Elucidation of the metabolism of undecaprenyl phosphate, a key lipid for bacterial cell wall polymers biosynthesis – BACTOPRENYL

The present project will in particular develop a multidisciplinary study of multiple integral membrane proteins. This will include detailed analyses of their physiological roles, catalytic reaction mechanisms, membrane topologies, 3D structure, and regulation of expression. The general knowledge on membrane proteins is still very limited because of the well-known intrinsic difficulty of studying such proteins. One of the ambitious goals of this project is that the data we will generate will contribute to an increase of our knowledge in this specific field

Identification and cloning of genes from different bacteria encoding proteins exhibiting undecaprenyl-pyrophosphate phosphatase activity that belong to two different protein families, BacA and PAP2. Study of the essential character of these genes and construction of conditional expression mutant strains. Purification to homogeneity of mg quantites of several of these enzymes which all are integral membrane proteins containing multiple transmembrane segments

The undecaprenyl-phosphate carrier lipid and all steps of its metabolism are absolutely required for the biosynthesis of peptidoglycan and other essential polymers constituting the bacterial cell wall and which are essential for maintaining the cell integrity. Therefore, they represent quite interesting potential targets that could be exploited in a search for new antibacterial compounds. The data generated in the present project, in particular the knowledge acquired on the catalytic mechanisms and substrate specificity of these enzymes, their membrane topology, and eventually their 3D structure will be very useful in this respect

No publication up to now

The biosynthesis of the various bacterial cell-wall polysaccharides, such as the peptidoglycan, requires the translocation of the glycan units across the cytoplasmic membrane. This translocation event necessitates a particular lipid carrier, undecaprenyl phosphate (C55-P). The glycan units are linked to the lipid carrier at the cytoplasmic side of the membrane, leading to the formation of a series of lipid intermediates of general C55-PP-sugar(s) structure. These intermediates are then translocated across the membrane (flip-flop) with the help of flippases and the glycan moieties are transferred to the final growing acceptor polymers. This reaction releases the lipid carrier in an inactive pyrophosphate form, which is then recycled. C55-P is involved in the synthesis of various polymers that are essential for cell integrity, as exemplified by the peptidoglycan. Therefore, the lipid carrier is itself essential. It is well established that the inhibition of C55-P synthesis causes rapid cell lysis due to the arrest of peptidoglycan synthesis. C55-P originates from the dephosphorylation of undecaprenyl pyrophosphate (C55-PP). The latter precursor is either generated by de novo synthesis or released (recycled) after the transfer of the glycan components to periplasmic acceptor molecules. De novo synthesis of C55-PP is catalyzed by the essential UppS synthase. 3-D structures of this enzyme together with several biochemical analyses have provided a high level of understanding of this enzymatic step. In contrast, the C55-PP dephosphorylation step leading to the active form of the lipid carrier has been largely overlooked until Partners 1 and 3 from the present project started investigating this research issue. Two unrelated families of proteins exhibiting C55-PP phosphatase (UppP) activity were identified: BacA on one hand and members of the PAP2 (type 2 phosphatidic acid phosphatase) super-family on the other hand. Escherichia coli was shown to possess several C55-PP phosphatases: a BacA enzyme and 3 phosphatases from the PAP2 super-family (PgpB, YbjG and YeiU). This redundancy of activity seems to be shared by most of the bacteria as suggested by a search for putative homologues in databases, raising the question of the significance of such a multiplicity. Furthermore, Partner 1 recently highlighted an unexpected and very important feature regarding the YeiU enzyme activity. This enzyme was indeed shown to catalyze both in vitro and in vivo the transfer of the C55-PP distal phosphate group onto lipid A, the lipidic moiety of lipopolysaccharides (LPS), yielding C55-P and a pyrophosphorylated form of lipid A. The significance of this LPS modification remains to be established, however, it suggests a tantalizing general hypothesis that the multiple other C55-PP phosphatases could also exhibit phospho-transferase activities with specific acceptor molecules. These reactions may be important in the regulation of cellular activities and/or in bacterial adaptation to stress conditions.
The present project aims at the complete elucidation of the metabolism of the essential undecaprenyl phosphate bacterial carrier lipid, using complementary and pluridisciplinary approaches and several Gram-negative and Gram-positive bacteria as models. In particular, the mechanistic and functional features of the multiple recently identified C55-PP phosphatases, their regulation pattern, and their respective roles in C55-P metabolism and in bacterial physiology in general will be investigated in detail. Two other enzymes (C55-P phosphatase and undecaprenol kinase) involved in this metabolism that have not been identified to date will also be studied. Only fundamental aspects will be developed in the present project. However, the acquired knowledge on this essential metabolism and on the specific enzymes involved could potentially be exploited further for a search of new antibiotics.