CONVERSION OF PRODUCT SPECIFICITY OF ARCHAEBACTERIAL GERANYLGERANYL-DIPHOSPHATE SYNTHASE - IDENTIFICATION OF ESSENTIAL AMINO-ACID-RESIDUES FOR CHAIN-LENGTH DETERMINATION OF PRENYLTRANSFERASE REACTION

Prenyltransferases catalyze the consecutive condensation of isopenteny l diphosphate with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme, To inve stigate the mechanism of the consecutive reaction and the determinatio n of the ultimate chain length, a random mutational approach was plann ed, A geranylgeranyl-diphosphate synthase gene from Sulfolobus acidoca ldarius was randomly mutagenized by NaNO2 treatment to construct a lib rary of mutated geranylgeranyl-diphosphate synthase genes on a yeast e xpression vector, The library was screened for suppression of a pet ph enotype of yeast C296-LH(3), which is deficient in hexaprenyl diphosph ate synthase, Five mutants that could grow on a YEPG plate, which cont ained only glycerol as an energy source instead of glucose, were selec ted from similar to 1,400 mutants, All selected mutated enzymes cataly zed the formation of polyprenyl diphosphates with prenyl chains longer than geranylgeranyl diphosphate. Especially mutants 1, 3, and 5 showe d the strongest elongation activity to produce large amounts of gerany lfarnesyl diphosphate with a concomitant amount of hexaprenyl diphosph ate. Sequence analysis revealed that each mutant contained a few amino acid substitutions and that the mutation of Phe-77, which is located on the fifth amino acid upstream from the first aspartate-rich consens us motif, is the most effective for elongating the ultimate product, A mino acid alignment of known prenyltransferases around this position a nd our previous observations on farnesyl-diphosphate synthase (Ohnuma, S.-i,, Nakazawa, T., Hemmi, H,, Hallberg, A.-M,, Koyama, T,, Ogura, K ,, and Nishino, T, (1996) J, Biol, Chem, 271, 10087-10095) clearly ind icate that the amino acid at the position of all prenyltransferases mu st regulate the chain elongation.