Crystal Structure of the C67A Mutant of Isopentenyl Diphosphate Isomerase Complexed With a Mechanism- Based Irreversible Inhibitor J. Wouters, 1,2 * Y. Oudjama, 1 V. Stalon, 1,2 L. Droogmans, 2 and C.D. Poulter 3 1 Institut de Recherches Microbiologiques J.M. Wiame, Bruxelles, Belgium 2 Laboratoire de Microbiologie, ULB Campus Ceria, Bruxelles, Belgium 3 Department of Chemistry, University of Utah, Salt Lake City, Utah ABSTRACT Isopentenyl diphosphate:dimethy- lallyl diphosphate (IPP:DMAPP) isomerase is a key enzyme in the biosynthesis of isoprenoids. The mechanism of the isomerization reaction involves protonation of the unactivated carbon-carbon double bond in the substrate. Analysis of the 1.97 Å crystal structure of the inactive C67A mutant of E. coli isopentenyl diphosphate:dimethylallyl diphos- phate isomerase complexed with the mechanism- based inactivator 3,4-epoxy-3-methyl-1-butyl diphos- phate is in agreement with an isomerization mechanism involving Glu 116, Tyr 104, and Cys 67. In particular, the results are consistent with a mechanism where Glu116 is involved in the protona- tion step and Cys67 in the elimination step. Proteins 2004;54:216 –221. © 2003 Wiley-Liss, Inc. INTRODUCTION The isoprenoid biosynthetic pathway produces a wide variety of compounds, including sterols, dolichols, ubiqui- nones, carotenoids, farnesylated and geranylgeranylated proteins, and many other isoprenoid-derived natural prod- ucts. The structures of the enzymes catalyzing the biosyn- thesis of these metabolites are of considerable interest from the perspective of drug design. For example, inhibi- tors of the enzyme hydroxymethylglutaryl CoA reductase are important as cholesterol lowering drugs, while inhibi- tors of farnesyl diphosphate synthase are important in treating osteoporosis, Paget’s disease, and hypercalcemia due to malignancy. Isopentenyl diphosphate-dimethylallyl diphosphate isomerase (EC 5.3.3.2) catalyses the isomer- ization of isopentenyl diphosphate (IPP (1, Scheme 1)) to its electrophilic allylic isomer dimethylallyl diphosphate (DMAPP (2)). DMAPP then condenses with additional molecules of IPP to form FPP, which is required for protein prenylation, cholesterol biosynthesis, and synthesis of a variety of higher molecular weight isoprenoids. Crystal structures of free and metal bound E .coli IPP isomerase show that the protein requires a divalent metal cation to fold into its active conformation, with the metal in a distorted octahedral coordination site composed of three histidines and two glutamates. 1 Recently, we solved the structures of wt E. coli IPP isomerase complexed with a transition state analogue (N,N-dimethyl-2-amino-1- ethyl diphosphate, NIPP (3) and a covalently attached irreversible inhibitor (3,4-epoxy-3-methyl-1-butyl diphos- phate, EIPP, (4). 2 We also obtained the structure of the complex between the bromohydrin of isopentenyl diphos- phate (5) and the inactive C67A mutant of E.coli IPP isomerase. 3 In all those structures of complexes, a second metal site was observed. The metal in this second site is coordinated to the carbonyl oxygen of residue 67 (Cys or Ala in wt or mutant structures respectively), a side chain oxygen (OE2) of Glu 87, two water molecules, and non- bridging oxygens on P1 and P2 of the diphosphate moieties in the inhibitors. Those structures provide insights about the isomerization of IPP to DMAPP and suggest that Glu 116, Tyr 104, and Cys 67 are involved in the antarafacial addition/elimination of protons during isomerization (Scheme 1). It is, however, of interest to study new structures of enzyme-inhibitor complexes in order to ob- tain additional information about the mechanism of the reaction. The structure of the inactive C67A mutant of E. coli IPP isomerase complexed with EIPP, which demon- strates that the protonation machinery is still intact, is now presented. EXPERIMENTAL PROCEDURES Crystal Structure Mutant (C67A) construction, enzyme characterization, protein production and purification were performed follow- ing described procedures. 1–3 Crystals of C67A mutant of E.coli IPP isomerase were grown at room temperature by the hanging drop method. Protein (10 mg/mL) was equili- brated against a reservoir containing PEG2000 (16%), Abbreviations: IPP, isopentenyl diphosphate; DMAPP, dimethylal- lyl diphosphate; EIPP, 3,4-epoxy-3-methyl-1-butyl diphosphate; FIPP, 3-(fluoromethyl)-3-butenyl diphosphate; NIPP N,N-2-dimethylamino- 1-ethyl diphosphate; wt, wild-type. Grant sponsor: the Belgian Fonds National de la Recherche Scienti- fique (FRFC and ISSN grant); Grnat sponsor: ‘Action de Recherche Concerte ´e’ (ARC) financed by the French Community of Belgium and from the Belgian Fund for Joint Basic research; Grant sponsor: National Institutes of Health; Grant number: GM25521. L. Droogmans is a Research Associate of the Belgian Fonds National de la Recherche Scientifique. *Correspondence to: Johan Wouters, Institut de Recherches Microbi- ologiques J.M. Wiame, 1 av E. Gryzon 1070 Bruxelles. E-mail: jwouters@dbm.ulb.ac.be Received 7 April 2003; Accepted 18 June 2003 PROTEINS: Structure, Function, and Bioinformatics 54:216 –221 (2004) © 2003 WILEY-LISS, INC.