Cloning, heterologous expression, and distinct substrate specificity of protein farnesyltransferase from Trypanosoma brucei

Protein prenylation occurs in the protozoan that causes African sleeping si ckness (Trypanosoma brucei), and the protein farnesyltransferase appears to be a good target for developing drugs. We have cloned the alpha- and beta- subunits of T. brucei protein farnesyltransferase (TB-PFT) using nucleic ac id probes designed from partial amino acid sequences obtained from the enzy me purified from insect stage parasites. TB-PFT is expressed in both bloods tream and insect stage parasites. Enzymatically active TB-PFT was produced by heterologous expression in Escherichia coli, Compared with mammalian pro tein farnesyltransferases, TB-PFT contains a number of inserts of >25 resid ues in both subunits that reside on the surface of the enzyme in turns link ing adjacent alpha-helices, Substrate specificity studies with a series of 20 peptides SSCALX (where X indicates a naturally occurring amino acid) sho w that the recombinant enzyme behaves identically to the native enzyme and displays distinct specificity compared with mammalian protein farnesyltrans ferase. TB-PFT prefers Gin and Met at the X position but not Ser, Thr, or C ys, which are good substrates for mammalian protein farnesyltransferase, A structural homology model of the active site of TB-PFT provides a basis for understanding structure-activity relations among substrates and CAAX mimet ic inhibitors.