Wild-Type Phosphoribosylpyrophosphate Synthase (PRS) from Mycobacterium tuberculosis: A Bacterial Class II PRS?

The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of Pi. ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of Pi would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of MtPRS to provide a solid foundation for the rational design of specific inhibitors of this enzyme

Dimethyl Sulfide is a Chemical Attractant for Reef Fish Larvae

Transport of coral reef fish larvae is driven by advection in ocean currents and larval swimming. However, for swimming to be advantageous, larvae must use external stimuli as guides. One potential stimulus is "odor" emanating from settlement sites (e.g., coral reefs), signaling the upstream location of desirable settlement habitat. However, specific chemicals used by fish larvae have not been identified. Dimethyl sulfide (DMS) is produced in large quantities at coral reefs and may be important in larval orientation. In this study, a choice-chamber (shuttle box) was used to assess preference of 28 pre-settlement stage larvae from reef fish species for seawater with DMS. Swimming behavior was examined by video-tracking of larval swimming patterns in control and DMS seawater. We found common responses to DMS across reef fish taxa - a preference for water with DMS and change in swimming behavior - reflecting a switch to "exploratory behavior". An open water species displayed no response to DMS. Affinity for and swimming response to DMS would allow a fish larva to locate its source and enhance its ability to find settlement habitat. Moreover, it may help them locate prey accumulating in fronts, eddies, and thin layers, where DMS is also produced