The 1-deoxy-D-xylulose-5-phosphate pathway for isoprenoid synthesis;
new roles for iron-sulfur proteins

 

In nature, there are two pathways found for the synthesis of the isoprene precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) [1]. These two compounds are very important as the building blocks for the essential biological molecules called isoprenoids, which include vitamins, cholesterol, steroid hormones, carotenoids and quinones. Mammals, including humans, use the mevalonate pathway to synthesize the isoprene precursors, while eubacteria and some other microorganisms use the DOXP pathway as the sole pathway for isoprene synthesis. Several of the microorganisms that utilize the DOXP pathway are pathogens, causing, for example, malaria, multidrug resistant tuberculosis (MDR-TB), anthrax, plague, methicillin-resistant Staphylococcus aureus (MRSA) infections, gastro-intestinal ulcers and venereal diseases [2]. This makes the DOXP pathway an attractive target for the development of new anti-infective drugs. Since this pathway is not present in humans these inhibitors should demonstrate very low toxicity.

 

Detailed knowledge of the mechanism and regulation of the DOXP pathway is a prerequisite for the rational design of inhibitors that are potential candidates for new anti-infective drugs; however, due to its recent discovery the function and catalytic mechanism of some of the proteins in this pathway are not well understood. The goal of the proposed research is to understand the reaction mechanism of the last two proteins in the DOXP pathway, GcpE ((E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; also known as IspG) [3] and LytB ((E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; also known as IspH) [4].

 

We recently found that the GcpE enzyme contains a highly oxygen-sensitive [4Fe-4S] cluster in its active site [5]. In kinetic studies, a transient species was detected in electron paramagnetic resonance (EPR) spectroscopy. The electronic and magnetic properties of this species indicate that its origin is probably an iron-sulfur-cluster-bound reaction intermediate. Interestingly, similar results were obtained for the LytB enzyme. This enzyme also contains a [4Fe-4S] cluster and also displayed a very similar transient EPR-active species in kinetic studies. Therefore, we hypothesize that both enzymes contain a [4Fe-4S] cluster in their respective active sites that is directly involved in the binding of reaction intermediates during the reaction cycle (Fig. 2).

 

Currently we work on understanding how the reaction intermediates are bound to the iron-sulfur clusters in the GcpE and LytB enzymes. Additionally, kinetic studies with substrate analogs and inhibitors are performed to elucidate the reaction mechanisms of these enzymes

 

 

 

 

Fig. 1: Coenzymes and cofactors of methyl-coenzyme M reductase.

 

 

 

 

Fig. 2: Two hypothetical reaction mechanisms for the reaction catalyzed by the GcpE enzyme. In the first mechanism (top) the 4Fe cluster has a direct role in the reaction. In the second mechanism (bottom) the cluster functions only as an internal electron source (adapted from [6]).

 

 

Students working on this project:
  • Weiya Xu


Selected Reading:

  1. Rohmer, M. (1999) The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. Nat. Prod. Rep., 16:565-574

  2. Rohdich, F., Bacher, A., and Eisenreich, W. (2005) Isoprenoid biosynthetic pathways as anti-infective drug targets. Biochem. Soc. Trans., 33:785-791

  3. Kollas, A.-K., Duin, E. C., Eberl, M., Altincicek, B., Hintz, M., Reichenberg, A., Henschker, D., Henne, A., Steinbrecher, I., Ostrovsky, D. N., Hedderich, R., Beck, E., Jomaa, H., and Wiesner, J. (2002) Functional characterization of GcpE, an essential enzyme of the non-mevalonate pathway of isoprenoid biosynthesis. FEBS Lett., 532:432-436.

  4. Altincicek, B., Duin, E. C., Reichenberg, A., Hedderich, R., Kollas, A.-K., Hintz, M., Wagner, S., Wiesner, J., Beck, E., and Jomaa, H. (2002) LytB protein catalyzes the terminal step of the 2-C-metyhl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis. FEBS Lett., 532:437-440.

  5. Adedeji, D., Hernandez, H., Wiesner, J., Köhler, U., Jomaa, H., and Duin, E. C. (2007) Possible direct involvement of the active-site [4Fe–4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP. FEBS Lett., 581:279-283. 

  6. Seemann, M., Bui, B. T. S., Wolff, M., Tritsch, D., Campos, N., Boronat, A., Marquet, A., and Rohmer, M. (2002) Isoprenoid biosynthesis through the methylerythritol phosphate pathway: The (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) is a [4Fe-4S] protein. Angew. Chem. Int. Ed. Engl., 41:4337-4339