A PATHWAY WHERE POLYPRENYL DIPHOSPHATE ELONGATES IN PRENYLTRANSFERASE- INSIGHT INTO A COMMON MECHANISM OF CHAIN-LENGTH DETERMINATION OF PRENYLTRANSFERASES

Prenyltransferases catalyze the consecutive condensations of isopenten yl diphosphate to produce linear polyprenyl diphosphates. Each enzyme forms the final product with a specific chain length. The product spec ificity of an enzyme is thought to be determined by the structure arou nd the unknown path through which the product elongates in the enzyme. To explore the path, we introduced a few mutations at the 5th, the 8t h, and/or the 11th positions before the first aspartate-rich motif of geranylgeranyl-diphosphate synthase or farnesyl-diphosphate synthase, The side chains of these amino acids are situated on the same side of an alpha-helix. In geranylgeranyl-diphosphate synthase, a single mutat ed enzyme (F77S) mainly produces a C-25 product (Ohnuma, S.-I., Hirook a, K., Hemmi, H., Ishida, C., Ohto, C., and Nishino, T. (1996) J. Biol . Chem. 271, 18831-18837). A double mutated enzyme (L74G and F77G) mai nly produces a C-35 compound with significant amounts of C-35 and C-40 . A triple mutated enzyme (I71G, L74G, and F77G) mainly produces a C-4 0 compound with C-35 and C-45. Mutated farnesyl-diphosphate synthases also show similar patterns. These findings indicate that the elongatin g product passages on a surface of the side chains of the mutated amin o acids, the original bulky amino acids had blocked the elongation, an d the path is conserved in prenyltransferases. Moreover, the fact that some double and triple mutated enzymes can also form small amounts of products longer than C-50 indicates that the paths in these mutated e nzymes can partially access the outer surface of the enzymes.