Construction of Functional Monomeric Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

Abstract

Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the isoprenoid biosynthetic pathway. The enzyme from Streptomyces pneumoniae (spIDI-2) is a homotetramer in solution with behavior, including a substantial increase in the rate of FMN reduction by NADPH in the presence of IPP, suggesting that substrate binding at one subunit alters the kinetic and binding properties of another. We now report the construction of catalytically active monomeric spIDI-2. The monomeric enzyme contains a single-point mutation (N37A) and a six-residue C-terminal deletion that preserves the secondary structure of the subunits in the wild-type (wt) homotetramer. UV-vis spectra of the enzyme-bound flavin mononucleotide (FMN) cofactor in FMNox, FMNred, and FMNred·IPP/DMAPP states are the same for monomeric and wt homotetrameric spIDI-2. The mutations in monomeric IDI-2 lower the melting temperature of the protein by 20 °C and reduce the binding affinities of FMN and IDI by 40-fold but have a minimal effect on kcat. Stopped-flow kinetic studies of monomeric spIDI-2 showed that the rate of reduction of FMN by NADH (k = 1.64 × 10(-3) s(-1)) is substantially faster when IPP is added to the monomeric enzyme (k = 0.57 s(-1)), similar to behavior seen for wt-spIDI-2. Our results indicate that cooperative interactions among subunits in the wt homotetramer are not responsible for the increased rate of reduction of spIDI-2·FMN by NADH, and two possible scenarios for the enhancement are suggested.

Comparative Proteomic Analysis of Dipsacus asperoides Roots from Different Habitats in China

“…Isopentyl diphosphate isomerase 2 (A0A059PYD4, EC 5.3.3.2, IDI-2) is an enzyme required for the synthesis of isoprenoid metabolites via the mevalonic acid (MVA) pathway [35]. IDI-2 catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the isoprenoid biosynthetic pathway [36] (Figure 7). Triterpenoid saponin in plants is composed of isopentenyl diphosphate (IPP) and C5 isoprene unites, which are primarily supplied from the cytosolic MVA pathway and play a major role in the production of triterpenoidal sapogenin backbones [37].…”

Vibrationally resolved absorption and fluorescence spectra of flavins: A theoretical simulation in the gas phase

“…At present, there have been many studies on the UV-visible absorption spectra of flavins. [11][12][13][14] The influence of solvent and protein environment on the UV-visible absorption spectra of five flavin forms has been discussed. [15] Besides, fluorescence spectra of flavins have also been widely studied as another tool to characterize their photophysical properties, especially for oxidized flavins.…”

Mechanistic Studies of the Protonation–Deprotonation Reactions for Type 1 and Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

“…Several lines of evidence, including mechanism-based inhibition, site-directed mutagenesis, and studies with substrate analogues and transition state inhibitors, indicate that IDI-1 and IDI-2 catalyze isomerization via a [1.3] protonation–deprotonation mechanism. − Active-site acids and bases were identified from X-ray structures of IDI-1 complexed with an ammonium transition-state/​reactive intermediate analogue and both enzymes with covalently attached mechanism-based irreversible inhibitors. ,, In the active site of IDI-1, a Zn 2+ -Glu/Tyr motif protonates the C3–C4 double bond in IPP, and a cysteine removes the proton at C2 . In contrast, a zwitter­ionic tautomer of reduced FMN (FMNH 2 ) is thought to be the acid–base catalyst for protonation of the double bond in IPP and abstraction of a proton from C2 …”