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

Abstract

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 mechanismbased 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.enzyme structure | mechanism of action I sopentenyl diphosphate isomerase (IDI) catalyzes the interconversion between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (1). IDI activity is essential in those organisms that synthesize isoprenoid compounds from mevalonic acid and, although not essential, is found in those that synthesize isoprenoid metabolites from methylerythritol phosphate. Two convergently evolved forms of IDI, which share no sequence or structural homology, are known. Type-1 IDI (IDI-1) is found in eukarya and some bacteria, whereas type-2 IDI (IDI-2) occurs in archaea and other bacteria. There is no apparent correlation between the occurrence of the IDI isoforms and the use of mevalonate or methylerythritol phosphate in isoprenoid biosynthesis.IDI-1 catalyzes isomerization between IPP and DMAPP by a protonation-deprotonation reaction through a 3°carbocationic intermediate (1). The mechanism of the reaction and identification of essential active cysteine and glutamate residues were confirmed by inhibition studies using active-site-directed irreversible inhibitors (2-5). In contrast, IDI-2 is a flavoprotein that requires reduced FMN for activity (6, 7). UV-visible spectroscopic studies indicate that the anionic flavin in IDI-2 · FMN red − is protonated upon substrate binding to give a catalytically competent IDI-2 · FMN red · IPP complex (8). Recent studies indicated that the mechanism for the IDI-2 is similar to the protonation-deprotonation sequence used by IDI-1. These include measurement of deuterium kinetic isotopic effects in D 2 O or with (R)-[2-2 H]-IPP (9), linear free energy studies with seven-and eight-substituted FMN analogues (10), and studies with alkyne and allene analogues of IPP and DMAPP (11). In contrast, at...

Substrate-Induced Change in the Quaternary Structure of Type 2 Isopentenyl Diphosphate Isomerase from Sulfolobus shibatae

“…Therefore, revealing the electron density in the N-terminal region of the new structure is thought to be a result of improvement of the crystal quality and the resolution. Moreover, the orientation and conformation of the N-terminal region in the new structure distinctly differ from those of the previously reported substratecomplex structures (22,33), in which the N-terminal region forms an ␣-helix to construct a part of the substrate-binding site. A superimposed structure of the type 2 IDI-FMN-IPP ternary complex in the reduced state (PDB code 3B05) showed that the N-terminal ␣-helix in the new structure partially unwinds to interact with the ␣-helix (␣11=) and loop regions between ␣11= and ␣12= in the neighboring tetramer unit (Fig.…”

“…As a result, we succeeded in solving the new structure of S. shibatae type 2 IDI at a resolution of 1.7 Å, which is the highest level yet obtained (Table 1, PDB code 3VKJ). Although the new structure contained no electron density of NADH, a remarkable difference between the new and the previously reported structures (22,33) was found in the N-terminal region (Fig. 1A and B).…”

“…Similarly with PFK-2, the enzyme activity of S. shibatae type 2 IDI can be inhibited by octamer formation. The substrate-complex tetrameric structures of the archaeal enzyme (22,33) suggest that unwinding of the N-terminal ␣-helix observed in the octameric structure precludes substrate binding because the helix is involved in active-site formation. If so, octamer dissociation proceeds when the production of IPP via the mevalonate pathway is enhanced and upregulates isoprenoid biosynthesis initiated from the isomerization of IPP.…”

“…Because type 2 IDI is a flavin mononucleotide (FMN)-and NA-DPH-dependent flavoenzyme, it has been expected to catalyze the same reaction with a different mechanism from that used by type 1 IDI, which is a nonflavoenzyme without any homology with type 2 IDI (15). However, recent studies have shown that the reaction mechanism of type 2 IDI is surprisingly similar to that of type 1 IDI (22,24,25,29,30,33). Both types of enzymes catalyze the isomerization between IPP and DMAPP via a protonation-deprotonation mechanism, probably through the same carbocation intermediate.…”

“…The structures of type 2 IDIs from Bacillus subtilis (27) and Thermus thermophilus (7) have been reported as homooctamers, but type 2 IDI from thermophilic archaeon Sulfolobus shibatae has a homotetrameric configuration (22,33). Interestingly, a boundary sedimentation experiment for B. subtilis type 2 IDI showed that the octamer dissociates into a smaller quaternary structure in the presence of FMN, NADPH, and a substrate (18).…”