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IS Superfamily

Isoprenoid Synthase (IS) Superfamily

Project period : 2010 - present

C. Dale Poulter, University of Utah


  • prenyl transfer via generation of allylic carbocations and subsequent condensation/rearrangement to form elongated and/or cyclized isoprene products


  • mostly all α-helical with a core bundle of anti-parallel α-helices

Challenges for Function Assignment

  • basic principles attributable to all members must be established due to lack of comprehensive study in the superfamily context
  • many eukaryotic members impedes adequate coverage of sequence space due to limitations in gene/protein acquisition
  • thousands of possible products complicates functional characterization

Value to Integrated Strategy

  • provides a scenario complementary to other superfamilies under study by requiring prediction of product instead of prediction of substrate
  • serves as a test case for functional assignment in superfamilies previously only thoroughly characterized at the individual enzyme level
  • extensive mechanistic study allows informed generation and analysis of bioinformatic and computational predictions

Members of the isoprenoid synthase (IS) superfamily are involved in the biosynthesis of a staggeringly diverse range of isoprenoid products.  While all characterized isoprenoid synthases generate C(5)n allylic carbocation species which subsequently alkylate nucleophilic centers, several distinct superfamilies exist which vary in fold and substrate/product specificity.  Type I IS superfamily members serve as major catalysts in isoprenoid biosynthesis, and with > 13,000 members in sequence databases, the IS (I) superfamily is an excellent choice for study by the EFI.


Figure IS1. Active site of farnesyl pyrophospate synthetase from E. coli showing inert analogue dimethylallyl S-thiolodiphosphate )S-DMAPP) and isopentyl prophosphate (IPP) as well as the hydrogen bonding network between the conserved Asp regions and the catalytically essential magnesium ions.


A hallmark of the IS (I) superfamily is conservation of two Asp rich regions (DDxxD) found within the active site.  These regions are involved in the binding of three Mg2+ ions essential for formation of the allylic carbocation intermediate (Figure IS1).  The IS (I) superfamily fold is comprised of an almost entirely α-helical structure that contains a core of bundled anti-parallel α-helices (Figure IS2).  The substrate pocket holds accepter and donor sites with the size commensurate to the length and shape of the final product.  Members of the IS (I) superfamily are highly selective for E double bond stereochemistry while processing of Z bonds is carried out by a distinct α/β fold superfamily. While Type II isoprenoid synthases are also structurally distinct and carry out protein prenylation as well as other prenyl transfer reactions, some multi-domain proteins harbor the signature folds of both the Type I and Type II IS superfamilies.


Figure IS2. The IS (I) superfamily a-heical core bundle as a monomer (left) and dimer (right). S-DMAPP is shown in dark grey to delinate the active site.


IS (I) superfamily members typically catalyze condensation of isoprenoid units through metal assisted dissociation of pyrophosphate to generate a carbocation intermediate (Figure IS3).  Condensation of the carbocation with an allylic acceptor and subsequent loss of a proton results 1 of 4 different connectivity patterns, and an additional 4 can be formed via rearrangement of a common intermediate (Figure IS3).  Standard nomenclature to describe the product (e.g.1’-4 and c1’-2-3) is based on the structures of the hemiterpenes dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) and assigns the prime designation to the donor (carbocation) unit to distinguish it from the acceptor (allylic) unit.  Linear chain elongation products synthesized by members of the IS (I) superfamily include C10-50 all-trans isoprenoid diphosphates such as the prototypical C15 product, farnesyl diphosphate.  Examples of cyclopropyl formation occur in members involved in sterol and carotenoid biosynthesis where c1’-2-3 is formed as an intermediate with subsequent rearrangement to give products such as squalene and phytoene.  Additional reactions catalyzed in the IS (I) superfamily include cyclization of geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) to result in mono-, sesqui-, and diterpenes.


Figure IS3. Representative reaction mechansism in the IS (I) superfamily showing carbocation formation and condensation of isoprenoid units during the biosynthesis of farnesyl diphospate (above). Simplified structures showing condensation and rearrangement patterns where the acceptor is shown in black, the donor in red, and the newly formed bonds in blue (below).


With the exception of a small group of parasitic bacteria, all organisms are known to carry out de novo biosynthesis of isoprenoids.  As such, more than 55,000 unique isoprenoid compounds have been described to date.  These molecules play critical roles in a wide range of cellular metabolism as the entry for all isoprenoid derived metabolites found in nature.  Although the wealth of recent sequencing data is a tremendous opportunity to identify and study new members of the IS (I) superfamily and further explore the isoprenoid class of compounds, the large number of poorly annotated sequences stifles the utility of such data.  Decades of fruitful mechanistic, structural, and mutagenesis studies has provided the basis for understanding individual enzymes and will complement a comprehensive examination of the sequence/structure relationships governing the entire IS (I) superfamily.  In conjunction with helping to build and refine the EFI’s strategy for functional assignment, collaboration between the IS Briding Project and the Superfamily/Genome, Computation, and Structure Cores will undoubtlably lead to characerization of unknown IS (I) enzymes and families and potentially reveal novel isoprenoid pathways. 

Representative References

  • Farnesyl pyrophosphate synthetase.  A stepwise mechanism for the 1'-4 condensation reaction. Poulter CD, Wiggins PL, Le AT. (1981) J Am Chem Soc 103, 3926-7.
  • Structural basis for bisphosphonate-mediated inhibition of isoprenoid biosynthesis. Hosfield DJ, Zhang Y, Dougan DR, Broun A, Tari LW, Swanson RV, Finn J. (2004) J Biol Chem 279, 8526-9.
  • Structural biology and chemistry of the terpenoid cyclases. Christianson DW. (2006) Chem Rev 106, 3412-42.
  • Chimeras of two isoprenoid synthases catalyze all four coupling reactions in isoprenoid biosynthesis. Thulasiram HV, Erickson HK, Poulter CD. (2007) Science 316, 73-6.