Cytokinin oxidase/dehydrogenase inhibitors: outlook for selectivity and high efficiency.

Inhibitors of cytokinin oxidase/dehydrogenase (CKX) reduce the degradation of cytokinins in plants, and this effect can be exploited in agriculture and in plant tissue culture. In this study, we examine the structure-activity relationship of two series of CKX inhibitors based on diphenylurea. The compounds of Series I were derived from the recently published CKX inhibitors 3TFM-2HM and 3TFM-2HE, and we identified key substituents with increased selectivity for maize ZmCKX1 and ZmCKX4a over AtCKX2 from Arabidopsis. Series II contained compounds that further exceled in CKX inhibitory activity as well as in the ease of their synthesis. The best inhibitors exhibited half-maximal inhibitory concentration (IC50) values in low nanomolar ranges with ZmCKX1 and especially with ZmCKX4a, which is generally more resistant to inhibition. The activity of the key compounds was verified in tobacco and lobelia leaf-disk assays, where N6-isopentenyladenine was protected from degradation and promoted shoot regeneration. All the prepared compounds were further tested for toxicity against Caenorhabditis elegans, and the assays revealed clear differences in toxicity between compounds with and without a hydroxyalkyl group. In a broader perspective, this work increases our understanding of CKX inhibition and provides a more extensive portfolio of compounds suitable for agricultural and biotechnological research.

High Throughput Screening Method for Identifying Potential Agonists and Antagonists of Arabidopsis thaliana Cytokinin Receptor CRE1/AHK4.

The CRE1/AHK4 cytokinin receptor is an important component of plants' hormone signaling systems, and compounds that can alter its activity have potential utility for studying the receptor's functions and/or developing new plant growth regulators. A high throughput method was developed for screening compounds with agonist or antagonist properties toward the CRE1/AHK4 cytokinin receptor in a single experiment using the Nanodrop II liquid handling system and 384-well plates. Potential ligands are screened directly, using a reporter system in which receptor signaling activity triggers expression of ß-galactosidase in Escherichia coli. This enzyme generates a fluorescent product from a non-fluorescent substrate, allowing the agonistic/antagonistic behavior of tested compounds to be assayed in relation to that of an internal standard (here the natural ligand, trans-zeatin). The method includes a robust control procedure to determine false positive or false negative effects of the tested compounds arising from their fluorescent or fluorescent-quenching properties. The presented method enables robust, automated screening of large libraries of compounds for ability to activate or inhibit the Arabidopsis thaliana cytokinin receptor CRE1/AHK4.

An automated method to evaluate the enzyme kinetics of ß-glucosidases.

Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize ß-glucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and time-consuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity of ß-glucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols.

Combining rational and random strategies in ß-glucosidase Zm-p60.1 protein library construction.

Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins' structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations' effects. Thus, in the presented study we applied the technique to randomize position W373 in ß-glucosidase Zm-p60.1, which is highly conserved among ß-glucosidases. Unexpectedly, ß-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.