Cloning, heterologous expression, and distinct substrate specificity of protein farnesyltransferase from Trypanosoma brucei.
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.
Buckner
F S
FS
Departments of Medicine and Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195-1700, USA.
Yokoyama
K
K
Nguyen
L
L
Grewal
A
A
Erdjument-Bromage
H
H
Tempst
P
P
Strickland
C L
CL
Xiao
L
L
Van Voorhis
W C
WC
Gelb
M H
MH
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*
D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase