Overexpression, physicochemical characterization, and modeling of a hyperthermophilic <i>pyrococcus furiosus</i> type 2 IPP isomerase

Dutoit, Raphaël; Ruyck, Jérôme de; Durisotti, Virginie; Legrain, Christianne; Jacobs, Eric J.

doi:10.1002/prot.21863

Cited by 10 publications

(6 citation statements)

References 49 publications

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“…The mass of purified His-tagged sp- IDI-2 was 39874 Da (calculated 39868 Da) as determined by ESI/MS. The mass of the native protein determined by gel filtration chromatography of 173.6 kDa corresponds to a homotetramer, as previously reported for Thermus thermophilus (tt -), Pyrococcus furiosus ( pf -) [15] and sa- IDI-2. [16] …”

Section: Resultssupporting

confidence: 65%

Determination of Kinetics and the Crystal Structure of a Novel Type 2 Isopentenyl Diphosphate: Dimethylallyl Diphosphate Isomerase fromStreptococcus pneumoniae

et al. 2014

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Isopentenyl diphosphate dimethylallyl diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI-1) is a metalloprotein and is found in eukaryotes, while the type-2 isoform (IDI-2) is a flavoenzyme found in bacteria and completely absent from human. IDI-2 from the pathogenic bacterium Streptococcus pneumoniae was recombinantly expressed in E. coli. Steady state kinetic studies of the enzyme indicated that FMNH2 (KM= 0.3 μM) bound before isopentenyl diphosphate (KM= 40 μM) in an ordered binding mechanism. An X-ray crystal structure at 1.4 Å resolution was obtained for the holo-enzyme, in the closed conformation with reduced flavin cofactor and two sulfate ions in the active site. These results helped to further approach the enzymatic mechanism of IDI-2 and, thus, open new possibilities for the rational design of antibacterial compounds against closely sequence and structure related pathogens such as E. faecalis or S. aureus.

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Section: Resultssupporting

confidence: 65%

Determination of Kinetics and the Crystal Structure of a Novel Type 2 Isopentenyl Diphosphate: Dimethylallyl Diphosphate Isomerase fromStreptococcus pneumoniae

et al. 2014

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“…The western blot analysis of purified enzyme (Figure 8B) pointed out that it corresponds to a dimeric state of Rxyl_2847. Such phenomenon was already reported for other thermophilic enzymes [41,42]. The activity of the purified enzyme was examined in the physiological, catabolic direction, i.e.…”

Section: Resultssupporting

confidence: 57%

Identifying reaction modules in metabolic pathways: bioinformatic deduction and experimental validation of a new putative route in purine catabolism

et al. 2013

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BackgroundEnzymes belonging to mechanistically diverse superfamilies often display similar catalytic mechanisms. We previously observed such an association in the case of the cyclic amidohydrolase superfamily whose members play a role in related steps of purine and pyrimidine metabolic pathways. To establish a possible link between enzyme homology and chemical similarity, we investigated further the neighbouring steps in the respective pathways.ResultsWe identified that successive reactions of the purine and pyrimidine pathways display similar chemistry. These mechanistically-related reactions are often catalyzed by homologous enzymes. Detection of series of similar catalysis made by succeeding enzyme families suggested some modularity in the architecture of the central metabolism. Accordingly, we introduce the concept of a reaction module to define at least two successive steps catalyzed by homologous enzymes in pathways alignable by similar chemical reactions. Applying such a concept allowed us to propose new function for misannotated paralogues. In particular, we discovered a putative ureidoglycine carbamoyltransferase (UGTCase) activity. Finally, we present experimental data supporting the conclusion that this UGTCase is likely to be involved in a new route in purine catabolism.ConclusionsUsing the reaction module concept should be of great value. It will help us to trace how the primordial promiscuous enzymes were assembled progressively in functional modules, as the present pathways diverged from ancestral pathways to give birth to the present-day mechanistically diversified superfamilies. In addition, the concept allows the determination of the actual function of misannotated proteins.

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“…6). The conserved amino acids were characteristic of type 2 IDI enzymes (Dutoit et al 2008). It had been suggested that a conserved cysteine in NxxCxHP consensus sequence and a conserved glutamate in ExE consensus sequence make the active site of type 1 IDI (Hahn et al 1996).…”

Section: Discussionmentioning

confidence: 99%

Type 2 IDI performs better than type 1 for improving lycopene production in metabolically engineered E. coli strains

Rad

Zahiri

Noghabi

et al. 2011

World J Microbiol Biotechnol

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In this study a comparison was made between type 1 and type 2 isopentenyl diphosphate isomerases (IDI) in improving lycopene production in Escherichia coli. The corresponding genes of Bacillus licheniformis and the host (i(Bl) and i(Ec), respectively) were expressed in lycopene producing E. coli strains by pTlyci(Bl) and pTlyci(Ec) plasmids, under the control of tac promoter. The results showed that the overexpression of i(Ec) improved the lycopene production from 33 ± 1 in E. coli Tlyc to 68 ± 3 mg/gDCW in E. coli Tlyci(Ec). In contrast, the expression of i(Bl) increased the lycopene production more efficiently up to 80 ± 9 mg/gDCW in E. coli Tlyci(Bl). The introduction of a heterologous mevalonate pathway to elevate the IPP abundance resulted in a lycopene production up to 132 ± 5 mg/gDCW with i(Ec) in E. coli Tlyci(Ec)-mev and 181 ± 9 mg/gDCW with i(Bl) in E. coli Tlyci(Bl)-mev, that is, 4 and 5.6 times respectively. When fructose, mannose, arabinose, and acetate were each used as an auxiliary substrate with glycerol, lycopene production was inhibited by different extents. Among auxiliary substrates tested, only citrate was an improving one for lycopene production in all strains with a maximum of 198 ± 3 mg/gDCW in E. coli Tlyci(Bl)-mev. It may be concluded that the type 2 IDI performs better than the type 1 in metabolic engineering attempts for isoprenoid production in E. coli. In addition, the metabolic engineering of citrate pathway seems a promising approach to have more isoprenoid accumulation in E. coli.

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Overexpression, physicochemical characterization, and modeling of a hyperthermophilic pyrococcus furiosus type 2 IPP isomerase

Cited by 10 publications

References 49 publications

Determination of Kinetics and the Crystal Structure of a Novel Type 2 Isopentenyl Diphosphate: Dimethylallyl Diphosphate Isomerase fromStreptococcus pneumoniae

Determination of Kinetics and the Crystal Structure of a Novel Type 2 Isopentenyl Diphosphate: Dimethylallyl Diphosphate Isomerase fromStreptococcus pneumoniae

Identifying reaction modules in metabolic pathways: bioinformatic deduction and experimental validation of a new putative route in purine catabolism

Type 2 IDI performs better than type 1 for improving lycopene production in metabolically engineered E. coli strains

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