Partial purification of a farnesyl diphosphate synthase from whole-body Manduca sexta.
Farnesyl diphosphate synthase (FPP synthase) is a ubiquitous enzyme that is required for the biosynthesis of sesquiterpenes, dolichols ubiquinones, and prenylated proteins in insects. We report on the partial purification and characterization of an FPP synthase, obtained from whole-body preparations of the lepidopteran insect, Manduca sexta. The larval enzyme was separated from isopentenyl diphosphate (IPP) isomerase, phosphatase, and GGPP synthase by preparative isoelectric focusing, and was further purified by DEAE Sepharose, hydroxyapatite, and size exclusion chromatography. Whole-body M. sexta FPP synthase has a native molecular weight of 60.5+/-3.5 kDa and consists of two subunits of 28.5+/-0.5 kDa. As seen with other prenyltransferases, the enzyme has an absolute requirement for divalent cation and both Mn(2+) and Mg(2+) stimulated activity, although the former was inhibitory at higher concentrations. Insect FPP synthase catalyzes the condensation of IPP (K(m)=2.9+/-1.2 microM) with both dimethylallyl diphosphate and geranyl diphosphate (K(m)=0.8+/-0.4 microM). The enzyme requires the presence of detergent, glycerol, and non-specific protein-protein interactions for stability and maximum catalytic activity.
Sen
Stephanie E
SE
Department of Chemistry, Indiana University--Purdue University at Indianapolis, 402 North Blackford Street, Indianapolis, IN 46202, USA. sen@chem.iupui.edu
Sperry
Andrea E
AE
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002845:Chromatography; D004795:Enzyme Stability; D051228:Geranyltranstransferase; D007525:Isoelectric Focusing; D018402:Manduca / Q000201:enzymology*; D008970:Molecular Weight; D021122:Protein Subunits; D013045:Species Specificity
D021122:Protein Subunits; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase