Yeast protein geranylgeranyltransferase type-I: overproduction, purification, and characterization.

Protein geranylgeranyltransferase type-I (PGGTase-I) catalyzes alkylation of the cysteine residue in proteins containing a consensus C-terminal CaaX sequence ending in leucine by the C20 hydrocarbon moiety in geranylgeranyl diphosphate (GGPP). The Saccharomyces cerevisiae genes encoding the alpha (RAM2) and beta (CDC43) subunits of PGGTase-I were translationally coupled by overlapping the RAM2-CDC43 stop-start codons and by locating a ribosome-binding site near the 3' end of RAM2. Recombinant PGGTase-I was overproduced in Escherichia coli to give approximately 8% of total cellular protein and purified 12-fold to > 95% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The purified heterodimer contained alpha- and beta-subunits with molecular masses of 34 and 42 kDa, respectively. A continuous fluorescence assay was developed to measure PGGTase-I activity. The recombinant enzyme showed maximal activity at pH 7.5 and required both Mg2+ and Zn2+. Michaelis constants for GGPP (1.0 microM) and dansyl-Gly-Cys-Ile-Ile-Leu (2.4 microM) were similar to those reported for yeast protein farnesyltransferase (PFTase) with farnesyl diphosphate and dansyl-Gly-Cys-Val-Ile-Ala; Vmax = 0.20 mumol min-1 mg-1 for recombinant yeast PGGTase-I was similar to that reported for yeast PFTase.