Phenolic herbicides binding

Herbicides that inhibit photosynthetic electron flow prevent reduction of plastoquinone by the photosystem II acceptor complex. The properties of the photosystem II herbicide receptor proteins have been investigated by binding and displacement studies with radiolabeled herbicides. The herbicide receptor proteins have been identified with herbicide-derived photoaffinity labels. Herbicides, similar in their mode of action to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) bind to a 34 kDa protein, whereas phenolic herbicides bind to the 43-51 kDa photosystem II reaction center proteins. At these receptor proteins, plastoquinone/herbicide interactions and plastoquinone binding sites have been studied, the latter by means of a plastoquinone-deriv-ed photoaffinity label. For the 34 kDa herbicide binding protein, whose amino acid sequence is known, herbicide and plastoquinone binding are discussed at the molecular level. 

The phenolic photoaffinity label azidodinoseb (Figure 4) binds less specifically than either azidoatrazine or azidotriazinone (14). In addition to other proteins, it labels predominantly the photosystem II reaction center proteins (spinach 43 and 47 kDa Chlamydomo-nas 47 and 51 kDa) (17). Because of the unspecific binding of azidodinoseb, this can best be seen in photosystem II preparations (17). Thus, the phenolic herbicides bind predominantly to the photosystem II reaction center, which might explain many of the differences observed between "DCMU-type" and phenolic herbicides (9). The photosystem II reaction center proteins and the 34 kDa herbicide binding protein must be located closely to and interact with each other in order to explain the mutual displacement of both types of herbicides (8,12,21). Furthermore, it should be noted that for phenolic herbicides, some effects at the donor side of photosystem II (22) and on carotenoid oxidation in the photosystem II reaction center have been found (23). 

Changes in binding affinities for herbicides in the Leu275 Phe mutant are marginal. It should only be stressed that triazinones are rendered resistant in this mutant and supersensitivity is observed against the phenolic herbicide ioxynil. 

This approach has been used primarily to study photosynthesis, for example to introduce tolerance to a variety of stress conditions. Many studies have been successful in verifying the effea of single amino acid modifications on herbicide binding affinity. High affinity binding to the D1 protein is a useful property for the detection of herbicides. A fluorescence biosensor based on mutants resistant to various herbicide subclasses was developed, it makes possible to distinguish between subclasses of herbicides (e.g., triazines from urea and phenolic type herbicides). ... 

The first photosynthesis-inhibiting herbicides such as arylurea (e.g., diuron) and triazines derivatives (e.g., atrazine) were identified in 1956 even before the photosynthetic reactions and two photosystems were known and before plastoquinone had been discovered. Surprisingly, this group of herbicides still dominates the field. The second group, which includes phenolic compounds such as bromoxynil and ioxynil, were recognized later. Although phenolic herbicides inhibit the PS II reaction centre differently from triazine herbicides, they also interfere with the Qg function and bind the DI protein. 

Due to their difference in chemistry, all PSII-inhibiting herbicides demonstrate different binding properties. For example, urea/triazine type inhibitors were proposed to be oriented towards Set 264, triazinones towards Ala 251 and phenolic herbicides were oriented towards His 215 (Table 1). ... 

Two families of inhibitors interfere with the plastoquinone or herbicide binding site on the D-1 polypeptide, i.e. on one of the reaction center subunits of PS II. The phenol and urea/triazinone family of PS II inhibitors are different in their functional inhibitory pattern (reviewed in [1]), although they both bind to the D-1 polypeptide and displace each other from the binding site (1). Both QSAR studies (2) and - more refined - quantum mechanical calculations... 

Nevertheless, there is a functional difference between metribuzin or DCMU when compared with a phenol inhibitor or the hydroxyquinolines. This is seen in tris-treated chloroplasts where a modification of the donor side of the herbicide binding protein affects the... 

Photoaffinity labels are an efficient tool for identification of inhibitor binding proteins in the photosynthetic electron transport chain. [ H]Azido-dinoseb, an azido-deri-vative of the phenolic herbicide dinoseb, was synthesized almost a decade ago and was shown to bind primarily to a 41 kDa protein (1,2). Contrary, labeling with azido-deri-vates of diuron-type herbicides revealed that these herbicides bind to a 32 kDa protein, which has now been recognized as the D-1 protein of the photosystem II reaction center core complex (see references in (3)). Tyrosine residues in positions 237 and 254 of the D-1 sequence were demonstrated to be the primary target of [ CJazido-monuron (3). The phenolic herbicide [ I]azido-ioxynil also labels predominantly the D-1 protein in position of Val249 and only in trace amounts a 41 kDa protein (4). 

The phenolic derivatives indicated in Figure 8.1 are also bound to the same binding niche on PS II as the triazines (Oettmeier, 1992). However, they have a somewhat different inhibition pattern than the classical family of PS II herbicides (e.g., triazines and ureas) and, therefore, were regarded as a separate family with a somewhat different mode of action (Van Rensen et al., 1978 Trebst and Draber, 1986). It is now clear that they just orient somewhat differently in the same binding niche, as discussed below. Although the phenolics are photosynthesis inhibitors, dinoseb and the halogenated benzonitriles also inhibit respiration. 

Some good inhibitors of the Hill reaction, however, do not contain the carbonyl oxygen-nitrogen moiety. Examples are the dinitroanilines, diphenylethers, 2,4-dinitrophenols, halogenated benzonitriles, and pyridinols. Hence, the postulates proposed are not all inclusive. Three of these herbicides are phenols. Under physiological pH s, the molecules can be expected to be ionized, and it may be the ionized form of the molecule that binds to the receptor. 

Many commercial herbicides inhibit photosynthesis by displacing Qg from its binding site in D1 and thus block electron transport from to They belong to various chemical classes like triazines, ureas or phenols (For a review see ref. 29) and pollute soil and water due to their massive use in agriculture. This in turn can be harmful for human and animal health and necessitates the development of fast and sensitive detection methods. Coincidentally, the herbicidal target itself is part of the PSII complex, which represents a reporter system directly coupled to an analyte binding site. Thus the most obvious application of the Dl protein in association with other central PSII proteins is its use as a biosensor for herbicides. 

Photosynthetic herbicides fall into three main groups phenylureas, triazines, diazines and phenols, depending on their chemical structure and binding properties. Although both classes of herbicides replace the Qp acceptor on the D1 protein, they interact with different amino acid residues on Dl. ... 

A large number of commercial herbicides such as arylureas, triazines, triazinones and phenolic compounds act as competitors to plastoquinones (Fig. 1). They occupy the Qp-binding site of the D1 protein, thereby displacing from its binding niche and prevent the oxidation of reduced Q/v. The displacement of electron mediator Qp from the D1 protein leads to interruption of the electron flow and, consequently, results in plant s death. 

As the primary mechanism of action of the. -triazines involves inhibition of PS II electron transport, attention was also directed at analysis of chloroplast reactions in resistant weed biotypes (10, 11, 12). These studies can be summarized as follows (a) in aTl cases studied to date, there is a modification in the chloroplast membranes of resistant biotypes that changes the characteristics of s-triazine binding (b) this modification results in altered bincfTng characteristics of other classes of herbicides, (i.e., only slight resistance to ureas, but increased sensitivity to phenols) (see for review), and (c) the alteration of the herbicide receptor in resistant weeds is accompanied... 

The distinctly different behavior of the phenol-type herbicides following trypsin treatment suggests that different determinants within the PS II protein complex establish the "domains" that regulate the binding properties of these inhibitors. In spite of the fact that phenol-type herbicides will displace bound radiolabeled herbicides such as diuron, these inhibitors show noncompetitive inhibition (29, 30). At present, there are three lines of evidence which favor TH e involvement of two domains within the PS II complex that participate in creating the binding sites for these herbicides (a) isolated PS II particles can be selectively depleted of a polypeptide with parallel loss of atrazine sensitivity, but not dinoseb inhibition activity (33) (b) in resistant weed biotypes, chloroplast membranes that exhibit extreme triazine resistance have increased sensitivity to the phenol-type herbicides (13) and (c) experiments with azido (photoaffinity) derivatives of phenol and triazine herbicides result in the covalent labeling of different PS II polypeptides (, 31). 

Quantitative Structure Activity Relationships. The goal of our structure activity studies was not the prediction of more active compounds in order to finally get new hints for the development of a herbicide. Instead, we wanted to corroborate our concept that phenols with the appropriate substitution enter the binding niche in the D1 protein just as the classical herbicidal inhibitors like atrazine and diuron do. 

Thus far, no mention has been made of the inhibitory binding characteristics of certain phenol-type herbicides such as the hydroxybenzonitriles, DNOC, and dinoseb. There are clear chemical differences between these... 

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