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Bioorganometallic mechanism of action, and inhibition, of IspH

We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K M , V max ), EPR and ¹H, ²H, ¹³C, ³¹P, and ⁵⁷Fe-electron-nuclear double resonance (... Full description

Journal Title: Proceedings of the National Academy of Sciences of the United States of America 9 March 2010, Vol.107(10), pp.4522-4527
Main Author: Wang, Weixue
Other Authors: Wang, Ke , Liu, Yi-Liang , No, Joo-Hwan , Li, Jikun , Nilges, Mark J. , Oldfield, Eric , Halpern, Jack
Format: Electronic Article Electronic Article
Language: English
Subjects:
ID: ISSN: 00278424
Link: https://www.jstor.org/stable/25664830
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recordid: jstor_archive_2325664830
title: Bioorganometallic mechanism of action, and inhibition, of IspH
format: Article
creator:
  • Wang, Weixue
  • Wang, Ke
  • Liu, Yi-Liang
  • No, Joo-Hwan
  • Li, Jikun
  • Nilges, Mark J.
  • Oldfield, Eric
  • Halpern, Jack
subjects:
  • Physical sciences -- Chemistry -- Chemical compounds
  • Physical sciences -- Chemistry -- Chemical compounds
  • Physical sciences -- Chemistry -- Chemical compounds
  • Physical sciences -- Chemistry -- Chemical compounds
  • Biological sciences -- Biochemistry -- Metabolism
  • Physical sciences -- Chemistry -- Chemical compounds
  • Applied sciences -- Laboratory techniques -- Spectroscopy
  • Physical sciences -- Chemistry -- Chemical compounds
  • Physical sciences -- Chemistry -- Chemical reactions
  • Physical sciences -- Chemistry -- Chemical compounds
ispartof: Proceedings of the National Academy of Sciences of the United States of America, 9 March 2010, Vol.107(10), pp.4522-4527
description: We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K M , V max ), EPR and ¹H, ²H, ¹³C, ³¹P, and ⁵⁷Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP "parent" molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on ⁵⁷Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a π/σ "metallacycle" or η²-alkenyl complex. The complex is poised to interact with H⁺ from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an η¹-allyl complex. After reduction, this forms an η³-allyl π-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by ¹H, ²H, and ¹³C ENDOR, where hyperfine couplings of approximately 6 MHz for ¹³C and 10 MHz for ¹H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe₄S₄-containing proteins, such as IspG.
language: eng
source:
identifier: ISSN: 00278424
fulltext: fulltext
issn:
  • 0027-8424
  • 00278424
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titleBioorganometallic mechanism of action, and inhibition, of IspH
creatorWang, Weixue ; Wang, Ke ; Liu, Yi-Liang ; No, Joo-Hwan ; Li, Jikun ; Nilges, Mark J. ; Oldfield, Eric ; Halpern, Jack
ispartofProceedings of the National Academy of Sciences of the United States of America, 9 March 2010, Vol.107(10), pp.4522-4527
identifierISSN: 00278424
subjectPhysical sciences -- Chemistry -- Chemical compounds ; Physical sciences -- Chemistry -- Chemical compounds ; Physical sciences -- Chemistry -- Chemical compounds ; Physical sciences -- Chemistry -- Chemical compounds ; Biological sciences -- Biochemistry -- Metabolism ; Physical sciences -- Chemistry -- Chemical compounds ; Applied sciences -- Laboratory techniques -- Spectroscopy ; Physical sciences -- Chemistry -- Chemical compounds ; Physical sciences -- Chemistry -- Chemical reactions ; Physical sciences -- Chemistry -- Chemical compounds
descriptionWe have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K M , V max ), EPR and ¹H, ²H, ¹³C, ³¹P, and ⁵⁷Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP "parent" molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on ⁵⁷Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a π/σ "metallacycle" or η²-alkenyl complex. The complex is poised to interact with H⁺ from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an η¹-allyl complex. After reduction, this forms an η³-allyl π-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by ¹H, ²H, and ¹³C ENDOR, where hyperfine couplings of approximately 6 MHz for ¹³C and 10 MHz for ¹H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe₄S₄-containing proteins, such as IspG.
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titleBioorganometallic mechanism of action, and inhibition, of IspH
descriptionWe have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K M , V max ), EPR and ¹H, ²H, ¹³C, ³¹P, and ⁵⁷Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP "parent" molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on ⁵⁷Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a π/σ "metallacycle" or η²-alkenyl complex. The complex is poised to interact with H⁺ from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an η¹-allyl complex. After reduction, this forms an η³-allyl π-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by ¹H, ²H, and ¹³C ENDOR, where hyperfine couplings of approximately 6 MHz for ¹³C and 10 MHz for ¹H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe₄S₄-containing proteins, such as IspG.
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abstractWe have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K M , V max ), EPR and ¹H, ²H, ¹³C, ³¹P, and ⁵⁷Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP "parent" molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on ⁵⁷Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a π/σ "metallacycle" or η²-alkenyl complex. The complex is poised to interact with H⁺ from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an η¹-allyl complex. After reduction, this forms an η³-allyl π-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by ¹H, ²H, and ¹³C ENDOR, where hyperfine couplings of approximately 6 MHz for ¹³C and 10 MHz for ¹H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe₄S₄-containing proteins, such as IspG.
pubNational Academy of Sciences
doi10.1073/pnas.0911087107
eissn10916490
date2010-03-09
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