Purification of a prenyltransferase that elongates cis-polyisoprene rubber from the latex of Hevea brasiliensis.
We have purified "rubber transferase" from latex of the commercial rubber tree Hevea brasiliensis and find that it is a dimer with a monomeric molecular mass of 38,000 Da, requires Mg2+, and is stabilized by thiols in agreement with studies of a partially purified preparation previously described (Archer, B. L., and Cockbain, E. G. (1969) Methods Enzymol. 15, 476-480). Greater than 90% of the [1-14C]isopentenyl pyrophosphate which is incorporated into deproteinated rubber particles by the purified prenyltransferase is added to high molecular mass polyisoprene (greater than 20,000 Da). Purified prenyltransferase and deproteinated rubber particles reconstitute 40-60% of the biosynthetic activity of whole latex in samples matched for rubber content. Incorporation is linear with added rubber particles up to at least 10 mg/ml rubber or 20 microM rubber molecules (based on a number average molecular mass of 500,000 Da). Prenyltransferase concentrations estimated in whole latex (0.37% or 160 nM) are sufficient to saturate all elongation sites in whole latex, and addition of purified prenyltransferase does not increase [1-14C]isopentenyl pyrophosphate incorporation. Deproteinated rubber particles can be titrated with the pure enzyme (Kd = 9 nM) demonstrating that the fraction of rubber molecules available for addition is low (approximately 0.01%). An estimated 7,000 isoprene units are added per complex at a rate of 1/s in a typical assay. Hevea prenyltransferase catalyzes the formation of cis-isoprene in the presence of rubber particles. However, in the absence of rubber particles and in the presence of dimethylallyl pyrophosphate, the purified prenyltransferase catalyzes the formation of geranyl pyrophosphate and all trans-farnesyl pyrophosphate as demonstrated by thin layer chromatography, gas chromatography, and molecular exclusion chromatography.
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Department of Medicinal and Biomolecular Chemistry, Genentech, Inc., South San Francisco, California 94080.
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D000595:Amino Acid Sequence; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004122:Dimethylallyltranstransferase / Q000302:isolation & purification* / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D045782:Hemiterpenes; D007700:Kinetics; D007840:Latex / Q000032:analysis*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D012408:Rubber / Q000378:metabolism*; D012717:Sesquiterpenes; D013379:Substrate Specificity; D014166:Transferases / Q000302:isolation & purification*; D014197:Trees
D045782:Hemiterpenes; D007840:Latex; D046911:Macromolecular Substances; D009943:Organophosphorus Compounds; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004809:isopentenyl pyrophosphate; C043060:3,3-dimethylallyl pyrophosphate; C015234:geranyl pyrophosphate; C004808:farnesyl pyrophosphate; D012408:Rubber; D014166:Transferases; D004122:Dimethylallyltranstransferase; D004229:Dithiothreitol