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

Abstract

SummaryImidazoleglycerol-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.

Rutin, a Natural Inhibitor of IGPD Protein, Inhibits the Biofilm Formation inStaphylococcus xylosusATCC700404

“…IGPD is one of the most important metalloenzymes in L-histidine biosynthesis [29][30][31][32]. In our study, 0.8mg/ml rutin significantly inhibited the activity of IGPD, which may affect the biosynthesis of L-histidine.…”

“…As one of the pathways of nitrogen metabolism, Lhistidine synthesis pathway is related to biofilm formation [27,28]. IGPD catalyzes the sixth step of histidine biosynthesis which involves a dehydration reaction to produce imidazoleacetol phosphate (IAP) from imidazoleglycerol phosphate (IGP) and a concomitant water molecule [29][30][31][32], it is often used as a target for herbicides because of its unique biological characteristics [26,33]. The presence of two molecules of metal ion Mn 2+ at the active center of IGPD plays an important role in catalyzing substrate reaction [32].…”

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

“…These showed the same mode of mirror-image packing as observed in At IGPD2 (see Figure S8), thus suggesting that the ability of IGPD to accommodate opposite chiral forms of C348 is ag eneral feature of the wider enzyme superfamily,rather than apeculiarity of the Arabidopsis enzyme.W eh ave previously proposed that catalysis by IGPD involves conversion between an open conformation of the enzyme,w hich binds to imidazole-IGP (PDB:4 MU3), and ac losed conformation, which binds imidazolate-IGP (PDB:4 MU4), wherein ad istinctly different binding site for the substrate-phosphate moiety is utilized. [7] Comparison of the open and closed At IGPD2/ substrate complexes with those of the At IGPD2/C348 complexes show that neither enantiomer of C348 can access the phosphate binding site observed in the open enzyme/ substrate complex, as the backbone of the inhibitor is one carbon atom shorter than that of the substrate.R ather,b oth enantiomers of C348 utilize the phosphate binding site that is associated with the closed conformation of the enzyme/ substrate complex, with the ordered C-loop;t he conformation believed to be that adopted by the enzyme during catalysis,thus suggesting that both enantiomers of C348 may mimic reaction intermediates.D uring the reaction catalyzed by IGPD,t he adoption of an sp 2 geometry at C3 is required for the formation of the diazafulvene intermediate,aprocess which is facilitated by the planar arrangement of the imidazolate ring, C3 and C2 of imidazolate-IGP.T he next step in the reaction involves production of the D 2 -enol, which also requires the adoption of an sp 2 geometry at C2. This geometry necessitates that C1 moves into the plane defined by C3, C2 and the imidazolate (Figure 3c).…”

“…Theresulting electron density showed clear evidence for the ordering of the C-loop of IGPD2 (residues 193-206;s ee Figure S1 in the Supporting Information), with the enzyme adopting the same closed conformation as seen in the previously determined structure of an inactive mutant (E21Q) of At IGPD2 with its substrate,IGP (PDB:4MU4). [7] Difference density for C348 could be identified within the active site and the triazole ring could be modelled between the two manganese ions with the N2 and N4 atoms forming ligands to Mn1 and Mn2, respectively.T he C348 C2ÀOH group acts as an additional ligand to Mn1 and the phosphonate group is bound in ap ositively charged pocket, sur-rounded by the side chains of R99, R121, K177, S199, and K201, and by water-mediated hydrogen bonds to Q51 and H55. However,d espite the 1.85 resolution of the data, there was alack of electron density around C3 of the inhibitor (see Figure S2a).…”

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

“…Crystals of a ΔN construct of IGPD isoform 2 from Arabidopsis thaliana ( At ΔN IGPD2 construct A; see Supporting Information for experimental methods) were grown in the presence of a racemate of C348 and the structure of the enzyme/inhibitor complex was determined to 1.85 Å resolution. The resulting electron density showed clear evidence for the ordering of the C‐loop of IGPD2 (residues 193–206; see Figure S1 in the Supporting Information), with the enzyme adopting the same closed conformation as seen in the previously determined structure of an inactive mutant (E21Q) of At IGPD2 with its substrate, IGP (PDB: 4MU4) . Difference density for C348 could be identified within the active site and the triazole ring could be modelled between the two manganese ions with the N2 and N4 atoms forming ligands to Mn1 and Mn2, respectively.…”