EFFECTS OF RANDOM MUTAGENESIS IN A PUTATIVE SUBSTRATE-BINDING DOMAIN OF GERANYLGERANYL DIPHOSPHATE SYNTHASE UPON INTERMEDIATE FORMATION ANDSUBSTRATE-SPECIFICITY

Archaeal geranylgeranyl diphosphate (GGPP) synthase catalyzes the cons ecutive condensation of isopentenyl diphosphate (IPP) with allylic dip hosphates to produce GGPP with significant amounts of intermediates, T o obtain information about the amino acids involved in the condensatio n and the release of intermediates, we randomly mutagenized two proxim al regions, I and II, of the Sulfolobus acidocaldarius GGPP synthase g ene and created two degenerate libraries, I and II, respectively, Regi ons I and II correspond to amino acid residues 170-173 and 166-168, re spectively, The prenyltransferase activities of about 200 clones were analyzed using the in vivo red-white system and the conventional in vi tro assay, Although, in library I, no mutated enzymes that failed to c atalyze the formation of GGPP were found, as assayed with the red-whit e system, almost all the mutated enzymes exhibited weak GGPP synthesis activity, and many produced large amounts of intermediates, The forma tion ofintermediates increased as the concentration of IPP was decreas ed or as the concentration of the allylic substrate was increased, The se phenomena can be regarded as a reflection of the increased Km for I PP and the decreased affinity for products including intermediates, On the other hand, no mutants from library II showed such changes, These results suggest that the region from 170 to 173 is concerned in the r ecognition of both IPP and allylic diphosphates, and that the change i n responsiveness to prenyl diphosphates causes a change in intermediat e formation.