The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.

The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. α-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin–DXR complex. Our best inhibitor has an IC50 = 0.15 μM on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 μg/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.

1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzes the NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate, as the second step of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate pathway found in many bacteria and plants. The end product, isopentenyl diphosphate, is the precursor of various isoprenoids vital to all living organisms. The pathway is not found in humans; the mevalonate pathway is instead used for the formation of isopentenyl diphosphate. This difference, combined with its essentiality, makes the reductoisomerase an excellent drug target in a number of pathogenic organisms. The structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Mycobacterium tuberculosis (Rv2870c) was solved by molecular replacement and refined to a resolution of 1.9 A. The enzyme exhibited an estimated kcat of 5.3 s-1 and Km and kcat/Km values of 7.2 microM and 7.4x10(5) M-1 s-1 for NADPH and 340 microM and 1.6x10(4) M-1 s-1 for 1-deoxy-D-xylulose 5-phosphate. In the structure, a sulfate is bound at the expected site of the phosphate moiety of the sugar substrate. The M. tuberculosis enzyme displays a similar fold to the previously published structures from Escherichia coli and Zymomonas mobilis. Comparisons offer suggestions for the design of specific drugs. Furthermore, the new structure represents an intermediate conformation between the open apo form and the closed holo form observed previously, giving insights into the conformational changes associated with catalysis.

Peptidyl-prolyl cis-trans isomerases (EC 5.2.1.8) catalyse the interconversion of cis and trans peptide bonds and are therefore considered to be important for protein folding. They are also thought to participate in processes such as signalling, cell surface recognition, chaperoning and heatshock response. Here we report the soluble expression of recombinant Mycobacterium tuberculosis peptidyl-prolyl cis-trans isomerase PpiA in Escherichia coli, together with an investigation of its structure and biochemical properties. The protein was shown to be active in a spectrophotometric assay, with an estimated k cat /K m of 2.0 · 10 6 M )1 AEs )1 . The X-ray structure of PpiA was solved by molecular replacement, and refined to a resolution of 2.6 Å with R and R free values of 21.3% and 22.9%, respectively. Comparisons to known structures show that the PpiA represents a slight variation on the peptidyl-prolyl cis-trans isomerase fold, previously not represented in the Protein Data Bank. Inspection of the active site suggests that specificity for substrates and cyclosporin A will be similar to that found for most other enzymes of this structural family. Comparison to the sequence of the second M. tuberculosis enzyme, PpiB, suggests that binding of peptide substrates as well as cyclosporin A may differ in that case.

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