Structure of the inhibitor complex of old yellow enzyme from Trypanosoma cruzi

The structures of old yellow enzyme from Trypanosoma cruzi which produces prostaglandin F2α from PGH2 have been determined in the presence or absence of menadione.

Chagas disease, caused by Trypanosoma cruzi, affects more than 20 million people and poses a major public health and economic problem in South America (World Health Organization, 1990). This situation has been worsened by the lack of effective vaccines and undesirable side effects of anti-chagasic drugs, such as nifurtimox and benznidazole (Aldunate & Morello, 1993;Henderson et al., 1988;Docampo & Moreno, 1986;Docampo, 1990), in addition to the emergence of parasite resistance to these drugs. Therefore, the development of a new chemotherapeutic treatment is an urgent need.
OYE from T. cruzi (TcOYE) catalyzes prostaglandin F 2 synthesis ( Fig. 1) as well as the reduction of many kinds of trypanocidal drugs, such as naphtoquinone and nitrohetero-cyclic compounds . By electron spin resonance it was found that these compounds can undergo either one-or two-electron reduction. Naphtoquinone, such as menadione (K m = 0.82 mM) or -lapachone (K m = 0.17 mM), is reduced by one-electron reduction, generating semiquinone radicals which cause the cell death in susceptible parasites, while nitroheterocyclic compounds, such as 4-nitroquinoline-N-oxide (K m = 9.5 mM) or nifurtimox (K m = 19.0 mM), are reduced by two-electron reduction . Therefore, three-dimensional information of the structure in complex with the quinine compounds is valuable for the design of novel anti-chagasic drugs by which semiquinone radicals are more efficiently generated in T. cruzi.
Here we report the first X-ray structures of TcOYE which produces PGF 2 from PGH 2 in the presence or absence of menadione. The structural analysis of TcOYE/FMN/menadione ternary complex has provided the binding motif of menadione at the active site. These structures are useful for further drug design for Chagas disease.

Protein expression and crystallization
Chemicals were obtained from Sigma-Ardrich at the highest available purity. TcOYE was overexpressed in E. coli and purified, and TcOYE/FMN binary complex was crystallized according to previously reported methods Sugiyama et al., 2007). These crystals were flash-cooled in liquid nitrogen with cryo-protectant solution containing 3% (v/v) 2-methyl-2,4-pentanediol. The X-ray diffraction data were obtained up to 1.70 Å resolution at SPring-8 beamline BL41XU. The diffraction data were processed and scaled using the program HKL2000 (Otwinowski & Minor, 1997).

Structure determination and refinement
The structure of TcOYE was determined by molecular replacement with the program AMoRe (Navaza, 2001) using the structure of morphinone reductase from Pseudomonas putida (Protein Data Bank code 1gwj) (Barna et al., 2002) as a search model. Crystallographic refinement was carried out with the program CNS (Brü nger et al., 1998). The refinement procedure included simulated annealing, positional refinement, restrained temperature factor refinement, and maximum-likelihood algorithms as provided by the CNS program. Electron density maps based on the coefficients of 2F o À F c and F o À F c were used to build the atomic models in Coot (Emsley & Cowtan, 2004). Water molecules were inserted manually and then checked by inspecting the F o À F c map. FMN (cofactor) and menadione (ligand) were refined using atomic parameters extracted from the HIC-UP server (Kleywegt & Jones, 1998), and could be inserted into the clearly defined electron density model after some cycles of refinement.

Preparation of TcOYE complexed with menadione
In order to prepare the complex crystals with menadione, the crystals of the TcOYE/FMN complex were soaked in a menadione solution for 16 h and then the crystals were flash frozen in liquid nitrogen. The X-ray diffraction data were obtained up to 2.5 Å resolution by using Ultrax18 (Rigaku). Data collection, structure determination and refinement were carried out by the same methods as described above.

Overall structure of TcOYE
The structure of TcOYE/FMN binary complex was determined by molecular replacement methods using that of morphinone reductase (Barna et al., 2002) as the searching model and refined at 1.7 Å resolution with final R cryst /R free values of 18.5%/23.2%. Our crystallographic properties and refinement statistics of the structure are presented in Table 1.
TcOYE, like other members of the OYE family, folds into an (/) 8 barrel in which the cylindrical core composed of eight parallel -strands (1-8) is surrounded by eighthelices (1-8). In addition, an N-terminal -hairpin (S1 and S2) closes the bottom of the barrel and two extra helices (H1 and H2) lie in the loops connecting 4 to 4 and 8 to 8, respectively (Fig. 2). The loop regions from 105 to 165 (Loop 1) and 351 to 379 (Loop 2) exhibit the relatively higher Bfactors of 21.3 and 18.8 Å 2 , respectively, while the overall Bfactor of TcOYE without those two loop ranges was calculated to be 13.1 Å 2 . This difference in the B-factor shows the flexibility of these two loops.

FMN binding site
FMN is tightly bound at the C-terminal ends of the eightstrands in the barrel and exhibits quite low B-factors (Fig. 3). Most amino acid residues which bind to the riboflavin moiety of FMN are highly conserved among the different known OYE structures, except Ala61. Ala61 in TcOYE was conserved as alanine or glycine among the known OYE structures; however, the amide nitrogen atom of the corresponding amino acid interacts with O4 of FMN. On the other hand, three residues bound to the phosphate O atoms in FMN are not conserved: the side chain of Asn313 and the main Reduction of PGH 2 to PGF 2 by TcOYE. FMN (cofactor of TcOYE) is reduced to FMNH 2 by intravital nicotinamide adenine dinucleotide phosphate (NADPH), and the product PGF 2 is subsequently generated by FMNH 2 . chains of Leu314 and Lys338 interact with the phosphate O atoms through water molecules.

The binding motif of menadione
In order to develop more effective inhibitors we have solved the structure of TcOYE/FMN/menadione ternary complex at 2.5 Å resolution (Table 1). The whole structure of TcOYE does not change even upon binding of menadione. The binding motif of menadione is defined in the difference electron density maps even at low occupancy (Fig. 4). The cause of its low affinity (K m = 0.82 mM) is assumed to be because the ligand is stabilized by only ainteraction with the isoalloxazien ring of FMN. To increase the affinity of menadione, interactions with His195, Asn198 or Tyr 364 should be designed in the future.

Conclusion
We have solved the structures of TcOYE in the presence or absence of menadione. The binding motif of the inhibitor at the active site of TcOYE has been elucidated from TcOYE/ FMN/menadione ternary complex. Future work is in progress to obtain the structure in complex with other inhibitors to elucidate the reaction mechanism of TcOYE in detail, which can be expected to lead to the development of heroic antichagasic drugs. The overall structure of TcOYE. The -helices (cyan) and -sheets (orange) are separated among them by loops (light yellow). Loop 1 and Loop 2 are shown in magenta. FMN is shown as a ball-and-stick model. The image was created by using MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt & Murphy, 1994). FMN binding model with 2F o À F c omit map. The FMN electron density is calculated at 1.7 Å and contoured at 2.5 . O, N and P atoms are shown in red, blue and magenta, respectively. Labelled residues indicate those involved in FMN binding. The figure was drawn using the programs MOLSCRIPT and RASTER3D.

Figure 4
The binding site of menadione. Menadione (green) is positioned above the isoalloxazine ring of FMN. The menadione electron density is calculated at 2.5 Å and contoured at 1.2 . The figure was drawn using the program PyMOL (DeLano, 2005).