DOI: 10.5061/dryad.qrfj6q5sj
title: Data from: A monomer-dimer switch modulates the activity of plant adenosine kinase

description: Adenosine undergoes ATP-dependent phosphorylation catalyzed by adenosine
 kinase (ADK). In plants, ADK also phosphorylates cytokinin ribosides,
 transport forms of the hormone. Here, we investigated the substrate
 preferences, oligomeric states and structures of ADKs from moss
 (Physcomitrella patens) and maize (Zea mays) alongside metabolomic and
 phenotypic analyses. We showed that dexamethasone-inducible ZmADK
 overexpressor lines in Arabidopsis can benefit from a higher number of
 lateral roots and larger root areas under nitrogen starvation. We
 discovered that maize and moss enzymes can form dimers upon increasing
 protein concentration, setting them apart from the monomeric human and
 protozoal ADKs. Structural and kinetic analyses revealed a catalytically
 inactive unique dimer. Within the dimer, both active sites are mutually
 blocked. The activity of moss ADKs, exhibiting a higher propensity to
 dimerize, was tenfold lower compared to maize ADKs. Two monomeric
 structures in a ternary complex highlight the characteristic transition
 from an open to a closed state upon substrate binding. This suggests that
 the oligomeric state switch can modulate the activity of moss ADKs and
 likely other plant ADKs. Moreover, dimer association represents a novel
 negative feedback mechanism, helping to maintain steady levels of
 adenosine and AMP.
description: This collection of datasets is related to molecular properties,
 ligand interactions and enzyme kinetics. Data measurements are given in
 Materials and methods. Other data are part of the Supplement of the
 manuscript. <strong><em>Gel permeation
 chromatography</em></strong> Gel permeation
 chromatography of studied plant Aadenosine kinases was performed on an NGC
 Medium-Pressure Liquid Chromatography System (https://www.bio-rad.com)on a Superdex 200 10/30 HR column in 20 mM Tris-HCl buffer, pH 7.5, 100 mM NaCl, with calibration performed using a gel filtration standard (Bio-Rad). <strong><em>Affinity and thermal stability measurements</em></strong> The MST method was used to determine the binding affinity of various ribosides to ZmADK2 and PpADK1. Proteins were fluorescently labeled with RED-tris-NTA dye (www.nanotemper-technologies.com) using a 1:1 dye/protein molar ratio. The labeled protein was adjusted to 100-300 nM in 50 mM HEPES buffer pH 7.5, 1 mM MgCl<sub>2</sub> and 0.2% Tween. Measurements were performed in premium capillaries on a Monolith NT.115 instrument at 30°C with 5 sec/30 sec/5 sec laser off/on/off times, respectively, with continuous sample fluorescence recording. Thermal stability was measured by nano-differential scanning fluorimetry on a Prometheus NT.48 instrument (www.nanotemper-technologies.com) in various buffers covering a pH (7.0-9.5) and temperature range (25 to 95°C), and with a heating rate of 1 °C min<sup>-1</sup> and using NT melting control software. Protein unfolding was measured by detecting the temperature-induced change in tryptophan fluorescence intensity at emission wavelengths of 330 and 350 ± 5 nm. The melting temperature (<em>T</em><sub>m</sub>) was deduced from the maximum of the first derivative of the fluorescence ratios F350/F330. <strong><em>Sedimentation coefficient</em></strong> The analytical ultracentrifugation (AUC) in sedimentation velocity mode was performed using ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter, Indianapolis, IN, USA) equipped with an An-60 Ti rotor. Samples of ZmADK2 and PpADK1 were diluted in 20 mM Tris-HCl pH 7.5 with 150 mM NaCl, 10 mM MgCl<sub>2</sub>, 10 µM DTT and 1% glycerol and equilibrated at 4 °C overnight. Absorbance data were collected at 25 °C and at a rotor speed of 48000 rpm. Scans were collected at 280 nm in 5-min intervals and 0.003 cm spatial resolution in continuous scan mode. The partial specific volume of the protein and the solvent density and viscosity were calculated from the amino acid sequence and buffer composition, respectively, using the Sednterp software (http://bitcwiki.sr.unh.edu). The data were analyzed with the continuous c(s) distribution model implemented in the program SEDFIT 15.01b, using a confidence level of 0.95 for the regularization procedure. The plots of c(s) distributions were created in GUSSI 1.3.1. <strong><em>DLS measurements</em></strong> The hydrodynamic diameter was determined by DLS in 20 mM Tris-HCl pH 7.5 and 1 mM MgCl<sub>2</sub> alone and in the presence of 2 mM ATP or 2 mM AP5A at 22 °C. Measurement was performed using the Zetasizer Nano ZS (Malvern Instruments, UK). Hydrodynamic diameter values were calculated by Zetasizer Software v7.13 (173° angle measurement, approximation fit to the sphere).
description: # Data from: A monomer-dimer switch modulates the activity of plant
 adenosine kinase
 [https://doi.org/10.5061/dryad.qrfj6q5sj](https://doi.org/10.5061/dryad.qrfj6q5sj) ## Description of the data and file structure This supplementary dataset, integral to our research paper, contains data covering molecular properties and interactions of studied plant adenosine kinases. It includes final microscale thermophoresis data used to describe the binding curves and, thus, affinity to various riboside ligands. The dataset consists of gel permeation chromatography profiles of studied plant adenosine kinases as well as the dynamic light scattering (DLS) data used to determine the oligomeric state of studied enzymes. Thermal stability curves measured by nano-differential scanning fluorimetry are provided to show differences in stability in the presence of ATP. The dataset further contains kinetic data measured with maize ADK2 used to determine Km and Vmax values. Finally, quantitative PCR (qPCR) data are provided for the expression of maize ADK genes in dexamethasone-inducible overexpressor lines in Arabidopsis. ### Files and variables #### File: Thermal\_stability\_nanoDSF.csv **Description:** Thermal stability was measured by nano-differential scanning fluorimetry on a Prometheus NT.48 (Nanotemper) instrument with a heating rate of 1 °C min-1 and using NT melting control software. Five enzymes were measured: maize adenosine kinase 1, 2 and 3 (ZmADK1,2 and 3) and moss adenosine kinase 1 and 2 (PpADK1 and PpADK2) ##### Variables * Temperature (degrees C): Temperature in capillary during thermal unfolding * ZmADK1 (First derivative F350/F330): First derivative of ZmADK1 fluorescence measured at 350 nm and 330 nm * ZmADK2 (First derivative F350/F330): First derivative of ZmADK2 fluorescence measured at 350 nm and 330 nm * ZmADK3 (First derivative F350/F330): First derivative of ZmADK3 fluorescence measured at 350 nm and 330 nm * ZmADK1 + 4 mM ATP (First derivative F350/F330): First derivative of ZmADK1 fluorescence measured at 350 nm and 330 nm in the presence of ATP * ZmADK2 + 4 mM ATP (First derivative F350/F330): First derivative of ZmADK2 fluorescence measured at 350 nm and 330 nm in the presence of ATP * ZmADK3 + 4 mM ATP (First derivative F350/F330): First derivative of ZmADK3 fluorescence measured at 350 nm and 330 nm in the presence of ATP * Temperature 2 (degrees C): Temperature in capillary during thermal unfolding * PpADK1 (First derivative F350/F330): First derivative of PpADK1 fluorescence measured at 350 nm and 330 nm * PpADK1 + 4 mM ATP (First derivative F350/F330): First derivative of PpADK1 fluorescence measured at 350 nm and 330 nm in the presence of ATP * Temperature 3 (degrees C): Temperature in capillary during thermal unfolding * PpADK2 (First derivative F350/F330): First derivative of PpADK2 fluorescence measured at 350 nm and 330 nm * PpADK2 + 4 mM ATP (First derivative F350/F330): First derivative of PpADK2 fluorescence measured at 350 nm and 330 nm in the presence of ATP #### File: Gel\_permeation\_chromatorgraphy\_AtADK2.csv **Description:** Gel filtration of highly concentrated ZmADK2 (at 30 mg ml-1) on Superdex S200 column. The chromatogram shows the existence of a small peak corresponding to the double molecular weight of the monomer. ##### Variables * Elution volume (ml): Elution volume in milliliters * AtADK2 (Abs at 280 nm): Absorbance of protein at 280 nm * Elution volume 2 (ml): Elution volume in milliliters * Bio-Rad std (Abs at 280 nm): Absorbance of protein at 280 nm #### File: Gel\_permeation\_chromatography.csv **Description:**  Gel permeation chromatography profiles of five plant ADKs on a Superdex 200 10/30 HR column in 20 mM Tris-HCl buffer, pH 7.5, 100 mM NaCl, with calibration performed using a gel filtration standard (Bio-Rad). ##### Variables * Elution volume (ml): Elution volume in milliliters * ZmADK1 (Abs at 280 nm): Absorbance of protein at 280 nm * ZmADK2 (Abs at 280 nm): Absorbance of protein at 280 nm * ZmADK3 (Abs at 280 nm): Absorbance of protein at 280 nm * PpADK1 (Abs at 280 nm): Absorbance of protein at 280 nm * PpADK2 (Abs at 280 nm): Absorbance of protein at 280 nm * Bio-Rad standards (Abs at 280 nm): Absorbance of protein at 280 nm #### File: Binding\_curves\_MST\_PpADK1.csv **Description:** Binding affinity values and curves of PpADK1 for selected riboside ligands, ADP, ATP and AP5A. Data were measured by MST in 50 mM HEPES buffer pH 7.5, 1 mM MgCl2 and 0.2% Tween on Monolith NT.115 instrument. Fitted curves were obtained using MO Affinity Analysis software from Nanotemper. ##### Variables * ATP concentration (M): Molar concentration * ATP fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * ATP concentration fitted: Molar concentration obtained using MO Affinity Analysis software * ATP fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * ADP concentration (M): Molar concentration * ADP fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * ADP concentration fitted: Molar concentration obtained using MO Affinity Analysis software * ADP fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * iPR concentration (M): Molar concentration * iPR fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * iPR concentration fitted: Molar concentration obtained using MO Affinity Analysis software * iPR fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * BAPR concentration (M): Molar concentration * BAPR fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * BAPR concentration fitted: Molar concentration obtained using MO Affinity Analysis software * BAPR fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * Ado concentration (M): Molar concentration * Ado fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * Ado concentration fitted: Molar concentration obtained using MO Affinity Analysis software * Ado fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * AP5A concentration (M): Molar concentration * AP5A fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * AP5A concentration fitted: Molar concentration obtained using MO Affinity Analysis software * AP5A fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * AP5A* concentration fitted: Molar concentration obtained using MO Affinity Analysis software * AP5A* fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * tZR concentration (M): Molar concentration * tZR fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * tZR concentration fitted: Molar concentration obtained using MO Affinity Analysis software * tZR fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * AMP concentration (M): Molar concentration * AMP fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * AMP concentration fitted: Molar concentration obtained using MO Affinity Analysis software * AMP fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * AMP* concentration (M): Molar concentration * AMP* fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * AMP* concentration fitted: Molar concentration obtained using MO Affinity Analysis software * AMP* fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software #### File: Binding\_curves\_MST\_ribosides\_ZmADK2.csv **Description:** Binding affinity values and curves of ZmADK2 for selected riboside ligands. Data were measured by MST in 50 mM HEPES buffer pH 7.5, 1 mM MgCl2 and 0.2% Tween on Monolith NT.115 instrument. Fitted curves were obtained using MO Affinity Analysis software from Nanotemper. ##### Variables * Ado concentration (M): Molar concentration * Ado fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * Ado fraction bound st error: St error of bound fraction * Ado concentration fitted: Molar concentration obtained using MO Affinity Analysis software * Ado fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * Ado* concentration (M): Molar concentration * Ado* fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * Ado* fraction bound st error: St error of bound fraction * Ado* concentration fitted: Molar concentration obtained using MO Affinity Analysis software * Ado* fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * N6-IsopropylAdo concentration (M): Molar concentration * N6-IsopropylAdo fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * N6-IsopropylAdo fraction bound st error: St error of bound fraction * N6-IsopropylAdo Concentration fitted: Molar concentration obtained using MO Affinity Analysis software * N6-IsopropylAdo fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * N6-IsobutylAdo concentration (M): Molar concentration * N6-IsobutylAdo fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * N6-IsobutylAdo fraction bound st error: St error of bound fraction * N6-IsobutylAdo concentration fitted: Molar concentration obtained using MO Affinity Analysis software * N6-IsobutylAdo fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * iPR concentration (M): Molar concentration * iPR fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * iPR fraction bound st error: St error of bound fraction * iPR concentration fitted: Molar concentration obtained using MO Affinity Analysis software * iPR fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * 2-DeoxyAdo concentration (M): Molar concentration * 2-DeoxyAdo fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * 2-DeoxyAdo fraction bound st error: St error of bound fraction * 2-DeoxyAdo concentration fitted: Molar concentration obtained using MO Affinity Analysis software * 2-DeoxyAdo fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * 3-DeoxyAdo concentration (M): Molar concentration * 3-DeoxyAdo fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * 3-DeoxyAdo fraction bound st error: St error of bound fraction * 3-DeoxyAdo concentration fitted: Molar concentration obtained using MO Affinity Analysis software * 3-DeoxyAdo fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * 7-DeazaAdo concentration (M): Molar concentration * 7-DeazaAdo fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * 7-DeazaAdo fraction bound st error: St error of bound fraction * 7-DeazaAdo concentration fitted: Molar concentration obtained using MO Affinity Analysis software * 7-DeazaAdo fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * Inosine concentration (M): Molar concentration * Inosine fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * Inosine fraction bound st error: St error of bound fraction * Inosine concentration fitted: Molar concentration obtained using MO Affinity Analysis software * Inosine fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * Guanosine concentration (M): Molar concentration * Guanosine fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * Guanosine fraction bound st error: St error of bound fraction * Guanosine concentration fitted: Molar concentration obtained using MO Affinity Analysis software * Guanosine fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * Xanthosine concentration (M): Molar concentration * Xanthosine fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * Xanthosine fraction bound st error: St error of bound fraction * Xanthosine concentration fitted: Molar concentration obtained using MO Affinity Analysis software * Xanthosine fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * N6-MethylAdo concentration (M): Molar concentration * N6-MethylAdo fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * N6-MethylAdo fraction bound st error: St error of bound fraction * N6-MethylAdo concentration fitted: Molar concentration obtained using MO Affinity Analysis software * N6-MethylAdo fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * N6,N6 -DimethylAdo concentration (M): Molar concentration * N6,N6 -DimethylAdo fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * N6,N6 -DimethylAdo fraction bound st error: St error of bound fraction * N6,N6 -DimethylAdo concentration fitted: Molar concentration obtained using MO Affinity Analysis software * N6,N6 -DimethylAdo fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * Uridine concentration (M): Molar concentration * Uridine fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) #### File: Binding\_curves\_MST\_ZmADK2.csv **Description:** Binding affinity values and curves of ZmADK2 for selected riboside ligands, ADP, ATP and AP5A. Data were measured by MST in 50 mM HEPES buffer pH 7.5, 1 mM MgCl2 and 0.2% Tween on Monolith NT.115 instrument. Fitted curves were obtained using MO Affinity Analysis software from Nanotemper. ##### Variables * ATP concentration (M): Molar concentration * ATP fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * ATP concentration fitted: Molar concentration obtained using MO Affinity Analysis software * ATP fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * ADP concentration (M): Molar concentration * ADP fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * ADP concentration fitted: Molar concentration obtained using MO Affinity Analysis software * ADP fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * iPR concentration (M): Molar concentration * iPR fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * iPR concentration fitted: Molar concentration obtained using MO Affinity Analysis software * iPR fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * BAPR concentration (M): Molar concentration * BAPR fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * BAPR concentration fitted: Molar concentration obtained using MO Affinity Analysis software * BAPR fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * Ado concentration (M): Molar concentration * Ado fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * Ado concentration fitted: Molar concentration obtained using MO Affinity Analysis software * Ado fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * AP5A concentration (M): Molar concentration * AP5A fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * AP5A concentration fitted: Molar concentration obtained using MO Affinity Analysis software * AP5A fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * AP5A* concentration fitted: Molar concentration obtained using MO Affinity Analysis software * AP5A* fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * tZR concentration (M): Molar concentration * tZR fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * tZR concentration fitted: Molar concentration obtained using MO Affinity Analysis software * tZR fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * AMP concentration (M): Molar concentration * AMP fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * AMP concentration fitted: Molar concentration obtained using MO Affinity Analysis software * AMP fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software * AMP* concentration (M): Molar concentration * AMP* fraction bound: Fraction of bound lingad (from 0 to 1 corresponding to 0 -100%) * AMP* concentration fitted: Molar concentration obtained using MO Affinity Analysis software * AMP* fraction bound fitted: Fraction of bound lingad obtained using MO Affinity Analysis software #### File: Enzyme\_kinetics\_ZmADK2.csv **Description:** Saturation curves of various Ado derivatives measured in a coupled reaction with PK and LDH at 30°C in 50 mM Tris-HCl buffer pH 7.5 using 10 mM ATP. Dataset comprises measured values (nkat/mg) and std error plus calculated curves fitted to Michaelis-Menten equation by GraphPan Prism sofware. ##### Variables * Ado concentration: Concentration (micromol/l) * Ado specific activity: Specific activity in nkat/mg * Ado specific activity st error: St error of specific activity in nkat/mg * Ado fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * Ado fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * N6-IsopropylAdo concentration: Concentration (micromol/l) * N6-IsopropylAdo specific activity: Specific activity in nkat/mg * N6-IsopropylAdo specific activity st error: St error of specific activity in nkat/mg * N6-IsopropylAdo fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * N6-IsopropylAdo fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * N6-IsobutylAdo concentration: Concentration (micromol/l) * N6-IsobutylAdo specific activity: Specific activity in nkat/mg * N6-IsobutylAdo specific activity st error: St error of specific activity in nkat/mg * N6-IsobutylAdo fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * N6-IsobutylAdo fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * iPR concentration: Concentration (micromol/l) * iPR specific activity: Specific activity in nkat/mg * iPR specific activity st error: St error of specific activity in nkat/mg * iPR fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * iPR fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * 2-DeoxyAdo concentration: Concentration (micromol/l) * 2-DeoxyAdo specific activity: Specific activity in nkat/mg * 2-DeoxyAdo specific activity st error: St error of specific activity in nkat/mg * 2-DeoxyAdo fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * 2-DeoxyAdo fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * 3-DeoxyAdo concentration: Concentration (micromol/l) * 3-DeoxyAdo specific activity: Specific activity in nkat/mg * 3-DeoxyAdo specific activity st error: St error of specific activity in nkat/mg * 3-DeoxyAdo fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * 3-DeoxyAdo fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * 7-DeazaAdo concentration: Concentration (micromol/l) * 7-DeazaAdo specific activity: Specific activity in nkat/mg * 7-DeazaAdo specific activity st error: St error of specific activity in nkat/mg * 7-DeazaAdo fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * 7-DeazaAdo fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * Vidarabine concentration: Concentration (micromol/l) * Vidarabine specific activity: Specific activity in nkat/mg * Vidarabine specific activity st error: St error of specific activity in nkat/mg * Vidarabine fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * Vidarabine fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * Inosine concentration: Concentration (micromol/l) * Inosine specific activity: Specific activity in nkat/mg * Inosine specific activity st error: St error of specific activity in nkat/mg * Inosine fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * Inosine fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * Guanosine concentration: Concentration (micromol/l) * Guanosine specific activity: Specific activity in nkat/mg * Guanosine specific activity st error: St error of specific activity in nkat/mg * Guanosine fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * Guanosine fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * Xanthosine concentration: Concentration (micromol/l) * Xanthosine specific activity: Specific activity in nkat/mg * Xanthosine specific activity st error: St error of specific activity in nkat/mg * Xanthosine fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * Xanthosine fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * N6-methylAdo concentration: Concentration (micromol/l) * N6-methylAdo specific activity: Specific activity in nkat/mg * N6-methylAdo specific activity st error: St error of specific activity in nkat/mg * N6-methylAdo fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * N6-methylAdo fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * N6,N6-dimethylAdo concentration: Concentration (micromol/l) * N6,N6-dimethylAdo specific activity: Specific activity in nkat/mg * N6,N6-dimethylAdo specific activity st error: St error of specific activity in nkat/mg * N6,N6-dimethylAdo fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * N6,N6-dimethylAdo fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting * Cytidine concentration: Concentration (micromol/l) * Cytidine specific activity: Specific activity in nkat/mg * Cytidine specific activity st error: St error of specific activity in nkat/mg * Cytidine fitted concentration: Molar concentration values obtained upon Michaelis-Menten equation fitting * Cytidine fitted spec activity: Specific activity values (nkat/mg) obtained upon Michaelis-Menten equation fitting #### File: Dynamic\_light\_scattering\_AtADK2.csv **Description:** The hydrodynamic diameter of Arabidopsis adenosine kinase AtADK2 determined by DLS, measured in 20 mM Tris-HCl pH 7.5 and 1 mM MgCl2 at 22°C at two concentrations (0.5 and 5.0 mg ml-1). ##### Variables * Measurement: Number of measurement * AtADK2 (0.5 mg ml-1) hydrodynamic diameter [nm]: Hydrodynamic diameter [nm] at protein concentration of 0.5 mg/ml * AtADK2 (5 mg ml-1) hydrodynamic diameter [nm]: Hydrodynamic diameter [nm] at protein concentration of 5 mg/ml #### File: Dynamic\_light\_scattering.csv **Description:** Hydrodynamic diameter determined by DLS for selected adenosine kinases. Measured for apoforms in 20 mM Tris-HCl pH 7.5 and 1 mM MgCl2 with 2 mM ATP or 2 mM AP5A at 22 °C. Measurement was performed using the Zetasizer Nano ZS (Malvern Instruments, UK) and values were calculated by Zetasizer Software v7.13. ##### Variables * Measurement: Number of measurement * ZmADK2: Hydrodynamic diameter [nm] * ZmADK2 with ATP: Hydrodynamic diameter [nm] in presence of ATP * ZmADK2 with ATP and Ado: Hydrodynamic diameter [nm] in presence of ATP and Ado * ZmADK2 with AP5A: Hydrodynamic diameter [nm] in presence of AP5A * PpADK1: Hydrodynamic diameter [nm] * PpADK1 with ATP: Hydrodynamic diameter [nm] in presence of ATP * PpADK1 with ATP and Ado: Hydrodynamic diameter [nm] in presence of ATP and Ado * PpADK1 with AP5A: Hydrodynamic diameter [nm] in presence of AP5A #### File: qPCR\_overexpressors\_dexamethasone\_induction.csv **Description:** Dexamethasone-inducible expression of *ZmADK* genes in homozygous Arabidopsis seedlings after 48 hours at 14 days after germination. Epression values determined at 0 and 48 hours. qPCR conducted with the RT Luna Universal Probe qPCR Master Mix on a Quant Studio 5 Real-Time PCR System. ##### Variables * Plant line: Name of the overespressor line * 0 hours: Gene copies of overexpressed ZmADK gene per 1 ng of RNA * 0 hours st error: St error of gene copies of overexpressed ZmADK gene per 1 ng of RNA * 48 hours: Gene copies of overexpressed ZmADK gene per 1 ng of RNA * 48 hours st error: St error of g ene copies of overexpressed ZmADK gene per 1 ng of RNA #### File: Sedimentation\_coefficient.csv **Description:** The analytical ultracentrifugation plots of c(s) distributions for ZmADK2 and PpADK1 as analyzed in sedimentation velocity mode. Performed in 20 mM Tris-HCl pH 7.5 with 150 mM NaCl, 10 mM MgCl2, 10 µM DTT and 1% glycerol. Scans were collected on a ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter, Indianapolis, IN, USA) equipped with an An-60 Ti rotor, at a rotor speed of 48000 rpm at 280 nm in 5-min intervals and 0.003 cm spatial resolution in continuous scan mode. ##### Variables * Sedimentation coefficient (S): Sedimentation coefficient * PpADK1 (0.01 mg ml-1): Normalized sedimentation coefficient distribution c(s) for PpADK1 at concentration of 0.01 mg/ml * PpADK1 (0.02 mg ml-1): Normalized sedimentation coefficient distribution c(s) for PpADK1 at concentration of 0.02 mg/ml * PpADK1 (0.03 mg ml-1): Normalized sedimentation coefficient distribution c(s) for PpADK1 at concentration of 0.03 mg/ml * PpADK1 (0.09 mg ml-1): Normalized sedimentation coefficient distribution c(s) for PpADK1 at concentration of 0.09 mg/ml * PpADK1 (0.27 mg ml-1): Normalized sedimentation coefficient distribution c(s) for PpADK1 at concentration of 0.27 mg/ml * PpADK1 (0.82 mg ml-1): Normalized sedimentation coefficient distribution c(s) for PpADK1 at concentration of 0.82 mg/ml * ZmADK2 (0.09 mg ml-1): Normalized sedimentation coefficient distribution c(s) for ZmADK2 at concentration of 0.09 mg/ml * ZmADK2 (0.27 mg ml-1): Normalized sedimentation coefficient distribution c(s) for ZmADK2 at concentration of 0.27 mg/ml * ZmADK2 (0.82 mg ml-1): Normalized sedimentation coefficient distribution c(s) for ZmADK2 at concentration of 0.82 mg/ml * ZmADK2 (1.6 mg ml-1): Normalized sedimentation coefficient distribution c(s) for ZmADK2 at concentration of 1.6 mg/ml * ZmADK2 (3.3 mg ml-1): Normalized sedimentation coefficient distribution c(s) for ZmADK2 at concentration of 3.3 mg/ml * ZmADK2 (6.6 mg ml-1): Normalized sedimentation coefficient distribution c(s) for ZmADK2 at concentration of 6.6 mg/ml