Enantiospecific (+)- and (-)-germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif

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Prosser, I., Altug, I. G., Phillips, A. L., Konig, W. A., Bouwmeester, H. J. and Beale, M. H. 2004. Enantiospecific (+)- and (-)-germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif. Archives of Biochemistry and Biophysics. 432 (2), pp. 136-144.

AuthorsProsser, I., Altug, I. G., Phillips, A. L., Konig, W. A., Bouwmeester, H. J. and Beale, M. H.
Abstract

The naturally occurring, volatile sesquiterpene hydrocarbon germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant–insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-germacrene D synthase and Sc19 as (−)-germacrene D synthase. Thus, the enantiomers of germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D, E), Sc11 is unusual in that this motif occurs as 303NDTYD. Mutagenesis of this motif to 303DDTYD gave rise to an enzyme that fully retained (+)-germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.

KeywordsGermacrene D; Enantiomers; Enantiospecificity; Terpene cyclase; Solidago; Sesquiterpene
Year of Publication2004
JournalArchives of Biochemistry and Biophysics
Journal citation432 (2), pp. 136-144
Digital Object Identifier (DOI)doi:10.1016/j.abb.2004.06.030
Open accessPublished as non-open access
Funder project or code504
Output statusPublished
Publication dates
Online28 Oct 2004
Copyright licensePublisher copyright
ISSN0003-9861
PublisherElsevier Science Inc

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