(E, Z, E)‐GGPP: A Potential Novel Bactericidal Compound

Mycobacterium tuberculosis (Mtb), a pathogenic Actinobacterium, has infected one quarter of the world’s population, according to the World Health Organization. The standard treatment of Mtb is a regimen of antibiotics, which has resulted in a mutated strain, XDRTb, that is resistant to every known antibiotic. The key to this antibiotic resistance is thought to be the fastidious cell wall. Most bacteria, including Mtb, utilize an isoprenoid glycosyl carrier for transfer of proteoglycan structures to the extracellular side of the membrane; however, unique to Mtb is the flexibility of its isoprenoid metabolism. Mtb synthesizes a carrier composed of 10 stereospecific isoprene units that is produced by elongation of the two‐unit (E)‐GPP by the (Z)‐FPP synthase, Rv1086, to produce (Z,E)‐FPP which then has 35 carbons added in a cis‐orientation by Rv2361c, resulting in (Z 8 ,E)‐decaprenyl diphosphate (DPP). Although this pathway is well characterized, multiple additional isoprenoid synthases are encoded in the Mtb genome, and previous in vitro studies have indicated Rv2361c can utilize multiple alternate substrates. While attempting to understand the apparent flexibility of DPP metabolism, it was observed that heterologous co‐expression of either of the encoded (E,E,E)‐GGPP synthases, Rv0562 or Rv3383c, with Rv1086 in the heterologous host, Escherichia coli (E. coli), results in complete cell death. Computational modeling of both Rv0562 and Rv3383c suggest that both active sites may accept (Z,E)‐FPP as a substrate, and if this is true, the product would be the novel compound (E,Z,E)‐GGPP. Protein purifications of Rv0562 and Rv3383c individually combined with synthetic (Z,E)‐FPP have resulted in the synthesis of the potentially novel terpenoid product, (E,Z,E)‐GGPP. Subsequent coupled assays of Rv0562 or Rv3383c and Rv1086 resulted in higher product formation than the synthetic substrate, suggesting complex formation. Purification of the proposed novel product will be confirmed spectroscopically and has implications for synthesis of a new antibiotic. Support or Funding Information University of Wisconsin ‐ Parkside College of Natural and Health Sciences’ Summer Undergraduate Research Fellowship and the SC Johnson Integrated Sciences Laboratory