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.
Stirtan
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WG
Department of Chemistry, University of Utah, Salt Lake City 84112, USA.
Poulter
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D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D008274:Magnesium; D015032:Zinc