Expression and characterization of protein geranylgeranyltransferase type I from the pathogenic yeast Candida albicans and identification of yeast selective enzyme inhibitors.
Protein geranylgeranyltransferase type I (GGTase I) is a heterodimeric zinc metalloenzyme catalyzing protein geranylgeranylation at cysteine residues present in C-terminal signature sequences referred to as CaaX (X=Leu) motifs. We have studied GGTase I as a potential antifungal target and recently reported its purification and cloning from the yeast Candida albicans (Ca GGTase I), an important human pathogen. Here, we report the high yield bacterial expression of Ca GGTase I by coexpression of maltose binding protein fusion proteins of both the alpha (Ram2p) and beta (Cdc43p) subunits. The cleaved and purified recombinant Ca GGTase I was demonstrated to be functional and structurally intact as judged by the presence of one equivalent of a tightly bound zinc atom and the near stoichiometric formation, isolation and catalytic turnover of a geranylgeranyl pyrophosphate-GGTase I complex. Kinetic analysis was performed with a native substrate protein, Candida Cdc42p, which exhibited significant pH dependent substrate inhibition, a feature not observed with other Ca GGTase I substrates. Prenyl acceptor substrate specificity was studied with a series of peptides in which both the CaaX motif, and the sequence preceding it, were varied. The prenyl acceptor K(M)s were found to vary nearly 100-fold, with biotinyl-TRERKKKKKCVIL, modeled after a presumably geranylgeranylated Candida protein, Crl1p (Rho4p), being the optimal substrate. A screen for inhibitors of Ca GGTase I identified compounds showing selectivity for the Candida versus human GGTase I. The most potent and selective compound, L-689230, had an IC(50) of 20 nM and >12,500-fold selectivity for Ca GGTase I. The lack of significant anti-Candida activity for any of these inhibitors is consistent with the recent finding that GGTase I is not required for C. albicans viability [R. Kelly et al., J. Bacteriol. 182 (2000) 704-713].
Smalera
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Department of Biochemistry, Merck Research Laboratories, P.O. Box 2000, RY80M-136, Rahway, NJ 07065, USA.
Williamson
J M
JM
Baginsky
W
W
Leiting
B
B
Mazur
P
P
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002176:Candida albicans / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I