The type II isopentenyl Diphosphate:Dimethylallyl diphosphate isomerase (IDI-2): A model for acid/base chemistry in flavoenzyme catalysis

Abstract

The chemical versatility of the flavin coenzyme is nearly unparalleled in enzyme catalysis. An interesting illustration of this versatility can be found in the reaction catalyzed by the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) - an enzyme that interconverts the two essential isoprene units (isopentenyl pyrophosphate and dimethylallyl pyrophosphate) that are needed to initiate the biosynthesis of all isoprenoids. Over the past decade, a variety of biochemical, spectroscopic, structural and mechanistic studies of IDI-2 have provided mounting evidence that the flavin coenzyme of IDI-2 acts in a most unusual manner - as an acid/base catalyst to mediate a 1,3-proton addition/elimination reaction. While not entirely without precedent, IDI-2 is by far the most extensively studied flavoenzyme that employs flavin-mediated acid/base catalysis. Thus, IDI-2 serves as an important mechanistic model for understanding this often overlooked, but potentially widespread reactivity of flavin coenzymes. This review details the most pertinent studies that have contributed to the development of mechanistic proposals for this highly unusual flavoenzyme, and discusses future experiments that may be able to clarify remaining uncertainties in the chemical mechanism of IDI-2.

Figure 1

Biochemical studies of the Staphylococcus aureus IDI-2 enzyme. A) UV-visible absorption spectra of reduced IDI-2:FMN in the absence (blue) and presence (magenta) of IPP. The λ_max value of ~ 425 nm in the IDI-2:FMN:IPP complex is consistent with the neutral 1,5-dihydro-FMNH₂ tautomeric state of the reduced flavin. B) Pre-steady state accumulation and decay of the FMNH₂ intermediate under single turnover conditions. After the rapid accumulation phase (k_fast = 37 s⁻¹), the intermediate decays to an equilibrium level at a kinetically competent rate (k_slow = 2.1 s⁻¹ > k_cat = 1.3 s⁻¹), suggesting that the FMNH₂ species could be catalytically relevant. C) Pre-steady state burst experiment. The lack of a detectable pre-steady state burst in DMAPP formation suggests that the rate-determining step in the kinetic mechanism occurs prior to or concomitant with DMAPP formation, yielding a velocity (k_ss = 1.5 s⁻¹) that is very similar to k_cat (1.3 s⁻¹). This observation is consistent with rate-limiting isomerization chemistry step(s). Please note that the theoretical maximum burst amplitude of 1.0 would not be expected for the isomerization reaction catalyzed by IDI-2 where IPP and DMAPP would be in equilibrium at the active site. The depicted curve is only meant to illustrate the diagnostic utility of a pre-steady state burst experiment. D) An ¹H-NMR assay was used to measure a primary substrate deuterium kinetic isotope effect on the reaction velocity at saturating IPP concentrations as an approximation of ^Dk_cat. The observed isotope effect (^Dk_cat = 2.2) is consistent with cleavage of the IPP pro-R C2-H/D bond in a partially rate-determining step.

Figure 2

Linear free energy relationship (LFER) studies of IDI-2. A) Hammett plots correlating the steady state kinetic parameters of IDI-2 enzymes reconstituted with the flavins shown at the bottom of the figure. LFERs were observed when the rate constants were plotted vs. either the sum of the Hammett σ_m and σ_p substituent constants or the estimated pK_a of the N5 position of the flavin (data not shown). The negative slopes in the Hammett plot (ρ = −2.1) are consistent with the accumulation of positive charge on the flavin N5 atom in the transition state(s) that limit steady state turnover. B) Combined KIE/LFER study. The steady state reaction velocities for IDI-2 reconstituted with each flavin analogue were measured at saturating concentrations of either IPP or (R)-[2-²H]-IPP. The KIE was found to be approximately constant across the series, suggesting that the flavin analogues are neither perturbing the transition state structure nor altering the expression of the KIE by exerting effects on the energetic barriers of other steps in the mechanism.

Figure 3

Active site views of IDI-2 from Sulfolobus shibatae (PDB ID 2ZRY). Panel A shows the constellation of conserved amino acid residues in the immediate vicinity of the flavin and bound isoprene. Mutations of these residues perturb IDI-2 catalysis, but none of them are suitably positioned to mediate proton transfers to and from IPP. In contrast, the flavin N5 atom appears to be optimally positioned to both C2 (3.19 Å) and C4 (3.52 Å) to mediate the suprafacial 1,3-proton addition/elimination suggested by stereochemical studies. Panel B shows an alternative view of the same structure to illustrate the putative π-stacking between the flavin and isoprene. The flavin N1 and O4′ are also in the vicinity of IPP C4 and C2, respectively. Roles for these residues in catalysis cannot yet be ruled out, but they are not positioned as optimally as N5.

Scheme 1

Biosynthetic pathways for the ubiquitous isoprene units, isopentenyl pyrophosphate (IPP, 1) and dimethylallyl pyrophosphate (DMAPP, 2). These compounds can be interconverted by isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI) enzymes.

Scheme 2

Chain initiation and elongation in isoprenoid biosynthesis. Note that both IPP and DMAPP are required in the initial condensation event (mediated by farnesyl pyrophosphate synthase). Thus, IDI enzymes serve an essential function for the growth and survival of most organisms.

Scheme 3

Putative chemical mechanism of IDI-1 from Escherichia coli.

Scheme 4

Stereochemical studies of IDI-2.

Scheme 5

Early mechanistic models for IDI-2 and the structures of the deazaflavin analogues.

Scheme 6

The radical clock IPP analogue 3-cyclopropyl-3-buten-1-yl diphosphate (cIPP, 9) is converted by IDI-2 into the corresponding DMAPP product (10), suggesting that a radical intermediate (e.g. 11) is not generated by IDI-2 during turnover. These data argue strongly against a single electron transfer mechanism for IDI-2.

Scheme 7

General considerations for IDI-2 catalyzed acid/base chemical mechanisms. Various roles for the reduced flavin in acid/base catalysis can be envisioned. The anionic flavin (13) could work in concert with an IDI-2 derived amino acid side chain (top path), or FMNH₂ could catalyze both proton transfers via the neutral 1,5-dihydro tautomer (14, middle path) or the zwitterionic 5,5-dihydro tautomer (15, bottom path). The reactions are drawn as occurring by stepwise mechanisms, but the paths involving FMNH⁻ (top) and 1,5-dihydro-FMNH₂ (middle) could also occur by concerted proton addition/elimination mechanisms.

Scheme 8

Mechanistic studies with fluoromethyl IPP/DMAPP analogues. Compounds 16-18 are isomerized by IDI-2 at rates much slower than the native substrate (IPP) – paralleling the reactivity trends seen in the hydrolysis of 19, which is believed to proceed by an S_N1 mechanism through a 3°cationic intermediate. This data is consistent with the formation of a carbocation intermediate or cation-like transition state in the IDI-2 catalyzed reaction.

Scheme 9

Suprafacial protonation/deprotonation by IDI-2 revealed by chiral methyl analysis. IPP analogues 20 and 21 yield DMAPP products with distinct enantiomeric configurations at the (E)-methyl group, suggesting that protonation at C4 and deprotonation at C2 occur from the face of the isoprene molecule that is exposed to the flavin in the crystal structure.

Scheme 10

Irreversible covalent inactivation of flavin by oIPP (3-oxiranyl-3-buten-1-yl pyrophosphate).

Scheme 11

Current models for IDI-2 mediated acid/base catalysis. See text for details.

Scheme 12

Chemical structure of 2-(dimethylamino)ethyl-pyrophosphate.