Kinetic and Binding Studies of Streptococcus pneumoniae Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase.

Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) converts isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP), the two fundamental building blocks of isoprenoid molecules. IDI-2 is found in many species of bacteria and is a potential antibacterial target since this isoform is non-homologous to the type 1 enzyme in Homo sapiens. IDI-2 requires a reduced flavin mononucleotide to form the catalytically active ternary complex, IDI-2·FMNH2·IPP. For IDI-2 from the pathogenic bacterium Streptococcus pneumoniae, the flavin can be treated kinetically as a dissociable cosubstrate in incubations with IPP and excess NADH. Under these conditions, the enzyme follows a modified sequential ordered mechanism where FMN adds before IPP. Interestingly, the enzyme shows sigmoidal behavior when incubated with IPP and NADH with varied concentrations of FMN in aerobic conditions. In contrast, sigmoidal behavior is not seen in incubations under anaerobic conditions where FMN is reduced to FMNH2 before the reaction is initiated by addition of IPP. Stopped-flow experiments revealed that FMN, whether bound to IDI-2 or without enzyme in solution, is slowly reduced in a pseudo-first-order reaction upon addition of excess NADH (k(red)(FMN) = 5.7 × 10(-3) s(-1) and k(red)(IDI-2·FMN) = 2.8 × 10(-3) s(-1)), while reduction of the flavin is rapid upon addition of NADH to a mixture of IDI-2·FMN, and IPP (k(red)(IDI-2·FMN·IPP) = 8.9 s(-1)). Similar experiments with dithionite as the reductant gave k(red)(FMN) = 221 s(-1) and k(red)(IDI-2·FMN) = 411 s(-1). Dithionite reduction of FMN in the IDI-2·FMN and IPP mixture was biphasic with k(red)(IDI-2·FMN·IPP (fast)) = 326 s(-1) and k(red)(IDI-2·FMN·IPP (slow)) = 6.9 s(-1) The pseudo-first-order rate constant for the slow component was similar to those for NADH reduction of the flavin in the IDI-2·FMN and IPP mixture and may reflect a rate-limiting conformational change in the enzyme.

Covalent modification of reduced flavin mononucleotide in type-2 isopentenyl diphosphate isomerase by active-site-directed inhibitors.

Evidence for an unusual catalysis of protonation/deprotonation by a reduced flavin mononucleotide cofactor is presented for type-2 isopentenyl diphosphate isomerase (IDI-2), which catalyzes isomerization of the two fundamental building blocks of isoprenoid biosynthesis, isopentenyl diphosphate and dimethylallyl diphosphate. The covalent adducts formed between irreversible mechanism-based inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor were investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of IDI-2 binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5, which had been proposed previously. In addition, the high-resolution crystal structures of IDI-2-substrate complexes and the kinetic studies of new mutants confirmed that only the flavin cofactor can catalyze protonation of the substrates and suggest that N5 of flavin is most likely to be involved in proton transfer. These data provide support for a mechanism where the reduced flavin cofactor acts as a general acid/base catalyst and helps stabilize the carbocationic intermediate formed by protonation.