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.