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IspH protein of Escherichia coli: studies on iron-sulfur cluster implementation and catalysis.
J Am Chem Soc. 2004 Oct 13; 126(40):12847-55.JA

Abstract

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.

Authors+Show Affiliations

Gräwert T
Contribution from the Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
Kaiser J
No affiliation info available
Zepeck F
No affiliation info available
Laupitz R
No affiliation info available
Hecht S
No affiliation info available
Amslinger S
No affiliation info available
Schramek N
No affiliation info available
Schleicher E
No affiliation info available
Weber S
No affiliation info available
Haslbeck M
No affiliation info available
Buchner J
No affiliation info available
Rieder C
No affiliation info available
Arigoni D
No affiliation info available
Bacher A
No affiliation info available
Eisenreich W
No affiliation info available
Rohdich F
No affiliation info available

MeSH

CatalysisElectron Spin Resonance SpectroscopyEnzyme ActivationEscherichia coliEscherichia coli ProteinsIron-Sulfur ProteinsMutagenesis, Site-DirectedNuclear Magnetic Resonance, BiomolecularOxidoreductasesPlasmidsRecombinant ProteinsSpectrophotometry, Ultraviolet

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15469281

Citation

Gräwert, Tobias, et al. "IspH Protein of Escherichia Coli: Studies On Iron-sulfur Cluster Implementation and Catalysis." Journal of the American Chemical Society, vol. 126, no. 40, 2004, pp. 12847-55.
Gräwert T, Kaiser J, Zepeck F, et al. IspH protein of Escherichia coli: studies on iron-sulfur cluster implementation and catalysis. J Am Chem Soc. 2004;126(40):12847-55.
Gräwert, 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. (2004). IspH protein of Escherichia coli: studies on iron-sulfur cluster implementation and catalysis. Journal of the American Chemical Society, 126(40), 12847-55.
Gräwert T, et al. IspH Protein of Escherichia Coli: Studies On Iron-sulfur Cluster Implementation and Catalysis. J Am Chem Soc. 2004 Oct 13;126(40):12847-55. PubMed PMID: 15469281.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - IspH protein of Escherichia coli: studies on iron-sulfur cluster implementation and catalysis. AU - Gräwert,Tobias, AU - Kaiser,Johannes, AU - Zepeck,Ferdinand, AU - Laupitz,Ralf, AU - Hecht,Stefan, AU - Amslinger,Sabine, AU - Schramek,Nicholas, AU - Schleicher,Erik, AU - Weber,Stefan, AU - Haslbeck,Martin, AU - Buchner,Johannes, AU - Rieder,Christoph, AU - Arigoni,Duilio, AU - Bacher,Adelbert, AU - Eisenreich,Wolfgang, AU - Rohdich,Felix, PY - 2004/10/8/pubmed PY - 2005/4/15/medline PY - 2004/10/8/entrez SP - 12847 EP - 55 JF - Journal of the American Chemical Society JO - J Am Chem Soc VL - 126 IS - 40 N2 - 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. SN - 0002-7863 UR - https://www.unboundmedicine.com/medline/citation/15469281/IspH_protein_of_Escherichia_coli:_studies_on_iron_sulfur_cluster_implementation_and_catalysis_ DB - PRIME DP - Unbound Medicine ER -