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Journal of Lipid Research, Vol. 45, 1140-1147, June 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology
,E,E-farnesyl diphosphate synthase from Mycobacterium tuberculosis

* Department of Microbiology, Colorado State University, Fort Collins, CO 80523-1677
Institut National de la Santé et de la Recherche Médicale U447, Institut Pasteur de Lille, 59019 Lille cedex, France
1 To whom correspondence should be addressed. e-mail: dean.crick{at}colostate.edu
We have identified an
,E,E-farnesyl diphosphate (
,E,E-FPP) synthase, encoded by the open reading frame Rv3398c, from Mycobacterium tuberculosis that is unique among reported FPP synthases in that it does not contain the type I (eukaryotic) or the type II (eubacterial)
,E,E-FPP synthase signature motif. Instead, it has a structural motif similar to that of the type I geranylgeranyl diphosphate synthase found in Archaea. Thus, the enzyme represents a novel class of
,E,E-FPP synthase. Rv3398c was cloned from the M. tuberculosis H37Rv genome and expressed in Mycobacterium smegmatis using a new mycobacterial expression vector (pVV2) that encodes an in-frame N-terminal affinity tag fusion with the protein of interest. The fusion protein was well expressed and could be purified to near homogeneity, allowing facile kinetic analysis of recombinant Rv3398c. Of the potential allylic substrates tested, including dimethylallyl diphosphate, only geranyl diphosphate served as an acceptor for isopentenyl diphosphate.
The enzyme has an absolute requirement for divalent cation and has a Km of 43 µM for isopentenyl diphosphate and 9.8 µM for geranyl diphosphate and is reported to be essential for the viability of M. tuberculosis.
Abbreviations: BLAST, Basic Local Alignment Search Tool; CLD, chain length-determining; DMAPP, dimethylallyl diphosphate; FARM, first aspartate-rich motif; FOH, farnesol; FPP, farnesyl diphosphate; GGOH, geranylgeraniol; GGPP, geranylgeranyl diphosphate; GPP, geranyl diphosphate; IPP, isopentenyl diphosphate; LB, Luria-Bertani; NCBI, National Center for Biotechnology Information
Supplementary key words dimethylallyl diphosphate mycobacterial expression vector short-chain prenyl diphosphate synthase
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