Mirror-Image Packing Provides a Molecular Basis for the Nanomolar Equipotency of Enantiomers of an Experimental Herbicide.

Programs of drug discovery generally exploit one enantiomer of a chiral compound for lead development following the principle that enantiomer recognition is central to biological specificity. However, chiral promiscuity has been identified for a number of enzyme families, which have shown that mirror-image packing can enable opposite enantiomers to be accommodated in an enzyme's active site. Reported here is a series of crystallographic studies of complexes between an enzyme and a potent experimental herbicide whose chiral center forms an essential part of the inhibitor pharmacophore. Initial studies with a racemate at 1.85 Šresolution failed to identify the chirality of the bound inhibitor, however, by extending the resolution to 1.1 Šand by analyzing high-resolution complexes with the enantiopure compounds, we determined that both enantiomers make equivalent pseudosymmetric interactions in the active site, thus mimicking an achiral reaction intermediate.

Crystal Structures Reveal that the Reaction Mechanism of Imidazoleglycerol-Phosphate Dehydratase Is Controlled by Switching Mn(II) Coordination.

Imidazoleglycerol-phosphate dehydratase (IGPD) catalyzes the Mn(II)-dependent dehydration of imidazoleglycerol phosphate (IGP) to 3-(1H-imidazol-4-yl)-2-oxopropyl dihydrogen phosphate during biosynthesis of histidine. As part of a program of herbicide design, we have determined a series of high-resolution crystal structures of an inactive mutant of IGPD2 from Arabidopsis thaliana in complex with IGP. The structures represent snapshots of the enzyme trapped at different stages of the catalytic cycle and show how substrate binding triggers a switch in the coordination state of an active site Mn(II) between six- and five-coordinate species. This switch is critical to prime the active site for catalysis, by facilitating the formation of a high-energy imidazolate intermediate. This work not only provides evidence for the molecular processes that dominate catalysis in IGPD, but also describes how the manipulation of metal coordination can be linked to discrete steps in catalysis, demonstrating one way that metalloenzymes exploit the unique properties of metal ions to diversify their chemistry.

Crystallization and preliminary crystallographic analysis of the putative sugar-binding protein Msmeg_0515 (AgaE) from Mycobacterium smegmatis.

Msmeg_0515, a gene from Mycobacterium smegmatis strain 155 encoding the ligand-binding domain, AgaE, of a putative ABC sugar transporter system, has been cloned into a pET-28a vector system, overexpressed in Escherichia coli and purified. The truncated protein lacking the first 27 residues, which correspond to a N-terminal signal sequence, was crystallized using the sitting-drop vapour-diffusion technique. The crystals of this protein diffracted to 1.48 Å resolution and belonged to space group P212121, with unit-cell parameters a = 64.06, b = 69.26, c = 100.74 Å, α = ß = γ = 90° and with one molecule in the asymmetric unit.

Structure and mechanism of imidazoleglycerol-phosphate dehydratase.

The structure of A. thaliana imidazoleglycerol-phosphate dehydratase, an enzyme of histidine biosynthesis and a target for the triazole phosphonate herbicides, has been determined to 3.0 A resolution. The structure is composed of 24 identical subunits arranged in 432 symmetry and shows how the formation of a novel dimanganese cluster is crucial to the assembly of the active 24-mer from an inactive trimeric precursor and to the formation of the active site of the enzyme. Molecular modeling suggests that the substrate is bound to the manganese cluster as an imidazolate moiety that subsequently collapses to yield a diazafulvene intermediate. The mode of imidazolate recognition exploits pseudosymmetry at the active site arising from a combination of the assembly of the particle and the pseudosymmetry present in each subunit as a result of gene duplication. This provides an intriguing example of the role of evolution in the design of Nature's catalysts.

Purification, crystallization and preliminary crystallographic analysis of Arabidopsis thaliana imidazoleglycerol-phosphate dehydratase.

Imidazoleglycerol-phosphate dehydratase catalyses the sixth step of the histidine-biosynthesis pathway in plants and microorganisms and has been identified as a possible target for the development of novel herbicides. Arabidopsis thaliana IGPD has been cloned and overexpressed in Escherichia coli, purified and subsequently crystallized in the presence of manganese. Under these conditions, the inactive trimeric form of the metal-free enzyme is assembled into a fully active species consisting of a 24-mer exhibiting 432 symmetry. X-ray diffraction data have been collected to 3.0 A resolution from a single crystal at 293 K. The crystal belongs to space group R3, with approximate unit-cell parameters a = b = 157.9, c = 480.0 A, alpha = beta = 90, gamma = 120 degrees and with either 16 or 24 subunits in the asymmetric unit. A full structure determination is under way in order to provide insights into the mode of subunit assembly and to initiate a programme of rational herbicide design.