Possible direct involvement of the active-site [4Fe–4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP

Adedeji, Dolapo; Hernandez, Heather; Wiesner, Jochen; Köhler, Uwe; Jomaa, Hassan; Duin, Evert C.

doi:10.1016/j.febslet.2006.12.026

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Volume 581, Issue 2 , Pages 279-283, 23 January 2007

Possible direct involvement of the active-site [4Fe–4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP

Edited by Hans Eklund

Dolapo Adedeji
- Department of Chemistry and Biochemistry, Auburn University, 179 Chemistry Building, Auburn, AL 36849, USA
Heather Hernandez
- Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA
Jochen Wiesner
- Institut für Klinische Chemie und Pathobiochemie, Universitätsklinkum Giessen und Marburg, D-35392 Giessen, Germany
Uwe Köhler
- Eurofins Medigenomix GmbH, Fraunhoferstrasse 22, D-82152 Martinsried, Germany
Hassan Jomaa
- Institut für Klinische Chemie und Pathobiochemie, Universitätsklinkum Giessen und Marburg, D-35392 Giessen, Germany
Evert C. Duin
- Department of Chemistry and Biochemistry, Auburn University, 179 Chemistry Building, Auburn, AL 36849, USA
- Corresponding author. Fax: +1 334 844 6959.

Received 7 November 2006; received in revised form 5 December 2006; accepted 8 December 2006. published online 19 December 2006.

Abstract

The GcpE enzyme converts 2-C-methyl-d-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the penultimate step of the DOXP pathway for isoprene biosynthesis. Purification of the enzyme under exclusion of air leads to a preparation that contains solely [4Fe–4S] clusters. Kinetic studies showed that in the presence of the artificial reductant dithionite and MEcPP a new transient iron–sulfur-based signal is detected in electron paramagnetic resonance (EPR) spectroscopy. Similarity of this EPR signal to that detected in ferredoxin:thioredoxin reductase indicates that during the reaction an intermediate is directly bound to the active-site cluster.

Keywords: GcpE, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate, [4Fe–4S], EPR spectroscopy, Resonance Raman spectroscopy, Isoprene synthesis