Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

NW12/2003G308

Crystal structures of 1-Deoxy-D-xylulose 5-phosphate reductoidomerase
complexed with bisphosphonate inhibitors
12Shunsuke YAJIMA*1, Kodai HARA, John SANDERS, Fenglin YIN3, Kanju
OHSAWA1, Jochen
442,3
WIESNER , Hassan JOMAA , Eric OLDFIELD
1
DepartmentofBioscience,
Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan2
Department of Chemistry and 3Department of Biophysics, University of Illinois at UrbanaChampaign, Urbana, IL61801, USA
4
Institute of Biochemistry, Academic Hospital Center, Justus-Liebig-University, D35392 Giessen,
Germany

Introduction
In all living organisms, isoprenoids such as steroid
hormones, carotenoids, and ubiquinone or menaquinone
play important roles. There are several routes to theproduction of isoprenoids, including the mevalonate
pathway, the mevalonate-independent or methylerythritol
phosphate (MEP) pathway, as well as less common
pathways involving leucine catabolism and the pentose
phosphate pathway. In recent work, it has been foundthat the MEP pathway is of particular importance in many
pathogenic bacteria and in addition, it is the pathway usedin the protozoan parasite Plasmodium falciparum. It wasalso shown that an old antibiotic, fosmidomycin was apotent inhibitor of the enzyme deoxyxylulose-5phosphate reductoisomerase (DXR) from bacteria such as
Escherichia coli and Pseudomonas aeruginosa, plantsand P. falciparum and in combination with clindamycin,
provided parasitological cures of uncomplicatedfalciparum malaria. These results show that DXR is avalid drug target and prompted us to search for additional
inhibitors.
In other work, we recently found that a variety ofbisphosphonates inhibited the growth of P. falciparum
both in vitro and in vivo, and those compounds inhibit
DXR activity. We thus initiated crystallographic structure
study to investigate the binding mode of thebisphosphonate inhibitors to assist the new drug design
against the enzyme.
Materials and Methods
E. coli DXR tagged with 6xHis at N-terminus wascrystallized with the mother liquor containing 1.65 M
ammonium sulfate, 0.06 M potassium sodium (+)-tartrate
in 0.1 M sodium citrate buffer, pH 5.6. The space group
was P21212 and the unit cell dimension was a = 182.4, b =59.0, c = 87.0 Å. The crystals were soaked with thebisphosphonate compound of either 0.2 M (3isoquinolylamine) methylene-1,1-bisphosphonate (1) or
0.2 M [{(5-chloro-2-pyridinyl)amino} methylene]-1,1bisphosphonate (2) for 15 min. The structures weresolved by the molecular replacement method with thecoordinate of 1JVS [1] as a target model.

The bisphosphonates dock into the same site as does
the hydroxamate moiety of fosmidomycin, there is close
register of two bisphosphonate oxygens with the two2+hydroxamate oxygens, which are bound to Mn [2]. The
fosmidomycin phosphonate-binding site is occupied inboth structures by a sulfate ion and there is no bound
NADPH. In each structure, the aromatic (isoquinoline orpyridine) side-chains of the bisphosphonate are located ina hydrophobic cleft containing Trp-211, Met-213, Pro273 and Met-275, while the bisphosphonate groups
appear to interact with Lys-124 and the conserved Asp149, Glu-151 and Glu-230 cluster. While it might beexpected that the bisphosphonate would bind to a divalent
metal in this active site region, as does fosmidomycin,
there is little evidence for electron density in this region
and indeed, these three residues have a generally similar
structural arrangement in the apo-enzyme structure,
where it was proposed that they could form a hydrogenbonding network, so it seems possible that thebisphosphonates may interact with this network. Whentaken together, these results are of general interest since
they represent a new class of inhibitors of the alternatemevalonate or MEP pathway, of importance because of toits presence in many pathogenic microorganisms andwhere, at present, there is only one published structure ofan enzyme-inhibitor complex. These results are also ofinterest since they suggest the possibility of synergistic
interactions of bisphosphonates with multiple enzymes
involved with isoprene biosynthesis, which could help
explain the potent activity of species such as the n-alkyl
bisphosphonates versus P. falciparume.

References[1] S. Yajima et al., J. Biochem. 131, 313 (2002).
[2] S. Yajima et al., J. Am. Chem. Soc. 126, 10824 (2004).
* yshun@nodai.ac.jp

Results and Discussion

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