Abstract
We have identified an omega,E,E-farnesyl diphosphate (omega,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) omega,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 omega,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 K(m) of 43 microM for isopentenyl diphosphate and 9.8 microM for geranyl diphosphate and is reported to be essential for the viability of M. tuberculosis.
Highlights
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 or the type II ,E,EFPP synthase signature motif
dimethylallyl diphosphate (DMAPP), geranylgeranyl diphosphate (GGPP), farnesol (FOH; mixed stereoisomers), ,E,E-FOH, ,E-geraniol, ,E,E,E-geranylgeraniol (GGOH), monoclonal anti-polyhistidine from mouse ascites fluid, anti-mouse IgG conjugated to alkaline phosphatase, and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium tablets were purchased from Sigma. ,E,E-FPP and geranyl diphosphate (GPP) were synthesized as previously described [14]. ,E,E,ZGGOH was a gift from Dr C
When the products of cytosol assays primed with DMAPP were compared, there was a small, but reproducible, increase in the synthesis of FOH (FPP before dephosphorylation) relative to GGOH (GGPP before dephosphorylation) in the preparation containing the recombinant protein
Summary
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,EFPP synthase signature motif. The CLD region and the FARM signatures were used to segregate the ,E,E-FPP synthases into two classes designated type I (eukaryotic) and type II (eubacterial) [1] Both of these classes of enzyme presumably evolved from a common precursor similar to the archaeal type I geranylgeranyl diphosphate (GGPP) synthase, which contains the FARM signature DDXXD and a single aromatic amino acid in the fifth position N-terminal to the FARM. Type II ,E,E-FPP synthases (eubacterial) contain the FARM signature DDXXXXD and a single aromatic amino acid in the fifth position N-terminal to the FARM This classification system is well established and is being used to identify putative prenyl diphosphate synthases as FPP or GGPP synthases for phylogenetic studies [9, 10] and genome annotation. This observation, combined with a report of sterol synthesis in mycobacteria [12], prompted us to try to identify the enzyme
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