title: Cloning, heterologous expression, and distinct substrate specificity of protein farnesyltransferase from Trypanosoma brucei.
abstract: Protein prenylation occurs in the protozoan that causes African sleeping sickness (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 acid probes designed from partial amino acid sequences obtained from the enzyme purified from insect stage parasites. TB-PFT is expressed in both bloodstream and insect stage parasites. Enzymatically active TB-PFT was produced by heterologous expression in Escherichia coli. Compared with mammalian protein farnesyltransferases, TB-PFT contains a number of inserts of >25 residues in both subunits that reside on the surface of the enzyme in turns linking adjacent alpha-helices. Substrate specificity studies with a series of 20 peptides SSCALX (where X indicates a naturally occurring amino acid) show that the recombinant enzyme behaves identically to the native enzyme and displays distinct specificity compared with mammalian protein farnesyltransferase. TB-PFT prefers Gln and Met at the X position but not Ser, Thr, or Cys, 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 mimetic inhibitors.
authors.affiliation: Departments of Medicine and Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195-1700, USA.
authors.forename: F S
authors.initials: FS
authors.lastname: Buckner
authors.forename: K
authors.initials: K
authors.lastname: Yokoyama
authors.forename: L
authors.initials: L
authors.lastname: Nguyen
authors.forename: A
authors.initials: A
authors.lastname: Grewal
authors.forename: H
authors.initials: H
authors.lastname: Erdjument-Bromage
authors.forename: P
authors.initials: P
authors.lastname: Tempst
authors.forename: C L
authors.initials: CL
authors.lastname: Strickland
authors.forename: L
authors.initials: L
authors.lastname: Xiao
authors.forename: W C
authors.initials: WC
authors.lastname: Van Voorhis
authors.forename: M H
authors.initials: MH
authors.lastname: Gelb
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051381:Rats; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics*
chemical_list: D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase