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Cloning, heterologous expression, and distinct substrate specificity of protein farnesyltransferase from Trypanosoma brucei.
Title | Cloning, heterologous expression, and distinct substrate specificity of protein farnesyltransferase from Trypanosoma brucei. |
Publication Type | Journal Article |
Year of Publication | 2000 |
Authors | Buckner, FS, Yokoyama, K, Nguyen, L, Grewal, A, Erdjument-Bromage, H, Tempst, P, Strickland, CL, Xiao, L, Van Voorhis, WC, Gelb, MH |
Journal | J Biol Chem |
Volume | 275 |
Issue | 29 |
Pagination | 21870-6 |
Date Published | 2000 Jul 21 |
ISSN | 0021-9258 |
Keywords | Alkyl and Aryl Transferases, Amino Acid Sequence, Animals, Cloning, Molecular, Molecular Sequence Data, Protein Conformation, Rats, Sequence Alignment, Sequence Analysis, Protein, Substrate Specificity, Trypanosoma brucei 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. |
DOI | 10.1074/jbc.M000975200 |
Alternate Journal | J. Biol. Chem. |
PubMed ID | 10749864 |