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. 2001 Apr 2;20(7):1530-7.
doi: 10.1093/emboj/20.7.1530.

Crystal structure of isopentenyl diphosphate:dimethylallyl diphosphate isomerase

V Durbecq  1 , G Sainz, Y Oudjama, B Clantin, C Bompard-Gilles, C Tricot, J Caillet, V Stalon, L Droogmans, V Villeret
Affiliations

Crystal structure of isopentenyl diphosphate:dimethylallyl diphosphate isomerase

V Durbecq et al. EMBO J. .

Abstract

Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway. This enzyme is responsible for the isomerization of the carbon-carbon double bond of IPP to create the potent electrophile DMAPP. DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature. The crystal structures of free and metal-bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism. The enzyme requires one Mn(2+) or Mg(2+) ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site. Two critical residues, C67 and E116, face each other within the active site, close to the metal-binding site. The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon-carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere. W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole- charge interaction.

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Figures

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Fig. 1. Interconversion of IPP into DMAPP by an antarafacial [1.3] transposition of hydrogen, as catalysed by IPP isomerase.
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Fig. 2. View of the overall structure of IPP isomerase by ribbon representation of secondary structure elements. (A) Metal-free structure. (B) Metal-bound structure, with two additional β-strands at the N-terminus. The location of the metal ion is shown (M). The sequence delimitation of secondary structure elements is as follows: H1, N76–L88; H2, L144–A153; H3, P160–T167; H4, R169–A177; S1, H5–L9; S2, P15–E20; S3, F35–F40; S4, L46–R51; S5, V61–N64; S6, V66–G68; S7, E96–Y99; S8, R103–T107; S9, V113–V117; S10, V120–R124; S11, V136–C142. S1 and S2 are only present in the metal-bound structures.
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Fig. 2. View of the overall structure of IPP isomerase by ribbon representation of secondary structure elements. (A) Metal-free structure. (B) Metal-bound structure, with two additional β-strands at the N-terminus. The location of the metal ion is shown (M). The sequence delimitation of secondary structure elements is as follows: H1, N76–L88; H2, L144–A153; H3, P160–T167; H4, R169–A177; S1, H5–L9; S2, P15–E20; S3, F35–F40; S4, L46–R51; S5, V61–N64; S6, V66–G68; S7, E96–Y99; S8, R103–T107; S9, V113–V117; S10, V120–R124; S11, V136–C142. S1 and S2 are only present in the metal-bound structures.
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Fig. 3. Stereo view of the metal-binding site in the Mn2+–IPP isomerase complex, refined at a resolution of 2.1 Å. The electron density map is contoured at the 1.2σ level. H25, H32, H69, E114 and E116 form a distorted octahedral coordination sphere, located in the active site.
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Fig. 4. Stereo view illustrating the location, within the active site, of the sulfate ion (SUL) and the imidazole molecule (IMD). The Mn2+ ion (in purple) and its coordination site composed of H25, H32, H69 and E114 and E116 are shown, as well as S36, Y104 and W161 at the bottom of the active site. K21, K55 and R51 interact with the sulfate ion. Buried water molecules are located between an ionic cluster composed of K55, E87, R83, E4 and K21 and the catalytic C67. The water molecule hydrogen bonded to E87 is indicated (WAT).
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Fig. 5. (A) Stereo view of IPP represented within the active site. (B) Possible contacts between IPP and the Mn2+-bound enzyme, and the proposed mechanism of IPP isomerization. C67 may protonate the C4 carbon of the allyl moiety of IPP, forming a carbocation stabilized by the indole π electrons of W161. E116 is positioned to accomplish the deprotonation at carbon C2, forming DMAPP.
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Fig. 5. (A) Stereo view of IPP represented within the active site. (B) Possible contacts between IPP and the Mn2+-bound enzyme, and the proposed mechanism of IPP isomerization. C67 may protonate the C4 carbon of the allyl moiety of IPP, forming a carbocation stabilized by the indole π electrons of W161. E116 is positioned to accomplish the deprotonation at carbon C2, forming DMAPP.
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