Isopentenyl-diphosphate isomerase: inactivation of the enzyme with active-site-directed irreversible inhibitors and transition state analogs

Muehlbacher, M.; Cd, Poulter

doi:10.1021/bi00419a021

Cited by 98 publications

(108 citation statements)

References 34 publications

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“…In the mevalonate pathway IPP and DMAPP are generated sequentially, the latter arising from the former in an equilibration reaction catalyzed by an isomerase that exploits a protonationdeprotonation mechanism (40)(41)(42). In the present case sequential formation of the two compounds seems unlikely for a variety of reasons.…”

Section: Discussionmentioning

confidence: 82%

Studies on the nonmevalonate terpene biosynthetic pathway: Metabolic role of IspH (LytB) protein

Rohdich

Hecht²,

Gärtner³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

315

231

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Isopentenyl diphosphate and dimethylallyl diphosphate serve as the universal precursors for the biosynthesis of terpenes. Although their biosynthesis by means of mevalonate has been studied in detail, a second biosynthetic pathway for their formation by means of 1-deoxy-D-xylulose 5-phosphate has been discovered only recently in plants and certain eubacteria. Earlier in vivo experiments with recombinant Escherichia coli strains showed that exogenous 1-deoxy-Dxylulose can be converted into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by the consecutive action of enzymes specified by the xylB and ispCDEFG genes. This article describes the transformation of exogenous [U-13 C5]1-deoxy-D-xylulose into a 5:1 mixture of [U-13 C5]isopentenyl diphosphate and [U-13 C5]dimethylallyl diphosphate by an E. coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes.T erpenes are one of the largest groups of natural products comprising numerous medically relevant compounds (e.g., vitamins, hormones, and antitumor agents such as Taxol) (1). Bloch, Lynen, Cornforth, and their coworkers showed that the universal terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), are biosynthesized by means of the mevalonate pathway in yeasts and animals (for review see refs. 2-5). These studies served as the basis for the development of metabolic inhibitors that are widely used for the treatment of hypercholesterolemia.For a period of several decades, these milestone achievements completely eclipsed the existence of a second terpenoid pathway for the biosynthesis of IPP and DMAPP. Recently, however, knowledge on that pathway has been unfolding rapidly on the basis of seminal discoveries by the research groups of . Briefly, the mevalonate independent pathway starts from 1-deoxy-D-xylulose 5-phosphate, which is assembled from pyruvate and D-glyceraldehyde 3-phosphate (13, 14) and was already known to serve as a biosynthetic precursor of vitamins B 1 (thiamine) and B 6 (pyridoxal) (Fig. 1) (15-17). 1-Deoxy-D-xylulose 5-phosphate is converted into 2C-methyl-D-erythritol 4-phosphate, the first committed intermediate of the nonmevalonate pathway, by isomerization followed by a two-electron reduction step catalyzed by 2C-methyl-D-erythritol 4-phosphate synthase specified by the ispC gene (formerly designated yaeM and then dxr) (18) (Fig. 1).In the next step, 2C-methyl-D-erythritol 4-phosphate is converted into the 2C-methyl-D-erythritol 2,4-cyclodiphosphate by the sequential action of three enzymes specified by the ispD, ispE, and ispF genes (19-24). The last known step of the sequence, the reductive transformation of the cyclic diphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate has been identified recently in work with a recombinant Escherichia coli strain engineered for hyperexpression of the ispG gene (previously designated gcpE) (25). Comparative genomics suggest that the protein specified by the lytB gene is involved in the conversion of ...

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Section: Discussionmentioning

confidence: 82%

Studies on the nonmevalonate terpene biosynthetic pathway: Metabolic role of IspH (LytB) protein

Rohdich

Hecht²,

Gärtner³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

315

231

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“…3). OMBPP is an effective inhibitor of yeast IPP isomerase activity (Muehlbacher and Poulter, 1988) and the addition of OMBPP inhibited the formation of FPP (Fig. 3).…”

Section: Resultsmentioning

confidence: 94%

“…IPP isomerase (Anderson et al, 1989) and 3,4-oxido-3-methyl-l-butyl diphosphate (OMBPP), a site-directed specific inhibitor of isomerase activity (Muehlbacher and Poulter, 1988) were generously supplied by C. Dale Poulter, University of Utah, USA.…”

Section: Enzymes and Isomerase Inhibitormentioning

confidence: 99%

The separate roles of plant cis and trans prenyl transferases in cis‐1,4‐polyisoprene biosynthesis

Cornish¹

1993

European Journal of Biochemistry

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In plants, the elongation of cis-1,4-polyisoprene (natural rubber, M, > lo6) requires a small transallylic diphosphate ( S C2J initiator. The trans-allylic diphosphates are hydrophilic cytosolic compounds, whereas cis-l,4-polyisoprene is hydrophobic and compartmentalised in subcellular rubber particles.In this paper, it is demonstrated that soluble trans-prenyl transferase from latex of Hevea brusiliensis functions solely as farnesyl diphosphate synthase, and plays no direct role in cis-1,4-polyisoprene elongation. The cis-l,4-prenyl transferase is firmly associated with the H. bvasiliensis rubber particle, as is also the case in other rubber-producing species

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“…5A), which resembles the reaction catalyzed by type 1 IDI, seems preferable to the carbanion-forming mechanism because the adduct formation between FMN and the epoxide substrate analogues such as eIPP is probably initiated by protonation of the epoxide oxygen, followed by nucleophilic attack by the N-5 nitrogen of reduced FMN (10,16,17), and because the proximity (3.42 Ϯ 0.05 Å in the IPP-IDI red structure) of the O-␦ oxygen of Glu 160 to the C-3 carbon of substrates in the complex structures implies stabilization of the 3°carbocation by the carbonyl oxygen. However, it should be noted that 2-(dimethylamino)ethyl diphosphate, which strongly inhibits type 1 IDI (K I Ͻ 10 Ϫ10 M when K m for IPP Ͼ 10 Ϫ6 M) as a transition state analogue (35,36), is a relatively mild inhibiter (K I ϭ 5.1 ϫ 10 Ϫ9 M, when K d for IPP ϭ 4.4 ϫ 10 Ϫ9 M) for type 2 IDI from T. thermophilus (9). Alternatively, if the N-5 nitrogen cannot act as the acid-base catalyst, the N-1 nitrogen and O-4 oxygen are likely to act as a pair of acid/base, along with the tautomeric rearrangement of reduced flavin.…”

Section: Discussionmentioning

confidence: 99%

New Role of Flavin as a General Acid-Base Catalyst with No Redox Function in Type 2 Isopentenyl-diphosphate Isomerase

Unno

Yamashita

Ikeda

et al. 2009

Journal of Biological Chemistry

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Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.Flavins are generally regarded as redox coenzymes because their primary function in redox-catalyzing flavoenzymes is donation and/or acceptance of electrons (1). As summarized in a review article (2), the redox activities of flavins also take part in the flavoenzymes that catalyze reactions with no net redox change. Most of these enzymes are thought to have redoxbased mechanisms, whereas flavins have only structural or stabilizing roles in a few exceptions. Recently, however, a report on UDP-galactopyranose mutase unexpectedly showed that flavin can act as a nucleophilic catalyst (3, 4). In UDP-galactopyranose mutase, a sugar carbon undergoes nucleophilic attack by the N-5 nitrogen of reduced FMN, concomitantly with the dissociation of UDP, forming an adduct intermediate. In this reaction, the flavin cofactor has no redox function because it is continuously in the reduced state.Type 2 isopentenyl-diphosphate isomerase (IDI) 4 is the flavoenzyme that catalyzes the interconversion between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that occurs with no net change in redox status (5). Both compounds are fundamental units for the biosynthesis of isoprenoids, a diverse family of Ͼ50,000 metabolites (6). Type 2 IDI requires FMN, NAD(P)H, and Mg 2ϩ to be active; however, NAD(P)H is used only for the reduction of FMN and can be replaced with Na 2 S 2 O 4 (7-9). The observation that reduced FMN is required for type 2 IDI activity allowed for development of various plausible reaction mechanisms, including redoxbased mechanisms. Based on the traditional interpretation of the results from the experiments that used cofactor analogues such as 5-deaza-FMN, radical-mediated mechanisms were proposed at first, negating both the hydride transfer mechanism and the mer...

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Isopentenyl-diphosphate isomerase: inactivation of the enzyme with active-site-directed irreversible inhibitors and transition state analogs

Cited by 98 publications

References 34 publications

Studies on the nonmevalonate terpene biosynthetic pathway: Metabolic role of IspH (LytB) protein

Studies on the nonmevalonate terpene biosynthetic pathway: Metabolic role of IspH (LytB) protein

The separate roles of plant cis and trans prenyl transferases in cis‐1,4‐polyisoprene biosynthesis

New Role of Flavin as a General Acid-Base Catalyst with No Redox Function in Type 2 Isopentenyl-diphosphate Isomerase

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