The HIS3+ gene of Saccharomyces cerevisiae was overexpressed in Escherichia coli and the recombinant imidazoleglycerol-phosphate dehydratase (IGPD) purified to homogeneity. Laser-desorption and electrospray m.s. indicated a molecular ion within 2 units of that expected (23833.3) on the basis of the protein sequence, with about half of the polypeptide lacking the N-terminal formylmethionine residue. IGPD initially purified as an apoprotein was catalytically inactive and mainly a trimer of M(r) 70,000. Addition of Mn2+ (but not Mg2+) caused this to assemble to an active (40 units/mg) enzyme (Mn-IGPD) comprising of 24 subunits (M(r) 573,000) and containing 1.35 +/- 0.1 Mn atoms/polypeptide subunit. An enzyme with an identical activity and metal content was also obtained when the fermenter growth medium of recombinant Escherichia coli was supplemented with MnCl2, and IGPD was purified through as Mn-IGPD rather than as the apoenzyme and assembled in vitro. Inhibition by EDTA indicated that the intrinsic Mn2+ was essential for activity. The retention of activity over time after dilution to very low concentrations of enzyme (< 20 nM) indicated that the metal remained in tight association with the protein. A novel continuous assay method was developed to facilitate the kinetic characterization of Mn-IGPD. At pH 7.0, the Km for IGP was 0.10 +/- 0.02 mM and the Ki value for inhibition by 1,2,4-triazole, 0.12 +/- 0.02 mM. In contrast with other reports, thiols had no influence on catalytic activity. The activity of Mn-IGPD varied with enzyme concentration in such a way as to suggest that it dissociates to a less active form at very low concentrations. Significant inhibition by the product, imidazole acetol phosphate, was inferred from the shape of the progress curve. Titration with, the potent competitive inhibitor, 2-hydroxy-3-(1,2,4-triazol-1-yl)propyl phosphonate indicated that Mn-IGPD contained 0.9 +/- 0.1 catalytic sites/protomer. The activity nearly doubled in the presence of high concentrations of Mn2+; the apparent Ks for stimulation was 20 microM. The basis of this effect was obscure, since there was no corresponding increase in the titre of active sites. Neither was there a discernable shift in the values of Km or Ki (above), although exogenous Mn2+ did reduce the optimum pH for kcat, from 7.2 to 6.8. On the basis of a single site/subunit, the maximum rate of catalytic turnover at 30 degrees C was 32 s-1.

Imidazoleglycerol phosphate dehydratase (IGPD) from Saccharomyces cerevisiae has been crystallized in the presence of a range of divalent cations using the hanging-drop method of vapour diffusion with ammonium sulfate or polyethylene glycol (PEG) 4000 as the precipitants. X-ray precession photographs have established that the crystals formed with ammonium sulfate (form A) belong to the space group F432, with cell parameter a = 177.5/~ and a single subunit in the asymmetric unit. A preliminary data set collected to 6 A resolution on a two-detector San Diego Multiwire area detector has established that the crystals formed with PEG 4000 (form B) belong to either of the special pair of space groups I23 or 12~3, with cell parameter a = 131.0 A,. A self-rotation function has been calculated using these data and indicates that the cell axes show pseudo fourfold symmetry consistent with a dimer in the asymmetric unit in this crystal form. Light-scattering studies indicate that in the presence of Mn 2÷ and a number of other divalent cations IGPD undergoes assembly to a particle of molecular weight approximately 500kDa. Given the subunit molecular weight of 23 kDa together with the symmetry of the crystals it would indicate that the most likely quaternary structure for this enzyme is based on a 24-mer in 432 symmetry.

In the pathway from aspartate semialdehyde. the first two enzymes, dihydrodipicolinate synthase and dihydrodipicolinate reductase as well as the last, meso diaminopimelate decarboxylase, have been reported in plant extracts [l].It has, however. remained unclear how plants convert the central intermediate, tetrahydrodipicolinate (THDP) through to mesodiaminopimelate (DAP), the immediate precursor of lysine. The supposition that the route is similar to that in most bacteria (i.e. v i a hydration of THDP. Nacetyl or N-succinyl transfer from CoA. transamination, N-deacylation and, epimerisation) was strengthened by the report [2] of DAP epimerase in maize leaf. However the reported [3] isolation of a functional DAP dehydrogenase gene from a soybean nuclear gene library suggested that, similar to some bacteria (41. the transformation in plants could also occur V f 8 reductive amination. We used a sensitive HPLC method [5] to reinvestigate [cf Fig 11.Pea extract was a broad 0-25% polyethylene glycol precipitate of pea seedlings [6]. Stromal extracts were made by bursting spinach or pea chloroplasts [7] in hypotonic buffer (pH 7.0) and centrifuging. Extracts were exchanged (625) into buffers and, if not used fresh, kept as frozen beads over liquid Nz. DD/LL and meso DAP (15% DDILL). their mono N-succinyl and acetyl derivatives and THDP were made [8.9].High levels of DAP epimerase and decarboxylase were found in all extracts (e.g. from 18 mM DD/LL DAP > 8 nmol lysine formed min-' m g ' ' in pea stroma). However, none of our extracts deacylated either N-acetyl or N-succinyl DD/LL DAP under a wide range of conditions (e.g. in fresh stroma at pH's 6 , 7 or 8 both with and without ZnZ* or Co2*). These experiments were partly obscured by lysine forming from the trace of unacylated material in the N-acyl DAP; within error, unacylated LL accounted for all the lysine detected. Thus. deacylase activity was less than 0.02 -0.20 nmol min-' mg-' (detection limits varying with [N-acyl DAP] between 0.4 and 10 mM). We considered the possibility LVDD-DAP 1 I Fig. 1. Conversion of LL to meso diaminoDimelate and lysine. 0 . 6 mg of pea extract in 0.1 M Kt Pi (pH 6 . 8 ) contained 10 mM EDTA, 10 mM p-mercaptoethanol, 1 mM pyridoxal phosphate and 0.4 mM LL/DD DAP. Aliquots were automatically pre-derivatized with 0phthaldialdehyde for HPLC [ 5 ] . Y-axis. fluoresence. Top chromatogram. zero time; lower. after 2 h at 3OOC.that, just as plants make ornithine [lo], there might be no deacylase but rather a direct transfer of the Nacyl group from DAP back to THDP. However, there was also no reaction in the presence of 4 mM THDP. Similarly, we found no evidence for the enzyme activities (THDP N-acyl transferase and N-acyl Z-oxopimelate/ glutamate transaminase) which bacteria use to form Nacyl DAP. The HPLC experiment provided sensitive (> 10 fold better than lysine) detection of all species of N-acyl DAP. However, none (< 0.3 nmol) was formed in any experiment (e.g. after 2 h in 0.4 mg of spinach stroma in HEPES buffer, pH 7.0, containing 0.2 m...

No abstract

:5-tert-Butyl-carbamoyloxy-3-(3-triÑuoromethyl) phenyl-4-thiazolidinone (compound 1), a representative of a novel class of thiazolidinone herbicides, shows potential for the pre-emergence control of grasses and smallseeded broad-leaved weeds in crops such as soyabean. This compound powerfully inhibited the growth and lateral branching of the roots of agar-grown seedlings of susceptible species but had no apparent e †ect on the growth of Escherichia coli, Aspergillus nidulans, Fusarium culmorum or of insects cells in liquid culture. It inhibited the growth of plant (Daucus carota and Zea mays) cells in liquid culture and this inhibition was not reversed by addition to the medium of mixtures of either amino acids or of nucleosides. It did not inhibit fatty acid biosynthesis, respiration, the biosynthesis of sterols, or the biosynthesis of protein in the systems examined herein. By contrast, it induced potent c.50 nM) and (IC 50 rapid inhibition of the incorporation of [3H]glucose into the acid-insoluble cellwall fraction of roots. Thus, compound 1 exerts its herbicidal e †ect, directly or indirectly, through inhibition of the biosynthesis of cellulose and cellulose-like polysaccharides, in a manner similar to isoxaben and dichlobenil. Mutant lines of Arabidopsis thaliana selected for resistance to isoxaben were cross-resistant to compound 1. Consistent with the selective herbicidal activity observed, the compound was a potent inhibitor of [3H]glucose incorporation into the polysaccharide of seedling roots of Zea mays and of Setaria viridis but only relatively weakly active on Glycine max and Ipomoea hederacea. Given that compound 1 appeared to be metabolised only slowly and that [3H]glucose incorporation experiments were conducted within a total period of less than 90 min, it seems probable that, as in the case of the acetyl CoA carboxylase-inhibitor graminicides, the observed selectivity is determined by species-dependent di †erences at the (in this case unknown) molecular target site for the herbicide.1998 Society ( of Chemical Industry Pestic. Sci., 54, 368È376 (1998)