RESUMO
4-Hydroxyphenylpyruvate dioxgenase (HPPD) enzymes from rat and from several plants contained only about a single inhibitor-binding active site per dimer which matched the content of iron in the purified Arabidopsis thaliana and Avena sativa enzymes. The dimeric HPPDs were about 10 fold more catalytically active than the tetrameric P. fluorescens enzyme with kcat/KmHPP values ranging from 0.8 to 2.5 s-1 µM-1. Most were also highly sensitive to herbicides with, for example, Ki values for mesotrione ranging from 25 to 100 pM. Curiously HPPDs from cool climate grasses were much less herbicide-sensitive. When likewise expressed in Nicotinia tabacum, Avena sativa HPPD, Ki value of 11 nM for mesotrione, conferred far greater tolerance to mesotrione (CallistoTM) than did any of the more sensitive HPPDs. Targeted mutagenesis of the Avena HPPD led to the discovery of 4 mutations imparting improved inherent tolerance, defined as the ratio of Ki to KmHPP, by about 16 fold without any loss of catalytic activity. The Nicotinia line with the highest expression of this quadruple mutant exhibited substantial resistance even up to a 3 kg/ha post-emergence application of mesotrione. The maximum observed expression level of heterologous plant HPPDs in tobacco was ca. 0.35% of the total soluble protein whereas the endogenous tobacco HPPD constituted only ca. 0.00075%. At such high expression even HPPDs with impaired catalytic activity could be effective. A quintuple mutant Avena sativa HPPD conferred substantial tolerance across a broad range of HPPD herbicide chemistries despite being only ca. 5 % as catalytically active as the wild type enzyme. Testing various wild type and mutant HPPDs in tobacco revealed that tolerance to field rates of herbicide generally requires about two order of magnitude increases in both inherent herbicide tolerance and expression relative to endogenous levels. This double hurdle may explain why target-site based resistance to HPPD-inhibiting herbicides has been slow to evolve in weeds.
Assuntos
4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Produtos Agrícolas/efeitos dos fármacos , Produtos Agrícolas/enzimologia , Cicloexanonas/farmacologia , Herbicidas/farmacologia , 4-Hidroxifenilpiruvato Dioxigenase/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Dados de Sequência Molecular , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/metabolismo , Ratos , Homologia de Sequência de AminoácidosRESUMO
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 1.85â Å resolution failed to identify the chirality of the bound inhibitor, however, by extending the resolution to 1.1â Å and 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.
RESUMO
Safeners such as metcamifen and benoxacor are widely used in maize to enhance the selectivity of herbicides through the induction of key detoxifying enzymes, notably cytochrome P450 monooxygenases (CYPs). Using a combination of transcriptomics, proteomics, and functional assays, the safener-inducible CYPs responsible for herbicide metabolism in this globally important crop have been identified. A total of 18 CYPs belonging to clans 71, 72, 74, and 86 were safener-induced, with the respective enzymes expressed in yeast and screened for activity toward thiadiazine (bentazon), sulfonylurea (nicosulfuron), and triketone (mesotrione and tembotrione) chemistries. Herbicide metabolism was largely restricted to family CYP81A members from clan 71, notably CYP81A9, CYP81A16, and CYP81A2. Quantitative transcriptomics and proteomics showed that CYP81A9/CYP81A16 were dominant enzymes in safener-treated field maize, whereas only CYP81A9 was determined in sweet corn. The relationship between CYP81A sequence and activities were investigated by splicing CYP81A2 and CP81A9 together as a series of recombinant chimeras. CYP81A9 showed wide ranging activities toward the three herbicide chemistries, while CYP81A2 uniquely hydroxylated bentazon in multiple positions. The plasticity in substrate specificity of CYP81A9 toward multiple herbicides resided in the second quartile of its N terminal half. Further phylogenetic analysis of CYP81A9 showed that the maize enzyme was related to other CYP81As linked to agrochemical metabolism in cereals and wild grasses, suggesting this clan 71 CYP has a unique function in determining herbicide selectivity in arable crops.
RESUMO
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.