Insect Cell Expression of Recombinant Imidazoleglycerolphosphate Dehydratase of Arabidopsis and Wheat and Inhibition by Triazole Herbicides

Abstract

lmidazoleglycerolphosphate dehydratase (ICPD; EC 4.2.1.1 9), which is involved in the histidine biosynthetic pathway of Arabidopsis fhaliana and wheat (Triticum aestivum), has been expressed in insect cells using the baculovirus expression vector system. Nterminal amino acid sequencing indicated that recombinant IGPDs (rlCPDs) were produced as mature forms via nonspecific proteolytic cleavages in the putative transit peptide region. The wheat rlCPD contained one M n atom per subunit, and the M n was involved in the assembly of the subunits to form active ICPDs. Protein-blotting analysis, using antibodies raised against the wheat rlCPD, indicated that ICPD was located in the chloroplasts of wheat. The rlCPDs of Arabidopsis and wheat, which were 86% identical in their primary structures deduced from the cDNAs, exhibited similar properties in terms of the molecular mass, pH optimum, and the K,,, for the substrate, imidazoleglycerolphosphate. However, the nonselective herbicides 3-amino-l,2,4-triazole and a newly synthesized triazole [(l R', 3Re)-[3-hydroxy-3-(2H-[1,2,4]triazole-3-yl)-cyclohexyl]-phosphonic acid], inhibited Arabidopsis and wheat ICPDs in a mixed-type and a competitive manner, respectively.

Histidine Biosynthesis

“…This work has demonstrated that Mn 2+ is also required for formation of the active site, and that the reaction mechanism of IGPD likely proceeds through the binding of the imidazole ring of IGP to a di-Mn 2+ cluster followed by conversion through diazafulvene and Δ2-enol intermediates to IAP (Glynn et al, 2005). Both the wheat germ and Arabidopsis enzymes also require a high concentration of 2-mercaptoethanol (100 mM) for maximal activity (Mano et al, 1993;Tada et al, 1995). The biochemical basis for this requirement is unclear as the mature IGPD proteins do not contain a Cys residue (Tada et al, 1994).…”

“…However, no hisn5b mutants have been characterized to date, so it is unclear whether HISN5A and HISN5B are functionally redundant. All IGPD enzymes characterized to date require Mn 2+ for activity (Mano et al, 1993;Tada et al, 1995), and for both the Arabidopsis and S. cerevisiae IGPD proteins it has been demonstrated that this requirement relates to the formation of catalytically active multimeric complexes. Removal of Mn 2+ causes the 24 subunit holoenzyme to dissociate into inactive trimers, while re-addition leads of Mn 2+ leads to reassembly of the active holoenzyme (Mano et al, 1993;Tada et al, 1995;Wilkinson et al, 1995;Glynn et al, 2005).…”

“…All the plant HISN proteins identified to date contain putative N-terminal plastid transit peptides, and several have been demonstrated to localize to the plastid (Nagai et al, 1993;Tada et al, 1995;Petersen et al, 2010), suggesting that His biosynthesis is confined to this organelle. However, His is mobile within the plant, as evidenced by its detection in both xylem and phloem exudates (Frommer et al, 1995;Krämer et al, 1996).…”

An Arabidopsis cDNA encoding a bifunctional glutamine amidotransferase/cyclase suppresses the histidine auxotrophy of a Saccharomyces cerevisiae his7 mutant

“…At least two plant His biosynthetic enzymes, IGPD [6] and HDH [27], have been shown to be localized at chloroplasts. The His biosynthesis is an energy consuming process, where 41 ATP molecules are utilized for the synthesis of each His molecule [28].…”

“…Recently, His biosynthetic genes have also been isolated from a variety of organisms including lower eukaryotes such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Neurospora crassa [3]. From higher plants, the cDNAs encoding the histidinol dehydrogenase (HDH) of Brassica oleracea [4] and the imidazoleglycerolphosphate dehydratases (IGPD) of Arabidopsis thaliana and Triticum aestivum [5,6] have been isolated.…”