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.