Publications

• Biosynthesis of terpenes: studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase.

  Adam P., Hecht S., Eisenreich W., Kaiser J., Graewert T., Arigoni D., Bacher A., Rohdich F.

  Earlier in vivo studies showed the involvement of IspH protein in the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). We have demonstrated now that cell extract of an Escherichia coli strain engineered for hyper ... >> More

  Earlier in vivo studies showed the involvement of IspH protein in the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). We have demonstrated now that cell extract of an Escherichia coli strain engineered for hyperexpression of the ispH (lytB) gene catalyzes the in vitro conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into IPP and DMAPP. The reaction requires NADH, FAD, divalent cations (preferably Co2+), and probably one or more as-yet-unidentified proteins. The low intrinsic catalytic activities of wild-type E. coli cell extract and isolated chromoplasts of red pepper (Capsicum annuum) are enhanced by the addition of purified recombinant IspH protein. << Less

  Proc. Natl. Acad. Sci. U.S.A. 99:12108-12113(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe-4S] protein.

  Wolff M., Seemann M., Tse Sum Bui B., Frapart Y., Tritsch D., Garcia Estrabot A., Rodriguez-Concepcion M., Boronat A., Marquet A., Rohmer M.

  The last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen-sensitive [4Fe-4S] cluster. This prosthe ... >> More

  The last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen-sensitive [4Fe-4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical. << Less

  FEBS Lett. 541:115-120(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli.

  Hintz M., Reichenberg A., Altincicek B., Bahr U., Gschwind R.M., Kollas A.K., Beck E., Wiesner J., Eberl M., Jomaa H.

  The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent ... >> More

  The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli DeltalytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, (1)H, (13)C and (31)P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10(4) times more potent in activating human Vgamma9/Vdelta2 T cells than isopentenyl pyrophosphate. << Less

  FEBS Lett 509:317-322(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• LytB protein catalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis.

  Altincicek B., Duin E.C., Reichenberg A., Hedderich R., Kollas A.-K., Hintz M., Wagner S., Wiesner J., Beck E., Jomaa H.

  Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was purified to apparent homogeneity. The purified LytB protein catalyzed the reduction of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in a defined in vitro system. The reaction produ ... >> More

  Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was purified to apparent homogeneity. The purified LytB protein catalyzed the reduction of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in a defined in vitro system. The reaction products were identified as isopentenyl diphosphate and dimethylallyl diphosphate. A spectrophotometric assay was established to determine the steady-state kinetic parameters of LytB protein. The maximal specific activity of 6.6+/-0.3 micromol x min(-1) x mg(-1) protein was determined at pH 7.5 and 60 degrees C. The k(cat) value of the LytB protein was 3.7+/-0.2 s(-1) and the K(m) value for HMBPP was 590+/-60 microM. << Less

  FEBS Lett. 532:437-440(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein.

  Rohdich F., Zepeck F., Adam P., Hecht S., Kaiser J., Laupitz R., Graewert T., Amslinger S., Eisenreich W., Bacher A., Arigoni D.

  Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-methyl-d-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. A ... >> More

  Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-methyl-d-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant Escherichia coli strain. The purified protein failed to transform 2C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an ispG-deficient E. coli mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deaza-isoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate from 2C-methyl-d-erythritol 2,4-cyclodiphosphate at a rate of 1 nmol x min(-1) x mg(-1). Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 micromol x min(-1) x mg(-1). IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol x min(-1) x mg(-1). The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions. << Less

  Proc. Natl. Acad. Sci. U.S.A. 100:1586-1591(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• IspH protein of Escherichia coli: studies on iron-sulfur cluster implementation and catalysis.

  Graewert T., Kaiser J., Zepeck F., Laupitz R., Hecht S., Amslinger S., Schramek N., Schleicher E., Weber S., Haslbeck M., Buchner J., Rieder C., Arigoni D., Bacher A., Eisenreich W., Rohdich F.

  The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene c ... >> More

  The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspH protein anaerobically purified from this strain by a factor of at least 200. For maximum catalytic activity, flavodoxin and flavodoxin reductase were required in molar concentrations of 40 and 12 microM, respectively. EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S](+) cluster in IspH protein. Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at the amino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced the catalytic activity by a factor of more than 70 000. << Less

  J. Am. Chem. Soc. 126:12847-12855(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum.

  Roehrich R.C., Englert N., Troschke K., Reichenberg A., Hintz M., Seeber F., Balconi E., Aliverti A., Zanetti G., Koehler U., Pfeiffer M., Beck E., Jomaa H., Wiesner J.

  In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid-like organelle (apicoplast) by the mevalonate independent 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a ... >> More

  In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid-like organelle (apicoplast) by the mevalonate independent 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe-4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin-NADP(+) reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin-NADP(+) reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum. << Less

  FEBS Lett. 579:6433-6438(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Studies on the nonmevalonate terpene biosynthetic pathway: metabolic role of IspH (LytB) protein.

  Rohdich F., Hecht S., Gaertner K., Adam P., Krieger C., Amslinger S., Arigoni D., Bacher A., Eisenreich W.

  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 ... >> More

  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-D-xylulose 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)C(5)]1-deoxy-D-xylulose into a 5:1 mixture of [U-(13)C(5)]isopentenyl diphosphate and [U-(13)C(5)]dimethylallyl diphosphate by an E. coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes. << Less

  Proc. Natl. Acad. Sci. U.S.A. 99:1158-1163(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.