Cloning, heterologous expression, and distinct substrate specificity of protein farnesyltransferase from Trypanosoma brucei Journal Article
| Authors: | Buckner, F. S.; Yokoyama, K.; Nguyen, L.; Grewal, A.; Erdjument-Bromage, H.; Tempst, P.; Strickland, C. L.; Xiao, L.; Van Voorhis, W. C.; Gelb, M. H. |
| Article 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 α- and β-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 α-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. |
| Keywords: | protein expression; nonhuman; protein conformation; mammalia; animals; trypanosoma brucei; trypanosoma; recombinant enzyme; structure activity relation; molecular cloning; cloning, molecular; amino acid sequence; molecular sequence data; enzyme analysis; sequence alignment; nucleotide sequence; escherichia coli; substrate specificity; rats; protein subunit; enzyme specificity; enzyme structure; protozoa; enzyme synthesis; enzyme active site; sequence analysis, protein; alpha helix; enzyme isolation; insecta; priority journal; article; alkyl and aryl transferases; trypanosoma brucei brucei; protein farnesyltransferase |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 275 |
| Issue: | 29 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2000-07-21 |
| Start Page: | 21870 |
| End Page: | 21876 |
| Language: | English |
| DOI: | 10.1074/jbc.M000975200 |
| PUBMED: | 10749864 |
| PROVIDER: | scopus |
| PMCID: | PMC2913713 |
| DOI/URL: | |
| Notes: | Export Date: 18 November 2015 -- Source: Scopus |
