First crystal structures of 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) from Mycobacterium tuberculosis indicate a distinct mechanism of intermediate stabilization

Abstract

The development of drug resistance by Mycobacterium tuberculosis and other pathogenic bacteria emphasizes the need for new antibiotics. Unlike animals, most bacteria synthesize isoprenoid precursors through the MEP pathway. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the first reaction of the MEP pathway and is an attractive target for the development of new antibiotics. We report here the successful use of a loop truncation to crystallize and solve the first DXPS structures of a pathogen, namely M. tuberculosis (MtDXPS). The main difference found to other DXPS structures is in the active site where a highly coordinated water was found, showing a new mechanism for the enamine-intermediate stabilization. Unlike other DXPS structures, a “fork-like” motif could be identified in the enamine structure, using a different residue for the interaction with the cofactor, potentially leading to a decrease in the stability of the intermediate. In addition, electron density suggesting a phosphate group could be found close to the active site, provides new evidence for the D-GAP binding site. These results provide the opportunity to improve or develop new inhibitors specific for MtDXPS through structure-based drug design.

Antibacterial Target DXP Synthase Catalyzes the Cleavage of d-Xylulose 5-Phosphate: a Study of Ketose Phosphate Binding and Ketol Transfer Reaction

“…In the open conformation (6OUW), the phosphoryl of X5P is indeed oriented toward the Arg pocket (Figure ), maintaining contacts with R423 (3.2 Å) and R480 (3.1 Å). Contacts between the phosphoryl group of X5P and Y395 (2.6 Å), which is also important for d -GAP acceptor substrate binding, , are also maintained in the open conformation. In contrast, docking to the closed conformation (6OUV) shows the phosphoryl of X5P interacting with R423 (3.2 Å), but not R480, and interaction with Y395 is not observed.…”

Not Every Hit‐Identification Technique Works on 1‐Deoxy‐d‐Xylulose 5‐Phosphate Synthase (DXPS): Making the Most of a Virtual Screening Campaign

“…[13] After the crystal structure of ΔMtDXPS (PDB: 7A9H) had been solved, Atomwise performed an AtomNet model virtual screen (VS) using the Enamine small-molecule library of several million compounds, as described previously. [8,10,14] The screen focused on the binding site of the second substrate d-GAP (gray mesh Figure 1). [15] Below the substrate pocket, thiamine diphosphate (ThDP) is bound to the protein (yellow and red sticks) and coordinates magnesium (green sphere).…”

“…Both MtDXPS enzymes (native and truncated) were not suitable for activity assays as their substrate turnover in comparison to the model enzyme Deinococcus radiodurans (Dr)DXPS was very slow and a change in reaction velocity in the presence of inhibitors could not be observed reliably. [8,16] Hence, we used the two Mt-homologues in SPR binding studies, and DrDXPS for the enzyme-activity assays.…”

“…[7] We have recently solved the first crystal structure of truncated M. tuberculosis DXPS (ΔMtDXPS, protein data bank (PDB): 7A9H). [8] MtDXPS is usually difficult to purify and crystallize. By removing a flexible loop from the native structure, we were able to crystallize the truncated enzyme at 1.85 Å resolution.…”

Structural analysis of 1-deoxy-D-xylulose 5-phosphate synthase from Pseudomonas aeruginosa and Klebsiella pneumoniae reveals conformational changes upon cofactor binding

“…Meyers et al devised the name spoon-fork motif to describe a loop movement involved in binding of a post-decarboxylation intermediate formed by interaction of pyruvate with the ThDP-bound enzyme followed by D/L-GAP 11 . Thus far, few experimentally determined structures of DXPS have been reported 14 , but due to the generally conserved active sites, the available crystal structures of DXPS are used as a model for structure-based drug design 12,15,16 . However, this has proven to be very limiting, as three-dimensional active sites can typically not be deduced from sequence homology alone.…”