FtsZ is a guanosine triphosphatase (GTPase) that mediates cytokinesis in bacteria. FtsZ is homologous in structure to eukaryotic tubulin and polymerizes in a similar head-to-tail fashion. The study of tubulin’s function in eukaryotic cells has benefited greatly from specific and potent small molecule inhibitors, including colchicine and taxol. Although many small molecule inhibitors of FtsZ have been reported, none has emerged as a generally useful probe for modulating bacterial cell division. With the goal of establishing a useful and reliable small molecule inhibitor of FtsZ, a broad biochemical cross-comparison of reported FtsZ inhibitors was undertaken. Several of these molecules, including phenolic natural products, are unselective inhibitors that seem to derive their activity from the formation of microscopic colloids or aggregates. Other compounds, including the natural product viriditoxin and the drug development candidate PC190723, exhibit no inhibition of GTPase activity using protocols in this work or under published conditions. Of the compounds studied, only zantrin Z3 exhibits good levels of inhibition, maintains activity under conditions that disrupt small molecule aggregates, and provides a platform for exploration of structure-activity relationships (SAR). Preliminary SAR studies have identified slight modifications to the two sidechains of this structure that modulate the inhibitory activity of zantrin Z3. Collectively these studies will help focus future investigations toward the establishment of probes for FtsZ that fill the roles of colchicine and taxol in studies of tubulin.

Predicting the binding mode of carbocations produced in sesquiterpene synthase enzymes is not unlike finding a piece of hay in a haystack. A new method for tackling this problem is described.

Terpene synthases comprise a family of enzymes that convert acyclic oligo-isoprenyl diphosphates to terpene natural products with complex, polycyclic carbon backbones via the generation and protection of carbocation intermediates. To accommodate this chemistry, terpene synthase active sites generally are lined with alkyl and aromatic, i.e., nonpolar, sidechains. Predicting the correct, mechanistically relevant binding modes for entire terpene synthase reaction pathways remains an unsolved challenge. Here we describe a method for identifying such modes: , a series of protocols to predict the orientation of carbon skeletons of substrates and derived carbocations relative to the bound diphosphate group in terpene synthase active sites. Using this recipe for bornyl diphosphate synthase, we have predicted binding modesthat are consistent with all current experimental observations, including the results of isotope labeling experiments and known stereoselectivity. In addition, the predicted binding modes recapitulate key findings of a seminal study involving more computationally demanding QM/MM molecular dynamics methods on part of this pathway. This work illustrates the value of the approach as a starting point for more involved calculations and sets the stage for the rational engineering of this family of enzymes.

Terpenes make up the largest class of natural products, with extensive chemical and structural diversity. Diterpenes, mostly isolated from plants and rarely prokaryotes, exhibit a variety of important biological activities and valuable applications, including providing antitumor and antibiotic pharmaceuticals. These natural products are constructed by terpene synthases, a class of enzymes that catalyze one of the most complex chemical reactions in biology: converting simple acyclic oligo-isoprenyl diphosphate substrates to complex polycyclic products via carbocation intermediates. Here we obtained the second ever crystal structure of a class II diterpene synthase from bacteria, tuberculosinol pyrophosphate synthase (i.e., Halimadienyl diphosphate synthase, MtHPS, or Rv3377c) from Mycobacterium tuberculosis (Mtb). This enzyme transforms (E,E,E)-geranylgeranyl diphosphate into tuberculosinol pyrophosphate (Halimadienyl diphosphate). Rv3377c is part of the Mtb diterpene pathway along with Rv3378c, which converts tuberculosinol pyrophosphate to 1-tuberculosinyl adenosine (1-TbAd). This pathway was shown to exist only in virulent Mycobacterium species, but not in closely related avirulent species, and was proposed to be involved in phagolysosome maturation arrest. To gain further insight into the reaction pathway and the mechanistically relevant enzyme substrate binding orientation, electronic structure calculation and docking studies of reaction intermediates were carried out. Results reveal a plausible binding mode of the substrate that can provide the information to guide future drug design and anti-infective therapies of this biosynthetic pathway.