Graduation Year


Document Type




Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department


Major Professor

Henry Lee Woodcock, Ph.D.

Committee Member

David J. Merkler, Ph.D.

Committee Member

Wayne C. Guida, Ph.D.

Committee Member

Yu Chen, Ph.D.


QM/MM, computational, 1-Deoxy-D-xylulose 5-Phosphate Synthase, 1-Deoxy-D-xylulose 5-Phosphate Reductosiomerase


Molecular drug design begins with the identication of a problem to solve. This work identies the growing resistance among human pathogens to current treatments. Once the problem is identied and understood, solutions must be proposed. This one is straight forward, we need new antimicrobial drugs. More specically, we need to identify novel targets to inhibit. A large portion of antibiotics focus on disruption of macromolecular production while only a few target metabolic systems. Finally, you need to propose solutions based on the information gathered. In order to avoid existing resistance, it is important to avoid the macromolecular route and focus on metabolic enzymes. Preferably, the pathway would have little overlap or similarity with pathways found in the treatment organism. With this in mind, the non-mevalonate (NMA) pathway poses as a very good target for drug design. Many pathogens have been found to be strictly dependent on this pathway while it is absent in humans. Additionally, fosmidomycin has already been shown to inhibit this pathway. Initially thought to just inhibit the 1-deoxy-D-xylulose 5- phosphate (DXP) reductoisomerase (DXR), it has been shown to inhibit several enzymes along the path to a lesser extent. Ideally, this could be repeated or improve upon for future drug design.

With this in mind, the initial stages of the rst two enzymes of the NMA pathway were examined utilizing quantum mechanical/molecular mechanical (QM/MM) techniques. The rst enzyme was DXP synthase (DXS), which catalyzes a transketolase-like condensation of pyruvate and glyceraldehyde-3-phosphate to produce DXP. DXS and other transketolases are dependent on the thiamine diphosphate (TDP) cofactor, which must be deprotonated of the imidazolium C2 atom producing a highly reactive ylide. A tautomerization occurs prior to this deprotonation to prime the pyrimidinium ring N4 atom to perform the C2 abstraction. The question at hand was the identity of a general base to perform the N4 abstraction. The results favored a water-mediate mechanism with a higher than usual Ez of 22.7 kcal/mol. An observation pertaining the tautomerization pertained to the aromaticity of the pyrimidine ring. Upon further investigation, aromaticity was found to play a signicant role in the ΔE observed. Aromaticity might contribute 14.2 kcal/mol to the barrier height. This high energy would drive the reaction forward producing the ylide.

Investigation of the DXR enzyme followed this work. Initially, the work was going to focus on the 2 mechanisms proposed for activity, alpha-ketol rearrangement and retroaldol/ aldol mechanism. Subsequent publications involving secondary kinetic isotope effects (KIEs) add to the pile of evidence supporting the retro-aldol/aldol mechanism. So the project was retooled to investigate the energetic dierences between two metal binding modes. The results of this work support a metal coordination across the C3-C4 bond, which eventually extends coordination to include the C2 oxygen. This conformation was help explain the tight binding eecting observation of the putative intermediates (transition states) and aldehyde intermediate. Additionally, as the C2-C3 mode consistently transfers a proton to the phosphate group of DXP or produces an elongated C-O bond, the C2-C3 mode would not be favorable.

Further investigations of these enzymes (e.g. completing the step begin, continuing through the reaction) could provide further illumination into the mechanism of action and possibly reveal new avenues of drug design. Examining the enzymes downstream in the NMA pathway might provide details of interest. Of particular interest is the radical reaction proposed for HDR/IspH. The nal step of the pathway produces IDP and DMADP in a 4:1 proportion, which corresponds to the general system requirements for production of the long chain, branched isoprenoids. It would be interesting to compute the mechanism to see if energetics could provide further insights. Additionally, normal mode analysis coupled with vibrational subsystem analysis could identify allosteric sites for feedback sensitivity.