Key message WRKY50 from A. thaliana requires WT-boxes at target gene promoters for activation and binding. Abstract Based on the genome-wide prediction of WRKY50 target genes and the similarity of a WRKY50 binding site to WT-boxes in microbe-associated molecular pattern (MAMP)-responsive cis-regulatory modules (CRM), four WT-box containing CRMs from the promoter region of three WRKY50 target genes were investigated for their interaction with WRKY50. These target genes are DJ1E, WRKY30 and ATBBE4. Two of the four CRMs, one from DJ1E and one from WRKY30, were able to activate reporter gene expression in the presence of WRKY50. Activation requires the WT-boxes GGACTTTT, GGACTTTG from DJ1E and GGACTTTC from WRKY30. WRKY50 does not activate a second CRM from WRKY30 and the CRM from ATBBE4, both containing the WT-box TGACTTTT. In vitro gel-shift assays demonstrate WT-box-specific binding of the WRKY50 DNA-binding domain to all four CRMs. This work shows a high flexibility of WRKY50 binding site recognition beyond the classic W-box TTGACC/T.

Despite the prevalence of ortho- and pyrophosphate in biochemistry, operationally simple and versatile high-throughput methodologies for their quantification are lacking. We herein introduce PUB, a module for p hosphate detection by continuous U V-spectroscopic monitoring of 5- b romouridine phosphorolysis. The PUB module uses cheaply available, bench-stable reagents and can be employed for continuous and discontinuous reaction monitoring in biochemical assays to detect (pyro-)­phosphate concentrations spanning almost 4 orders of magnitude, as demonstrated with representative use cases.

This report advises against the use of 5-iodoridine or 5-ethynyluridine as alternative assay reagents in the PUB module, primarily due to their lack of an isosbestic point of phosphorolysis under moderately alkaline conditions.

Biocatalytic nucleoside (trans-)glycosylations catalyzed by nucleoside phosphorylases have graduated to a practical and convenient approach to the preparation of modified nucleosides, which are important pharmaceuticals for the treatment of various cancers and viral infections. However, the obtained yields in these reactions are generally determined exclusively by the innate thermodynamic properties of the nucleosides involved, hampering the biocatalytic access to many sought-after target nucleosides. We herein report an orthogonal dimension for reaction engineering of these systems. We show how apparent equilibrium shifts in phosphorolysis and glycosylation reactions can be effected through entropically driven, biased esterification of nucleosides with inorganic borate. Our multifaceted analysis further describes the kinetic implications of this in situ reactant esterification for a model phosphorylase. Our results suggest an unusual pseudo-non-competitive inhibitory mechanism where reversible binding of the borate ester of the nucleoside substrate yields a non-productive inhibitor-enzyme complex. This complex exhibits constricted molecular dynamics and exists in a rapid equilibrium with the productive enzyme-substrate complex via hydrolytic interconversion. Collectively, this report presents a partial departure from the stringent thermodynamic constraints of nucleoside phosphorolysis reactions and shines light on the molecular processes regulating the activity of nucleoside-binding enzymes in the presence of borate.