Pentalenene Synthase: Analysis of Active Site Residues by Site-Directed Mutagenesis

M. Seemann, G. Zhai, J.W. de Kraker, C.M. Paschall, D.W. Christianson, D.E. Cane

Research output: Contribution to journalArticleAcademicpeer-review

106 Citations (Scopus)

Abstract

Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F mutants of pentalenene synthase, an enzyme from Streptomyces UC5319, yielded pentalenene (2), accompanied by varying proportions of (+)-germacrene A (7) with relatively minor changes in kcat and kcat/Km. By contrast, single H309 mutants gave rise to both (+)-germacrene A (7) and protoilludene (8) in addition to pentalenene (2). Mutation to glutamate of each of the three aspartate residues in the Mg2+-binding aspartate-rich domain, 80DDLFD, resulted in reduction in the kcat/Km for farnesyl diphosphate and formation of varying proportions of pentalenene and (+)-germacrene A (7). Formation of (+)-germacrene A (7) by the various pentalenene synthase mutants is the result of a derailment of the natural anti-Markovnikov cyclization reaction, and not simply the consequence of trapping of a normally cryptic, carbocationic intermediate. Both the N219A and N219L mutants of pentalenene synthase were completely inactive, while the corresponding N219D mutant had a kcat/Km which was 3300-fold lower than that of the wild-type synthase, and produced a mixture of pentalenene (2) (91%) and the aberrant cyclization product ß-caryophyllene (9) (9%). Finally, the F77Y mutant had a kcat/Km which was reduced by 20-fold compared to that of the wild-type synthase.
Original languageEnglish
Pages (from-to)7681-7689
JournalJournal of the American Chemical Society
Volume124
Issue number26
DOIs
Publication statusPublished - 2002

Keywords

  • farnesyl-diphosphate synthase
  • high-level expression
  • pre-steady-state
  • crystal-structure
  • aristolochene synthase
  • trichodiene synthase
  • 5-epi-aristolochene synthase
  • enzymatic cyclization
  • sterol biosynthesis
  • molecular-cloning

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