Kinetic and Spectroscopic Characterization of Type II Isopentenyl Diphosphate Isomerase from Thermus thermophilus:  Evidence for Formation of Substrate-Induced Flavin Species

Abstract

Type II isopentenyl diphosphate (IPP) isomerase catalyzes the interconversion of IPP and dimethylallyl diphosphate (DMAPP). Although the reactions catalyzed by the type II enzyme and the well-studied type I IPP isomerase are identical, the type II protein requires reduced flavin for activity. The chemical mechanism, including the role of flavin, has not been established for type II IPP isomerase. Recombinant type II IPP isomerase from Thermus thermophilus HB27 was purified by Ni 2+ affinity chromatography. Aerobically purified enzyme was inactive until the flavin cofactor was reduced by NADPH, dithionite, or photochemically. The inactive oxidized flavin-enzyme complex bound IPP in a Mg 2+ dependent manner with K D ~ K m IPP , suggesting that the substrate binds to the inactive oxidized and active reduced forms of the protein with similar affinities. N,Ndimethyl-3-amino-1-propyl diphosphate (NIPP), a transition state analog for the type I isomerase, competitively inhibits the type II enzyme, but with much lower affinity. pH dependent spectral changes indicate that the binding of IPP, DMAPP, and a saturated analogue isopentyl diphosphate promotes protonation of anionic reduced flavin. Electron paramagnetic resonance (EPR) and UVvisible spectroscopy show a substrate-dependent accumulation of the neutral flavin semiquinone during both the flavoenzyme reduction and re-oxidation processes in the presence of IPP and related analogues. Redox potentials of IPP-bound enzyme indicate that the neutral semiquinone state of the flavin is stabilized thermodynamically relative to free FMN in solution.Isopentenyl diphosphate (IPP) isomerase catalyzes the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP), the basic building blocks of isoprenoid molecules (Scheme 1). This family of natural products now consists of over 35,000 compounds (1), which comprise several classes of biologically important molecules, including sterols, carotenoids, prenylated proteins, dolichols, heme A, and ubiquinones. IPP isomerase is essential in organisms that generate IPP through the mevalonate pathway found in eukaryotes, archaea, and some gram-positive eubacteria (2). The enzyme, although not essential, is also found in most organisms that utilize the methylerythritol phosphate pathway, where isomerase activity is probably important for balancing the pools of IPP and DMAPP (3).Two structurally unrelated forms of IPP isomerase have been identified. The type I enzyme resides in eukaryotes and in some eubacteria, while the type II protein is found in archaea and other eubacteria (4). Type I IPP isomerase was discovered over 40 years ago and has been extensively characterized (5). The enzyme is a zinc metalloprotein (6) that catalyzes the isomerization of IPP and DMAPP by an antarafacial (7-9) protonation-deprotonation mechanism (10;11). Structural studies and site directed mutagenesis experiments have † This work was supported by NIH Grant GM25521. SCR was supported by NIH Postdoctoral Fellowship GM071114. *To who...

Evidence for the Involvement of Acid/Base Chemistry in the Reaction Catalyzed by the Type II Isopentenyl Diphosphate/Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus

“…The λ max value at 435 nm is unusual for flavoproteins but is most consistent with a neutral reduced FMN (as suggested previously for IDI-2 (22, 31)), which has been observed to have a λ max value anywhere from 390 to 450 nm, depending on the hydrophobicity and rigidity of the active site environment (32)(33)(34)(35). Consistent with previous photoreduction studies (22), a neutral semiquinone (E•FMN sem ) could be generated under anaerobic conditions if 2 mM IPP was pre-incubated with E•FMN ox prior to a 2 min photoreduction. On the basis of the ε 583 (3300 M −1 cm −1 ) value determined previously for the S. aureus IDI-2-bound FMN sem (21), the concentration of FMN sem in this photoreduced sample is estimated to be 25 µM (or about 31% of the total E•FMN concentration).…”

“…Furthermore, they suggested that the enzyme-bound FMN intermediate that forms during the normal reaction is consistent with a neutral reduced dihydroflavin (4, Scheme 2). Although the steady-state kinetic parameters have been reported for a few IDI-2 enzymes (13,15,18,21,22,25), the nature of the rate limiting step(s) in the catalytic mechanism is unknown. In this paper, we report the investigation of the FMN intermediate formed during the catalysis of S. aureus IDI-2 using UV-vis and EPR spectroscopies.…”

“…For example, it is not clear as to why 5-deazaFMN does not support catalysis, unless this cation-π interaction depends strongly on the N5 atom or a hydrogen bonding network maintained by the N5 atom. Also, it is not clear as to why the neutral form of the reduced coenzyme (4) accumulates upon the addition of substrate, as the anionic form should be better suited to stabilize a carbocation intermediate through electrostatic interactions.If the flavin does not simply perform a structural or electrostatic stabilization role, then it could be acting directly as the active site acid (Scheme 3B), the active site base (Scheme 3C), or both (Scheme 3D) (22,31). The acidic function may be fulfilled by the flavin N5-H group (Scheme 3B).…”

“…Subsequent biochemical and structural characterization of IDI-2 (13,(15)(16)(17)(18)(19)(20)(21)(22) has revealed that the IDI-2-FMN ox complex is reduced with stoichiometric amounts of NAD(P)H via stereospecific transfer of the pro-S hydride and that the resultant IDI-2-FMN red is capable of performing multiple turnovers (21). Recent redox titrations of IDI-2 from Staphylococcus aureus and Thermus thermophilus in the presence and absence of IPP (1) have shown that the apoenzyme thermodynamically stabilizes the neutral flavin semiquinone in the presence of IPP (21,22). It was also found that the apoenzyme reconstituted with 5-deazaFMN is inactive, while 1-deazaFMN supports catalysis (19,21).…”

“…Although the neutral FMN sem (5) has been observed in redox titration and photoreduction experiments (21,22), the catalytic relevance of FMN sem during turnover has not been established. Poulter and co-workers have recently shown that the semiquinone does not accumulate to significant levels in the steady state (22). Furthermore, they suggested that the enzyme-bound FMN intermediate that forms during the normal reaction is consistent with a neutral reduced dihydroflavin (4, Scheme 2).…”