The Crystal Structure of Human Isopentenyl Diphosphate Isomerase at 1.7 Å Resolution Reveals its Catalytic Mechanism in Isoprenoid Biosynthesis Wei Zheng 1 , Fei Sun 2 , Mark Bartlam 1,2 , Xuemei Li 2 Ran Li 1 and Zihe Rao 1,2 ⁎ 1 Tsinghua-Nankai-IBP Joint Research Group for Structural Biology, Tsinghua University , Beijing 100084, China 2 National Laboratory of Biomacromolecules, Institute of Biophysics (IBP), Chinese Academy of Sciences, Beijing 100101, China Isopentenyl diphosphate isomerase catalyses a crucial activation step in the biosynthesis of isoprenoids, one of the most ancient and diverse classes of natural products. This enzyme is responsible for an unusual isomerization of the inactive carbon-carbon double bond of isopentenyl diphosphate (IPP) to create its electrophilic allylic isomer dimethylallyl diphosphate (DMAPP). Here we report the crystal structure of human IPP isomerase at 1.7 Å resolution and the complex structure with its native substrate at 1.9 Å resolution. These structures reveal a mechanism wherein interconver- sion is catalyzed by a stereoselective antarafacial [1.3] transposition of a proton involving the indispensable residues Cys87, Glu149, Trp197 and Tyr137. A newly identified alternative conformation of Cys87 driven by Trp197 and the selectivity of different metal ions located in the active site provide further insight into the catalytic mechanism. Comparison with Escherichia coli IPP isomerase reveals a novel substrate entrance in human IPP isomerase. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: isopentenyl diphosphate isomerase; isoprenoid biosynthesis; IPP isomerase; isomerization; crystal structure Introduction Isopentenyl diphosphate isomerase (IPP isomer- ase) is a key enzyme which catalyzes a mandatory activation step in isoprenoid biosynthesis by iso- merization of the carbon-carbon double bond of isopentenyl diphosphate (IPP) 1 to create its electro- philic allylic isomer dimethylallyl diphosphate (DMAPP) (Scheme 1). 2 The isoprenoid biosynthetic pathway is the most chemically diverse pathway in nature and produces the largest group of over 30,000 contemporary natural metabolites 3 such as sterols, 4 carotenoids, 5 dolichols, 6 ubiquinones 7 and some prominent classes of prenylated proteins. 8 Strikingly, the structural complexity of these isoprenoid products is in contrast to their biosynthesis, since all of these compounds are synthesized from only two simple precursors: IPP and DMAPP. DMAPP then alkylates other molecules, including IPP, ultimately forming the extensive isoprenoid compounds in nature. In most eukaryotes, archaea and fungi, the isoprenoids are derived from acetyl-CoA via the well established mevalonate pathway leading to the synthesis of IPP, and IPP isomerase activity is essential for all of these organisms. More recently, it has been found that in higher plants and some bacteria and algae, IPP and DMAPP are synthesized by the methylerythritol phosphate (MEP) pathway, which utilizes pyruvate and dihydroxyacetone phosphate as starting points and branches in its final steps, resulting in the independent formation of IPP and DMAPP such that IPP isomerase is not an essential enzyme in these organisms. 9,10 Two non-homologous protein families 11,12 are reported to catalyze the isomerization reaction in isoprenoid biosynthesis. Type I IPP isomerases were first discovered in the late 1950 s 13 and are found in eukaryota and bacteria where they utilize a divalent metal for activity. Type II IPP isomerases are always found in archaea and bacteria and require reduced flavin and divalent metal cofactors to fulfill catalytic Abbreviations used: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate. E-mail address of the corresponding author: raozh@xtal.tsinghua.edu.cn doi:10.1016/j.jmb.2006.12.055 J. Mol. Biol. (2007) 366, 1447–1458 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.