ideal model system to study the cell cycle, since a strictly synchronous division of nuclei occurs in several stages of its development. Further, it can be driven into various differentiation stages in a defined way. Combining studies on the expression of NO synthase in the various stages of this myxomycete, with studies of the effects of specific inhibitors and effectors of NO synthase on the physiology and development of the organism, we hope to find the role of NO synthase in Physarum. Since many fundamental biochemical data on e.g. nuclear division were first characterized in Physarum, we hope to find a new role of NO, which we will then test for its significance for mammalian cells.

The myxomycete Physarum polycephalum is a well established model system for studying cell cycle- and differentiation-related cellular processes. In this project, we purified and cloned nitric oxide synthase (NOS) from this organism, the first characterized non-animal NOS so far, and defined its role for developing fruiting bodies (sporangia). While several motifs for binding the various cofactors required for NOS are highly conserved in Physarum NOS, its overall homology to NOSs from animal species is < 39 %. Limitation of glucosse, a pre- condition for obtaining the ability to differentiate into sporangia, caused a dramatic increase in NOS expression. We identified NO and cGMP as crucial signalling molecules in the light-induced development of mature sporangia and showed positive correlation between NOS expression, availability of NO and cGMP and the ability to sporulate with expression of lig1, an early sporulation-specific gene that is homologous to hus1, a cell cycle and DNA integrity checkpoint gene from yeast and mammals. In addition, we also characterized two further enzymes, i.e. GTP cyclohydrolase I and dihydropteridine reductase. GTP cyclohydrolase I is the key enzyme for biosynthesis of tetrahydrobiopterin, an essential cofactor of NOS. Dihydropteridine reductase is involved in recycling of tetrahydrobiopterin. Both enzymes are highly conserved between Physarum and mammals. Interestingly, the genomic structure and alternative mRNA splicing of GTP cyclohydrolase I are also well conserved between Physarum and humans. In summary, our work on the protist Physarum defined a novel role for NO/cGMP mediated cell differentiation and established conservation of tetrahydrobiopterin and NO biosynthesis at a low stage of evolution. Studying this model organism holds the potential to better understand the complex signalling pathways involved in cell differentiation that could also be operative in animals.