Heteromeric geranyl diphosphate synthase from mint: construction of a functional fusion protein and inhibition by bisphosphonate substrate analogs.
Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate (C(5)) with isopentenyl diphosphate (C(5)) to produce geranyl diphosphate (C(10)), the essential precursor of monoterpenes. The enzyme from peppermint and spearmint (Menthaxpiperita and Mentha spicata, respectively) functions as a heterodimer or heterotetramer consisting of a 40kDa subunit and 33kDa subunit. The DNAs encoding each subunit were joined with different sized linkers and in both possible orders, and expressed in Escherichia coli to yield the corresponding fused protein. The properties of the recombinant fused version, in which the small subunit was followed by the large subunit with a 10 amino acid linker, resembled those of the native heteromeric enzyme in kinetics, product chain-length specificity, and architecture, and this form thus provided a suitable single gene transcript for biotechnological purposes. Bisphosphonate substrate analogs of the type that inhibit farnesyl diphosphate synthase (C(15)) and geranylgeranyl diphosphate synthase (C(20)) also inhibited the fused geranyl diphosphate synthase, apparently by interacting at both the allylic and homoallylic co-substrate binding sites. The results of inhibition studies, along with the previously established role of the small subunit and related mutagenesis experiments, suggest that geranyl diphosphate synthase employs a different mechanism for chain-length determination than do other short-chain prenyltransferases.
Burke
Charles
C
Institute of Biological Chemistry, Graduate Program in Plant Physiology, Washington State University, Pullman, WA 99164-6340, USA.
Klettke
Karin
K
Croteau
Rodney
R
D000595:Amino Acid Sequence; D002250:Carbon Radioisotopes; D002849:Chromatography, Gas / Q000379:methods; D002850:Chromatography, Gel; D004122:Dimethylallyltranstransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D004164:Diphosphonates / Q000737:chemistry / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D045782:Hemiterpenes; D007093:Imidazoles / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007700:Kinetics; D027541:Mentha / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000439:Pentanols / Q000032:analysis; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D000077211:Zoledronic Acid
D002250:Carbon Radioisotopes; D004164:Diphosphonates; D004791:Enzyme Inhibitors; D045782:Hemiterpenes; D007093:Imidazoles; D000439:Pentanols; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C009034:prenol; D000077211:Zoledronic Acid; D004122:Dimethylallyltranstransferase