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International Journal of Molecular Sciences

Article Biochemical Characterization of Recombinant UDPG-Dependent IAA Glucosyltransferase from Maize (Zea mays)

Anna Ciarkowska 1,*, Maciej Ostrowski 1 and Anna Kozakiewicz 2

1 Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University in Toruń,87-100 Toruń,Poland; [email protected] 2 Faculty of Chemistry, Nicolaus Copernicus University in Toruń,87-100 Toruń,Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-56-611-4520

Abstract: Here, we report a biochemical characterization of recombinant maize indole-3-acetyl-β-D- glucose (IAGlc) synthase which glucosylates indole-3-acetic acid (IAA) and thus abolishes its auxinic activity affecting plant hormonal homeostasis. Substrate speciﬁcity analysis revealed that IAA is a preferred substrate of IAGlc synthase; however, the enzyme can also glucosylate indole-3-butyric

acid and indole-3-propionic acid with the relative activity of 66% and 49.7%, respectively. KM values determined for IAA and UDP glucose are 0.8 and 0.7 mM, respectively. 2,4-Dichlorophenoxyacetic acid is a competitive inhibitor of the synthase and causes a 1.5-fold decrease in the enzyme afﬁnity

towards IAA, with the Ki value determined as 117 µM, while IAA–Asp acts as an activator of the synthase. Two sugar-phosphate compounds, ATP and glucose-1-phosphate, have a unique effect on the enzyme by acting as activators at low concentrations and showing inhibitory effect at higher concentrations (above 0.6 and 4 mM for ATP and glucose-1-phosphate, respectively). Results of molecular docking revealed that both compounds can bind to the PSPG (plant secondary product Citation: Ciarkowska, A.; Ostrowski, M.; Kozakiewicz, A. Biochemical glycosyltransferase) motif of IAGlc synthase; however, there are also different potential binding sites Characterization of Recombinant present in the enzyme. We postulate that IAGlc synthase may contain more than one binding site for UDPG-Dependent IAA ATP and glucose-1-phosphate as reﬂected in its activity modulation. Glucosyltransferase from Maize (Zea mays). Int. J. Mol. Sci. 2021, 22, 3355. Keywords: auxin conjugate; indole-3-acetic acid; UDPG-dependent IAA glucosyltransferase; enzyme https://doi.org/10.3390/ijms22073355 modulators; inhibitor–enzyme interaction

Academic Editor: Stephan Pollmann

Received: 26 February 2021 1. Introduction Accepted: 22 March 2021 Glycosyltransferases (GTs, EC (Enzyme Commission number) 2.4.x.y) are enzymes Published: 25 March 2021 catalyzing transfer of a sugar moiety from an activated donor molecule to a wide range of acceptors with formation of glycoside linkages [1]. The majority of GTs are uridine Publisher’s Note: MDPI stays neutral diphosphate glycosyltransferases (UGTs) which utilize as a donor a nucleotide-activated with regard to jurisdictional claims in sugar molecule (UDP sugar) [2]. Plant UGTs are cytosolic soluble proteins that share some published maps and institutional afﬁl- iations. structural similarities. They all contain a highly conserved PSPG (plant secondary product glycosyltransferase) motif located near the C-terminus of the protein. The PSPG motif is a 44 amino acid sequence which binds the UDP moiety of the UDP sugar substrate [3–5]. The N-terminus of the UGTs is a less conserved domain of the enzyme and binds the sugar acceptor molecule. The high sequence diversity of the N-terminal domain is responsible Copyright: © 2021 by the authors. for the vast spectrum of substrates that are glycosylated by UGTs [4]. UGT-mediated Licensee MDPI, Basel, Switzerland. glycosylation of plant compounds affects their biological activity, solubility and other This article is an open access article properties [1]. Thus, phytohormone glycosylation is an important mechanism regulating distributed under the terms and conditions of the Creative Commons plant hormonal homeostasis. Attribution (CC BY) license (https:// Auxins are phytohormones regulating proper growth and development of plants [6]. creativecommons.org/licenses/by/ Local auxin concentration is modulated by different metabolic processes, such as de novo 4.0/). biosynthesis, degradation, hormone transportation and conjugation [7]. The latter results

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in the loss of biological auxinic activity of the phytohormone. In monocotyledonous plants, auxin is mostly conjugated to sugars or myo-inositol via ester bonds [8]. The ester conjugation pathway of indole-3-acetic acid (IAA), the main natural auxin, has been extensively studied in maize. The first step of ester–IAA conjugate synthesis is formation of 1-O-indole-3-acetyl-β-D-glucose (1-O-IAGlc) [9]. This reaction is catalyzed by IAA glucosyltransferase (1-O-IAGlc synthase) of the UGT family. As an energy-rich molecule, 1-O-IAGlc is subsequently utilized by various transferases as a donor of an indole-3-acetyl moiety for synthesis of other, more stable, IAA conjugates, such as indole-3- acetyl-myo-inositol [10,11]. Maize IAGlc synthase has been partially purified and characterized by Le´znickiand Bandurski [12,13]. Later, Kowalczyk and Bandurski [14] obtained a homogenous maize IAGlc synthase preparation which allowed identification of the IAGLU gene encoding this enzyme [15]. The authors confirmed identity of the IAGLU gene by its heterologous expression in Escherichia coli; however, characterization of the recombinant IAGlc synthase has not been performed. Few years later, glucosyltransferase UGT84B1 which conjugates glucose to IAA and other natural auxins was identified in Arabidopsis thaliana [16]. After that, a number of auxin glucosyltransferases has been identified in A. thaliana [17–20], as well as in other plant species, such as pea and rice [21,22]. Auxin ester conjugation has a great impact on a number of processes, such as seed development [23], seed germination [19], tiller formation [24], leaf positioning [25,26], hypocotyl elongation [20], gravitropic response [25], stress response [17,19] and more. As the role of IAA UGTs in plant development and stress response seems significant, there is a need to deeply explore properties of these enzymes. Maize is a model monocotyledonous plant for studying ester–auxin conjugate synthesis; however, IAA UGTs are mostly explored in A. thaliana, a dicotyledonous plant which conjugates IAA mainly to amino acids. In this study, we produced recombinant maize IAGlc synthase and performed its biochemical characterization to complete studies started by Le´znickiand Bandurski [13] and provide deeper understanding of this enzyme vital for maintenance of auxin homeostasis.

2. Results and Discussion 2.1. Production of Recombinant Maize IAGlc Synthase in Escherichia coli and Purification of the Enzyme The Zm-IAGLU gene encoding maize IAGlc synthase was introduced into the E. coli BL21-CodonPlus(DE3)-RIL strain using the pET-15b expression vector provided by Antoni Le´znickiform the Department of Biochemistry at Nicolaus Copernicus University who performed cloning of the gene based on Szerszen et al. [15]. The activity of IAGlc synthase was detected in the soluble protein fraction of the lysate. Recombinant maize IAGlc synthase was N-terminally His-tagged; thus, we purified the protein from the lysate by Ni2+ affinity chromatography using an ÄKTA start chromatography system. IAGlc synthase was eluted as two peaks with 77 mM and 170.5 mM imidazole. The purity of obtained fractions was determined by SDS-PAGE analysis with silver staining. As shown in Figure1a, an approximately 52 kDa-thick protein band which corresponds to His-tagged IAGlc synthase is present in the purified fraction. Western blot analysis with anti-polyHistidine antibodies (Figure1b) confirmed that the ~52 kDa protein band contains a His-tag. The purified IAGlc synthase fraction shows trace presence of protein contaminants; however, the purity of the enzyme preparation was sufficient for kinetic studies. Int. J. Mol. Sci. 2021, 22, 3355 3 of 23 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 24

Figure 1. SDS-PAGEFigure 1. analysis SDS-PAGE of IAGlc analysis synthase: of IAGlc M—molecular synthase: M—mo mass marker,lecular mass 1—lysate, marker, 2—puriﬁed 1—lysate, IAGlc 2—purified synthase fraction (a). WesternIAGlc blot analysis synthase of IAGlcfraction synthase: (a). Western M—molecular blot analysis mass of marker,IAGlc synthase: 1—puriﬁed M—molecular IAGlc synthase mass fraction marker, (b ). 1—purified IAGlc synthase fraction (b). 2.2. Kinetic Properties of Maize Recombinant IAGlc Synthase 2.2. Kinetic Properties2.2.1. Determination of Maize Recombinant of pH Effect IAGlc on Synthase IAGlc Synthase 2.2.1. DeterminationThe of effectpH Effect of pH on on IAGlc the IAGlc Synthase synthase activity was determined in the pH range from The effect5.8 of topH 8.8 on (pH the 5.8; IAGlc 6.2; synthase 6.6; 6.8; 7.0; acti 7.4;vity 7.6; was 8.0; determined 8.2; 8.6; 8.8) in as the shown pH range in Figure from2. The enzyme −1 −1 5.8 to 8.8 (pH 5.8;shows 6.2; high6.6; 6.8; activity 7.0; 7.4; (above 7.6; 8.0; 100 8.2; nmol 8.6; min 8.8) asmg shownprotein) in Figure in a 2. broad The pHenzyme range (pH 6.2–8.8). shows high activityCatalytic (above activity 100 nmol of IAGlc min− synthase1 mg−1 protein) visibly in decreasesa broad pH at range pH lower (pH 6.2–8.8). than 6.0. The highest Catalytic activityactivity of IAGlc was observed synthase at visibly pH 6.6 decreases and 7.6. The at pH optimal lower pH than for 6.0. native The IAGlc highest synthase activity activity was observedwas determined at pH 6.6 to and be 7.6. 7.1 byThe Michalczuk optimal pH and for native Bandurski IAGlc [27 synthase] and 7.3–7.6 activity by Le´znickiand was determinedBandurski to be 7.1 [ 12by], Michalczuk which is similar and Bandurski to the second [27] activityand 7.3–7.6 peak by of Le theźnicki recombinant and enzyme. Bandurski [12],Interestingly, which is simila nativer to IAGlc the second synthase activity exhibited peak noof activitythe recombinant at pH lower enzyme. than 6.7 [12], while Interestingly, thenative recombinant IAGlc synt enzymehase exhibited displays no the activity highest at activitypH lower at than pH 6.6 6.7 and [12], starts while losing catalytic the recombinantactivity enzyme at pH displays below the 6.0. highest This difference activity at could pH 6.6 be and a result starts of losing different catalytic folding conditions activity at pHoccurring below 6.0. in This bacterial difference and plantcould cellsbe a [result28]. UGT74D1, of different an folding IAA glycosyltransferase conditions from A. thaliana occurring in bacterial and, is plant also cells active [28]. in theUGT74D1, broad pH an spectrumIAA glycosyltransferase (pH 6.0–9.0) when from the A. HEPES (4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid) buffer is used, with the highest catalytic thaliana, is also active in the broad pH spectrum (pH 6.0–9.0) when the HEPES (4-(2-hy- activity at pH 6.0 and 7.0 [29]. Similar to maize IAGlc synthase, UGT74D1 exhibits rapid droxyethyl)-1-piperazineethanesulfonic acid) buffer is used, with the highest catalytic ac- decrease in activity at pH lower than 6.0. A. thaliana UGT75D1 was most active at pH 7.0 tivity at pH 6.0 and 7.0 [29]. Similar to maize IAGlc synthase, UGT74D1 exhibits rapid with most of the tested buffers; however, it also showed high activity at pH 6.0 and 9.0 [19]. decrease in activity at pH lower than 6.0. A. thaliana UGT75D1 was most active at pH 7.0 At pH 5.0 and 9.0, UGT75D1 exhibited no catalytic activity with most of the tested buffers. with most of the tested buffers; however, it also showed high activity at pH 6.0 and 9.0 Rice OsIAGT1 is also active at pH ranging from 6.0 to 9.0, with the highest activity at pH 8.0 [19]. At pH 5.0 and 9.0, UGT75D1 exhibited no catalytic activity with most of the tested and with the Tris buffer [22]. HEPES was not used for determination of the optimal pH of buffers. Rice OsIAGT1 is also active at pH ranging from 6.0 to 9.0, with the highest activity this enzyme. Catalytic activity of UGTs is often dependent on the buffer used for analysis. at pH 8.0 and with the Tris buffer [22]. HEPES was not used for determination of the op- As for native maize IAGlc synthase, the enzyme exhibited similar activity with both HEPES timal pH of this enzyme. Catalytic activity of UGTs is often dependent on the buffer used and Tris buffers; however, it was strongly inhibited by the 100 mM phosphate buffer [12]. for analysis. As for native maize IAGlc synthase, the enzyme exhibited similar activity UGTs are active in the broad pH spectrum and usually display the highest catalytic activity with both HEPESat pH and close Tris to 7.0,buff whichers; however, is consistent it was with strongly their cytoplasmic inhibited by localization the 100 mM [5, 26]. It should phosphate bufferbe noted [12]. UGTs that another are active IAA-conjugating in the broad pH enzyme, spectrum IAA–aspartate and usually synthetasedisplay the GH3 from pea highest catalyticseeds activity is also at localized pH close in to the 7.0, cytoplasm which is consistent [30]. with their cytoplasmic localization [5,26]. It should be noted that another IAA-conjugating enzyme, IAA–aspartate synthetase GH3 from pea seeds is also localized in the cytoplasm [30].

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Figure 2. Effect of pH on the IAGlc synthase activity. Enzyme activity was determined using [Figure14C]IAA 2. after Effect TLC of (thin pH layer on chromatography).the IAGlc synthase For thisactivi assay,ty. Enzyme 25 mM HEPES activity buffers was (pH determined 5.8; 6.2; using 6.6;[14C]IAA 6.8; 7.0; after 7.4; 7.6;TLC 8.0; (thin 8.2; layer 8.6 and chromatography). 8.8) were used. Catalytic For th activityis assay, was 25 expressedmM HEPES as the buffers means (pH± 5.8; 6.2; SD6.6; for 6.8; three 7.0; repetitions.7.4; 7.6; 8.0; 8.2; 8.6 and 8.8) were used. Catalytic activity was expressed as the means ± SD for three repetitions. 2.2.2. Determination of IAGlc Synthase Substrate Specificity 2.2.2.IAA Determination is the main natural of IAGlc auxin; Synthase however, Substrate other auxinic Specificity hormones are also present in plants and can form ester conjugates with glucose. As determined by Le´znickiand BandurskiIAA is [13 the], native main maize natural IAGlc auxin; synthase however, catalyzes other conjugation auxinic hormones of IAA to glucoseare also and present in doesplants not and utilize can oxidized form ester forms conjugates of IAA, oxindole-3-acetic with glucose. acidAs determined (OxIAA) and by 7-OH-OxIAA, Leźnicki and Ban- asdurski sugar [13], acceptors. native Kowalczyk maize IAGlc et al. synthase [31] analyzed catalyzes various conjugation non-indolic of auxins IAA andto glucose aromatic and does compoundsnot utilize oxidized as potential forms glucose of IAA, acceptors oxindole-3-acetic of native maize acid IAGlc (OxIAA) synthase. and Based7-OH-OxIAA, on as thesesugar results, acceptors. IAGlc Kowalczyk synthase effectively et al. [31] conjugated analyzed 1-naphtaleneacetic various non-indolic acid auxins (1-NAA) and and aromatic phenylaceticcompounds acid as potential (PAA) to theirglucose glucose acceptors conjugates of native (more thanmaize 70%, IAGlc and 30%synthase. of the relativeBased on these activityresults, inIAGlc comparison synthase to IAA, effectively respectively). conjugated 2,4-Dichlorophenoxyacetic 1-naphtaleneacetic acid acid (2,4-D) (1-NAA) was aand phe- poor acceptor of a glucosyl moiety. nylacetic acid (PAA) to their glucose conjugates (more than 70%, and 30% of the relative We have tested some natural auxins as well as synthetic auxinic herbicides as potential sugaractivity acceptors in comparison of maize to glucosyltransferase IAA, respectively). (Table 2,4-Dichlorophenoxyacetic1). Recombinant IAGlc synthase acid (2,4-D) was exhibitsa poor acceptor the highest of activitya glucosyl towards moiety. IAA; however, it can also utilize indole-3-butyric acid (IBA)We and have indole-3-propionic tested some natural acid (IPA) auxins as glucose as well acceptors, as synthetic though auxinic with lower herbicides activity as poten- (relativetial sugar activity acceptors is 66% of and maize 49.7%, glucosyltransferase respectively). Only (Table trace glucosyltransferase 1). Recombinant IAGlc activity synthase wasexhibits detected the highest towards activity indole-3-pyruvic towards IAA; acid (IPyA) howeve (relativer, it can activity also utilize of 2.76%). indole-3-butyric PAA and acid tryptophan,(IBA) and indole-3-propionic an amino acid which acid contains (IPA) the as indole glucose ring acceptors, characteristic though of IAA, with were lower not activity substrates(relative activity for IAGlc is synthase.66% and 49.7%, Moreover, respectively). IAGlc synthase Only showed trace glucosyltransferase no catalytic activity activity towards synthetic auxins, 2,4-D, Picloram and Dicamba. IAA is also a preferred substrate was detected towards indole-3-pyruvic acid (IPyA) (relative activity of 2.76%). PAA and of pea IAA glucosyltransferase; however, the enzyme can also glucosylate other auxins, IPAtryptophan, and 1-NAA, an amino with the acid relative which activity contains of 22% the and indole 24%, ring respectively characteristic [21]. A. of thaliana IAA, were not UGT84B1substrates has for similar IAGlc affinitysynthase. towards Moreover, IAA, IBA IAGlc and synthase IPA; however, showed IAA no is thecatalytic preferred activity to- substratewards synthetic of the enzyme auxins, [16 2,4-D,,32]. Another PicloramA. and thaliana Dicamba.UGT, UGT74E2, IAA is also strongly a preferred favors IBAsubstrate of aspea a substrate;IAA glucosyltransferase; however, it can also however, glucosylate, the enzyme with less can efficiency, also glucosylate IAA, 2,4-D, other NAA andauxins, IPA someand 1-NAA, other phytohormones with the relative [17]. Rice activity OsIAGT1 of 22% exhibits and high 24%, catalytic respectively activity [21]. towards A. thaliana IBA,UGT84B1 IPA and has IAA similar and lower affinity activity towards towards IAA, NAA IBA and and 2,4-D IPA; [22 ].however, The highest IAA preference is the preferred substrate of the enzyme [16,32]. Another A. thaliana UGT, UGT74E2, strongly favors IBA as a substrate; however, it can also glucosylate, with less efficiency, IAA, 2,4-D, NAA and some other phytohormones [17]. Rice OsIAGT1 exhibits high catalytic activity towards IBA, IPA and IAA and lower activity towards NAA and 2,4-D [22]. The highest preference towards IBA is also characteristic of A. thaliana UGT74D1 [29]. This enzyme can also quite

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towards IBA is also characteristic of A. thaliana UGT74D1 [29]. This enzyme can also quite efﬁciently glucosylate other auxins, such as IAA, IPA, NAA and, less effectively, 2,4-D. Additional studies revealed that UGT74D1 can also utilize OxIAA as a glucose acceptor and has higher afﬁnity to this substrate compared to IAA [18].

Table 1. Substrate speciﬁcity of recombinant IAGlc synthase. Enzyme activity was determined using the spectrophotometric method. Relative activity (%) was calculated taking the IAGlc synthase activity for IAA (nmol min−1 mg−1 protein) as 100%. IAA—indole-3-acetic acid; IBA—indole-3- butyric acid; IPA—indole-3-propionic acid; IPyA—indole-3-pyruvic acid; PAA—phenylacetic acid; 2,4-D—2,4-dichlorophenoxyacetic acid.

IAGlc Synthase Substrate Relative Activity (%) (nmol min−1 mg−1 protein) IAA 210.46 ± 0.5 100 IBA 129.53 ± 1.04 66 IPA 104.65 ± 3.8 49.7 IPyA 5.81 ± 0.2 2.76 PAA 0 0 2,4-D 0 0 Dicamba 0 0 Picloram 0 0 Tryptophan 0 0

Even though most auxinic UGTs have a relatively broad substrate speciﬁcity and, like maize IAGlc synthase, can utilize more than one glucose acceptor, there are enzymes which glucosylate only one type of auxin. A. thaliana UGT76F1 catalyzes conjugation of IPyA, but not of IAA and IBA, despite the latter two being the dominant forms of auxin present in the plant [20]. Moreover, A. thaliana UGT75D1 shows only trace activity towards IAA, IPA and NAA, glucosylating almost exclusively IBA [19].

2.2.3. Determination of Kinetic Parameters of IAGlc Synthase Maize IAGlc synthase displays the Michaelis–Menten hyperbolic kinetic characteristic for isosteric enzymes (Figure3a,c). Le´znickiand Bandurski [ 12] failed to determine KM for UDPG of native maize IAGlc synthase as the enzyme showed nonlinear kinetics. The authors calculated approximate affinity of the synthase to be 14 mM and postulated that nonlinear kinetics is an effect of the absence of the potential cofactor. Later, it was revealed that IAGlc synthase requires presence of divalent cations which were not used for KM determination [13]. Thus, the native IAGlc synthase KM for IAA, which was determined to be 1 mM in the absence of divalent cations [12], is also questionable. However, it is very similar to KM for IAA of the recombinant enzyme, which is 0.8 ± 0.1 mM (Table2) as determined using a Hanes–Woolf plot (Figure3b). Similar values of KM for IAA suggest that divalent cations are not required for binding of the sugar acceptor but only for binding of the sugar donor. KM for UDPG determined using a Hanes–Woolf plot (Figure3d) for the recombinant enzyme in the presence of Mg2+ was 0.7 ± 0.09 mM (Table2), which indicates that divalent cations increase affinity of the IAGlc synthase for UDPG. Other UGTs which prefer IAA as a substrate have similar KM values to IAGlc synthase, that is 0.52 mM for IAA glucosyltransferase from pea [21] and 0.24 mM for A. thaliana UGT84B1 [16]. However, pea IAA glucosyltransferase has KM = 2.67 mM for UDPG [21], which indicates lower affinity for the glucose donor compared to maize IAGlc synthase. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 24

Table 2. Kinetic parameters of recombinant maize IAGlc synthase.

Vmax Substrate Cosubstrate KM (mM) (nmol min−1 mg−1 protein) IAA 4 mM UDPG 0.8 ± 0.1 519 ± 27

Int. J. Mol. Sci. 2021, 22, 3355 UDPG 3 mM IAA 0.7 ± 0.09 243 ±6 18 of 23 The KM and Vmax parameters were calculated using a Hanes–Woolf plot and confirmed by the Michaelis–Menten equation. All values are expressed as the means ± SD (n = 3).

Figure 3. 3.Determination Determination of of kinetic kinetic parameters parameters of IAGlc of IAGlc synthase. synthase. Effect Effect of IAA of concentrationIAA concentration on on thethe IAGlcIAGlc synthase synthase activity: activity: Michaelis–Menten Michaelis–Menten plot plot (a) and (a) Hanes–Woolfand Hanes–Woolf plot (b ).plot Effect (b). of Effect UDPG of UDPG concentrationconcentration on on the the IAGlc IAGlc synthase synthase activity: activity: Michaelis–Menten Michaelis–Menten plot (c plot) and ( Hanes–Woolfc) and Hanes–Woolf plot (d). plot (d). The KM and Vmax values were calculated using Hanes–Woolf plots (IAA/v = f(IAA) and UDPG/v = The KM and Vmax values were calculated using Hanes–Woolf plots (IAA/v = f(IAA) and UDPG/v = f(UDPG), respectively). f(UDPG), respectively).

Table2.2.4. 2. EffectKinetic of parameters Divalent of Cations recombinant on IAGlc maize IAGlcSynthase synthase. UGT activity can be modulated by a number of different compounds. Maize IAGlc Vmax −1 −1 synthaseSubstrate requires presence Cosubstrate of divalent cations KtoM display(mM) its catalytic(nmol min activitymg [13]. The recombinant enzyme shows no activity if divalent cations are not presentprotein) in the reaction mixture IAAor when a chelating 4 mM agent, UDPG EDTA, is added 0.8 ± 0.1 (Figure 4). The 519highest± 27 activity of the enzymeUDPG was observed in the 3 mM presence IAA of Mn2+ ions 0.7 ± and0.09 a little less active 243 ± when18 the reaction 2+ 2+ mixtureThe KM and containedVmax parameters Ca and were calculatedMg ; this using is in aHanes–Woolf agreement plot with and the conﬁrmed native by enzyme the properties Michaelis–Menten equation. All values are expressed as the means ± SD (n = 3). [13].

2.2.4. Effect of Divalent Cations on IAGlc Synthase UGT activity can be modulated by a number of different compounds. Maize IAGlc synthase requires presence of divalent cations to display its catalytic activity [13]. The recombinant enzyme shows no activity if divalent cations are not present in the reaction mixture or when a chelating agent, EDTA, is added (Figure4). The highest activity of the enzyme was observed in the presence of Mn2+ ions and a little less active when the reaction mixture contained Ca2+ and Mg2+; this is in agreement with the native enzyme properties [13].

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14 FigureFigure 4. Effect 4. of Effect divalent of cationsdivalent on IAGlccations synthase. on IAGlc Enzyme synthase. activity wasEnzyme determined activity using was [ C]IAAdetermined after TLC using in the presence of 5 mM MgCl2, 5 mM CaCl2, 5 mM MnCl2 or 5 mM EDTA; the control mixture did not contain any divalent [14C]IAA after TLC in the presence of 5 mM MgCl2, 5 mM CaCl2, 5 mM MnCl2 or 5 mM EDTA; the cations. Catalytic activity was expressed as the means ± SD for three repetitions. control mixture did not contain any divalent cations. Catalytic activity was expressed as the means ± SD for three repetitions. 2.2.5. Effect of Modulators on IAGlc Synthase 2.2.5. Effect of ModulatorsSome on compounds IAGlc Synthase act as activators of recombinant maize IAGlc synthase (Figure5). Some compoundsAn amide act IAAas activators conjugate, of IAA–Asp, recombinant affected maize activity IAGlc of the synthase enzyme in(Figure a concentration- 5). dependent manner. IAA–Asp in the amount of 0.1 mM increased catalytic activity of IAGlc An amide IAA conjugate,synthase approximately IAA–Asp, affected twofold, activity while in of the the presence enzyme of 1in mM a concentration- conjugate, the activity dependent manner.of the IAA–Asp enzyme increasedin the amount 3.5-fold. of The 0.1 activating mM increased effect of catalytic IAA–Asp activity may have of deeper IAGlc synthase approximatelyimplications. IAA–Asp twofold, is while synthesized in the by presence amido synthetases of 1 mM conjugate, from the GH3 the family ac- (the tivity of the enzymeexpression increased thereof 3.5-fold. is auxin-inducible) The activating [8]. Thus,effect an of elevatedIAA–Asp level may of IAA–Asp have deeper signalizes a implications. IAA–Asphigh level is synthesized of endogenous by IAA amido and synthetases may indicate from a need the in aGH3 more family effective (the reduction ex- of pression thereof isthe auxin-inducible) free auxin pool, for [8]. example, Thus, an by itselevated ester conjugation. level of IAA–Asp IAGlc synthase signalizes activation a by IAA–Asp may act as the means of better control of auxin homeostasis. For verification high level of endogenous IAA and may indicate a need in a more effective reduction of of this hypothesis, we studied the effect of 0.1 mM IAA–Asp on the Vmax value and the free auxin pool,affinity for ofexample, IAGlc synthase by its foreste IAAr conjugation. (defined by the IAGlcKM value). synthase Surprisingly, activation kinetic by studies IAA–Asp may actrevealed as the means that IAA–Asp of better enhanced controlV ofmax auxinabout homeostasis. 1.6-fold, but also For increased verification the K ofM value this hypothesis, we(approximately studied the 2.4-fold). effect of This 0.1 findingmM IAA–Asp may suggest on thatthe IAA–AspVmax value competes and affinity with IAA for the catalytic site of IAGlc synthase and reduces affinity of the enzyme for IAA. Considering of IAGlc synthase for IAA (defined by the KM value). Surprisingly, kinetic studies revealed that IAGlc synthase catalyzes a two-substrate reaction, IAA–Asp might potentiate catalytic that IAA–Asp enhanced Vmax about 1.6-fold, but also increased the KM value (approxi- efficiency by an unknown mechanism. mately 2.4-fold). This finding may suggest that IAA–Asp competes with IAA for the catalytic site of IAGlc synthase and reduces affinity of the enzyme for IAA. Considering that IAGlc synthase catalyzes a two-substrate reaction, IAA–Asp might potentiate catalytic efficiency by an unknown mechanism.

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FigureFigure 5.5.Effect Effect ofof modulators modulators on on IAGlc IAGlc synthase. synthase. Enzyme Enzyme activity activity was was determined determined using using [ 14[14C]IAAC]IAA afterafter TLC;TLC; thethe controlcontrol mixture mixture did did not not contain contain any any modulators. modulators. Catalytic Catalytic activity activity was was expressed expressed as as thethe meansmeans± ± SDSD forforthree three repetitions. repetitions. ATP—adenosine ATP—adenosine triphosphate; triphosphate;β β-ME—-ME—ββ-mercaptoethanol;-mercaptoethanol; GSSG—oxidized glutathione; GSH—reduced glutathione; Glc-1-P—glucose-1-phosphate; PPi—py- GSSG—oxidized glutathione; GSH—reduced glutathione; Glc-1-P—glucose-1-phosphate; PP — rophosphate; IAA–Asp—indole-3-acetyl-aspartate. i pyrophosphate; IAA–Asp—indole-3-acetyl-aspartate.

TheThe activatingactivating effecteffect onon recombinantrecombinant IAGlcIAGlc synthasesynthase isis alsoalsodisplayed displayed byby oxidized oxidized glutathioneglutathione (GSSG) (GSSG) at at 10 10 mM mM concentration, concentration, which which activated activated the the enzyme enzyme fourfold fourfold (Figure (Figure5 ). Interestingly,5). Interestingly, reduced reduced glutathione glutathione (GSH) (GSH) at the at samethe same concentration concentration is an is inhibitor an inhibitor of the of enzymethe enzyme (relative (relative activity activity of 72%). of 72%). In theIn the study study on on the the native native enzyme, enzyme, GSH GSH acted acted as as an an activatoractivator ofof thethe synthasesynthase [[13].13]. MaizeMaize IAGlcIAGlc synthasesynthase containscontains eighteight cysteinecysteine residuesresidues inin thethe aminoamino acidacid sequence.sequence. AnalysisAnalysis performedperformed withwith thethe DiANNADiANNA (DiAminoacid(DiAminoacid NeuralNeural NetworkNetwork Application)Application) 1.11.1 webweb serverserver revealedrevealed thatthat Cys103Cys103 isis highlyhighly likelylikely toto formform aa disulﬁdedisulfide bondbond withwith Cys360.Cys360. TheThe recombinant recombinant enzyme enzyme obtained obtained in bacterialin bacteria cellsl cells can can exhibit exhibit some some structural structural differences differ- fromences the from native the native protein protein synthesized synthesized in plant in cells plant caused cells caused by disturbance by disturbance in disulﬁde in disulfide bond formationbond formation [33]. The [33]. further The evidencefurther evidence that the expressionthat the expression host may affecthost may protein affect properties protein isproperties that β-mercaptoethanol is that β-mercaptoethanol (β-ME), another (β-ME), reducing another agent, reducing has noagent, effect has on no recombinant effect on re- IAGlccombinant synthase IAGlc (Figure synthase5) but (Figure is an activator 5) but is ofan theactivator native of enzyme the native [13]. enzyme [13]. OfOf thethe testedtested compounds,compounds, threethree havehave aa completely completely inactivatedinactivated recombinantrecombinant IAGlcIAGlc synthase:synthase: 1 mM ATP, 44 mMmM pyrophosphate (PP ii)) and and 4 4 mM mM glucose-1-phosphate glucose-1-phosphate (Glc-1- (Glc- 1-P)P) (Figure (Figure 5).5). This is in agreementagreement withwith thethe inhibitoryinhibitory effecteffect of of phosphate phosphate compounds, compounds, suchsuch asas PPPPii, phosphate phosphate esters esters and and phospholipids, phospholipids, re reportedported before before and and with with inhibition inhibition by bythe the phosphate phosphate buffer buffer [13]. [13 On]. On the the contrary, contrary, A. A.thaliana thaliana UGT84B1UGT84B1 was was not not inhibited inhibited by byphospholipids phospholipids [16]. [16 ].Interestingly, Interestingly, when when effect effect of of different different concentrations ofof glucose-1-glucose-1- phosphatephosphate onon recombinantrecombinant maizemaize IAGlcIAGlc synthasesynthase waswas tested,tested, atat lower lower concentrations, concentrations, itit hadhad an an activating activating effect effect on on the the enzyme enzyme (Figure (Figure6). IAGlc6). IAGlc synthase synthase activity activity increased increased rapidly rap- inidly the in presence the presence of 2.5 of mM 2.5 mM glucose-1-phosphate, glucose-1-phosphate, but atbut 3 mM,at 3 mM, it was it was on aon similar a similar level level to theto the control. control. As As mentioned mentioned above, above, a highera higher 4 mM4 mM concentration concentration of of glucose-1-phosphate glucose-1-phosphate completelycompletely abolishedabolished IAGlcIAGlc activity.activity. It It is is well-known well-known that that glucose-1-phosphate glucose-1-phosphate is is not not a adonor donor of of a aglucosyl glucosyl moiety moiety for for glucosyltransferase glucosyltransferase activity, activity, but but is involved is involved in UDPG in UDPG bio- biosynthesissynthesis according according to to the the reaction reaction cataly catalyzedzed by by UDPG pyrophosphorylase: glucose-1-glucose-1- phosphatephosphate ++ UTPUTP ↔↔ UDPGUDPG ++ PPPPii.. InIn thisthis study,study, wewe analyzedanalyzed enzymeenzyme activityactivity of of UDPG UDPG

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pyrophosphorylase in the purified fraction of IAGlc synthase. No UDPG-synthesizing ac- pyrophosphorylase in the purified fraction of IAGlc synthase. No UDPG-synthesizing ac- pyrophosphorylasetivity was observed in(data the not puriﬁed shown). fraction A simila ofr IAGlc effect synthase.was also observed No UDPG-synthesizing for ATP, though tivity was observed (data not shown). A similar effect was also observed for ATP, though activityits activating was observed effect was (data more not prominent shown). A similarin a wider effect concentration was also observed range for(Figure ATP, though7). ATP its activating effect was more prominent in a wider concentration range (Figure 7). ATP itsactivated activating IAGlc effect synthase was more at 0.1–0.5 prominent mM concentr in a widerations concentration with the activity range peak (Figure observed7). ATP in activated IAGlc synthase at 0.1–0.5 mM concentrations with the activity peak observed in activatedthe presence IAGlc of synthase0.3 mM ATP. at 0.1–0.5 When mM the concentrationsconcentration used with was the activity0.6 mM peakor higher, observed ATP the presence of 0.3 mM ATP. When the concentration used was 0.6 mM or higher, ATP incompletely the presence abolished of 0.3 the mM IAGlc ATP. synthase When the acti concentrationvity. UGTs utilize used wasUDP 0.6glucose mM oras higher,a sugar completely abolished the IAGlc synthase activity. UGTs utilize UDP glucose as a sugar ATPdonor, completely therefore abolishedwe propose the that IAGlc at higher synthase concentrations, activity. UGTs ATP utilize competes UDP with glucose the nucle- as a donor, therefore we propose that at higher concentrations, ATP competes with the nucle- sugarotide donor,moiety thereforeof UDPG wefor proposebinding thatto the at PSPG higher motif concentrations, of the enzyme. ATP IAGlc competes synthase with may the otide moiety of UDPG for binding to the PSPG motif of the enzyme. IAGlc synthase may nucleotidepossess two moiety binding of UDPGsites for for ATP; binding one toof thethem PSPG is specific, motif of to the which enzyme. ATP IAGlc binds synthaseand acti- possess two binding sites for ATP; one of them is specific, to which ATP binds and acti- mayvates possess the enzyme, two binding and the sites PSPG for motif, ATP; onewhere of themATP binds is speciﬁc, as a competitive to which ATP inhibitor. binds and On vates the enzyme, and the PSPG motif, where ATP binds as a competitive inhibitor. On activatesthe other the hand, enzyme, it cannot and the be PSPGexcluded motif, that where IAGlc ATP synthase binds as has a competitive one binding inhibitor. site of OntheUDPG/ATP theother other hand, and hand, higherit itcannot cannot concentrations be be excluded excluded of thatATP that IAGlcbl IAGlcock thissynthase synthase site and has has reduce one bindingthe catalytic sitesite ac- ofof UDPG/ATPUDPG/ATPtivity. It is possible and and higher higher that theconcentrations concentrations mechanism of of IAATP ATPGlc bl synthase blockock this this siteactivity site and and modulationreduce reduce the the catalytic by catalytic glucose- ac- activity.tivity.1-phosphate ItIt is is possible possible is similar that that to the thethat mechanism mechanism of ATP, as of both of IA IAGlcGlc glucose synthase synthase and phosphateactivity activity modulation are present by in glucose- UDPG. 1-phosphate1-phosphateThus, high glucose-1-phosphate isis similarsimilar toto thatthat ofof ATP,ATP, concentration asas bothboth glucoseglucose may andand block phosphatephosphate binding are areof presentpresentUDPG intoin UDPG.UDPG.the en- Thus,Thus,zyme. high high glucose-1-phosphate glucose-1-phosphate concentration concentration may may block block binding binding of UDPG of UDPG to the to enzyme. the enzyme.

FigureFigure 6.6.Effect Effect ofof glucose-1-phosphate glucose-1-phosphate (Glc-1-P) (Glc-1-P) on on IAGlc IAGlc synthase. synthase. The The following following concentrations concentrations Figure 6. Effect of glucose-1-phosphate (Glc-1-P) on IAGlc synthase. The following concentrations ofof Glc-1-PGlc-1-P werewere usedused forfor thisthis assay:assay: 0;0; 0.5;0.5; 1.0;1.0; 1.5;1.5; 2.0;2.0; 2.5;2.5; 3.03.0 andand 4.04.0mM. mM. Enzyme Enzyme activity activitywas was ofdetermined Glc-1-P were using used [14C]IAA for this after assay: TLC. 0; Catalytic 0.5; 1.0; 1.5;activity 2.0; 2.5;was 3.0expressed and 4.0 asmM. the Enzymemeans ± activitySD for three was determined using [14C]IAA after TLC. Catalytic activity was expressed as the means ± SD for three determinedrepetitions. using [14C]IAA after TLC. Catalytic activity was expressed as the means ± SD for three repetitions.repetitions.

Figure 7. Effect of ATP on IAGlc synthase. The following concentrations of ATP were used for this FigureFigureassay: 7.0;7. Effect0.1;Effect 0.3; ofof 0.4; ATPATP 0.5;on on 0.6;IAGlc IAGlc 0.8 andsynthase. synthase. 1.0 mM. TheThe Enzyme followingfollowing activity concentrationsconcentrations was determined ofof ATPATP usingwere were [used 14usedC]IAA for for thisafter this 14 assay:assay:TLC. Catalytic 0;0; 0.1;0.1; 0.3;0.3; activity 0.4;0.4; 0.5;0.5; wa 0.6;0.6;s expressed 0.80.8 andand 1.01.0 as mM. mM. the Enzymemeans Enzyme ± activity SDactivity for three was was determined repetitions.determined using using [ 14[ C]IAAC]IAA afterafter TLC. Catalytic activity was expressed as the means ± SD for three repetitions. TLC. Catalytic activity was expressed as the means ± SD for three repetitions.

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Int. J. Mol. Sci. 2021, 22, 3355 10 of 23 2.2.6. Effect of Phytohormones on IAGlc Synthase The IAA glucosyltransferase activity may also be modulated by other phytohor- 2.2.6.mones. Effect Pea of IAA Phytohormones glucosyltransferase on IAGlc Synthase is inhibited by IBA, which is not a substrate of this enzymeThe IAA but glucosyltransferaseonly acts as a competitive activity may inhibitor also be modulated[21]. Native by maize other phytohormones. IAGlc synthase is also Peainhibited IAA glucosyltransferase by some phytohormones: is inhibited zeatin, by IBA, 2,4-D, which gibberellic is not a substrate acid (GA), of this abscisic enzyme acid and butkinetin only acts[13]. as However, a competitive cytokinin inhibitor had [21]. no Native inhibitory maize IAGlc effect synthase on A. isthaliana also inhibited UGT84B1 [16]. by some phytohormones: zeatin, 2,4-D, gibberellic acid (GA), abscisic acid and kinetin [13]. Maize IAGlc synthase was also inhibited by IAA conjugates: indole-3-acetyl-myo-inositol However, cytokinin had no inhibitory effect on A. thaliana UGT84B1 [16]. Maize IAGlc synthaseand IAA- wasβ-1,4-glucan. also inhibited As by IAGlc IAA conjugates:synthase is indole-3-acetyl-myo-inositol the first enzyme of the IAA and ester IAA- conjugationβ- 1,4-glucan.pathway, Asit is IAGlc inhibited synthase by the is the final first products enzyme of of the auxin IAA conjugation. ester conjugation As the pathway, phytohormonal it iseffect inhibited on IAA by the glucosyltransferases final products of auxin is conjugation.evident, we As tested the phytohormonal some plant hormones effect on IAA as potential glucosyltransferasesrecombinant IAGlc is evident,synthase we inhibitors tested some (Figure plant hormones8). We confirmed as potential that recombinant GA3 and kinetin IAGlchave synthasea slight inhibito inhibitorsry (Figure effect 8on). Wethe confirmed enzyme, thatwhile GA 2,4-D3 and strongly kinetin havedecreases a slight the IAGlc inhibitorysynthase effectactivity. on the enzyme, while 2,4-D strongly decreases the IAGlc synthase activity.

FigureFigure 8. 8.Effect Effect of of phytohormones phytohormones on on IAGlc IAGlc synthase. synthase. Phytohormones Phytohormones that that are are not not utilized utilized by by IAGlc IAGlcsynthase synthase as substrates as substrates were were used used for forthis this assay. assay. Enzyme Enzyme activity activity was was determined determined using [14C]IAA [14afterC]IAA TLC; after the TLC; control the controlmixture mixture did not did contain not contain any additional any additional phytoh phytohormones.ormones. Catalytic Catalytic activity was activityexpressed was as expressed the means as the ± SD means for ±threeSD forrepetition three repetitions.s. PAA–phenylacetic PAA–phenylacetic acid; acid;2,4-D—2,4-dichlorophe- 2,4-D—2,4-

dichlorophenoxyaceticnoxyacetic acid; GA3 acid;—gibberellic GA3—gibberellic acid. acid.

WeWe also also tested tested effect effect of PAA of PAA and auxinicand auxinic herbicides herbicides Dicamba Dicamba and Picloram and Picloram on the on the enzyme.enzyme. While While PAA PAA and and Picloram Picloram slightly slightly activated activated IAGlc IAGlc synthase, synthase, Dicamba Dicamba turned turned out out to be another inhibitor of the enzyme. As 2,4-D exhibited the strongest inhibitory to be another inhibitor of the enzyme. As 2,4-D exhibited the strongest inhibitory effect on effect on IAGlc synthase, we investigated its effect in a concentration-dependent manner inIAGlc the presence synthase, of 1.95 we mMinvestigated and 3 mM its IAA effect (Figure in 9a). concentration-dependent 2,4-D had a negative effect onmanner the in the IAGlcpresence synthase of 1.95 activity mM atand all 3 tested mM concentrations,IAA (Figure 9). with 2,4-D the had rapid a increasenegative in effect inhibition on the IAGlc abovesynthase the 250activityµM concentration.at all tested concentrations The 2,4-D IC50, with(50% the inhibition) rapid increase values for in 1.95inhibition mM above andthe 3250 mM μM IAA concentration. were 439 and 350Theµ 2,4-DM, respectively IC50 (50% (Tableinhibition)3). We values determined for 1.95 that mM 2,4-D and 3 mM displaysIAA were a competitive 439 and 350 type μ ofM, inhibition respectively as it is(Table structurally 3). We similar determined to IAA andthat causes 2,4-D an displays a approximatelycompetitive type 1.5-fold of inhibition decrease in as afﬁnity it is structurally of the enzyme similar towards to IAA IAA (Table and3 causes). an approximately 1.5-fold decrease in affinity of the enzyme towards IAA (Table 3).

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FigureFigure 9. 9.Effect Effect of of 2,4-D 2,4-D (2,4-dichlorophenoxyacetic (2,4-dichlorophenoxyacetic acid) acid) on on IAGlc IAGlc synthase. synthase. The The assay assay was was per- per- formedformed in in the the presence presence of of 1.95 1.95 or or 3.0 3.0 mM mM IAA. IAA. Inhibition Inhibition (%) (%) was was expressed expressed as as the the means means± ±SD SD for for threethree repetitions. repetitions. The The following following concentrations concentrations of of 2,4-D 2,4-D were were used used for for this this assay: assay: 0; 0; 75; 75; 250; 250; 375; 375; 500 500 and 675 μM. Enzyme activity was determined using [14C]IAA after TLC. and 675 µM. Enzyme activity was determined using [14C]IAA after TLC.

Table 3. Inhibition of IAGlc synthase by 2,4-dichlorophenoxyacetic acid (2,4-D). Table 3. Inhibition of IAGlc synthase by 2,4-dichlorophenoxyacetic acid (2,4-D). Inhibition InhibitorInhibitor Inhibition TypeKi (μM) K (µM)IC50 (μM) IC (µM) KMKappapp (mM)(mM) Type i 50 M 350 ± 28 350 ± 28 1.22 ± 0.08 (for 3 mM IAA) Competitive (1.53-fold 2,4-D Competitive 117 ± 10(for 3 mM IAA)439 ± 33 1.22 ± 0.08 (1.53-fold 2,4-D (IAA) 117 ± 10 decrease of (IAA) 439 ± 33(for 1.95 mM decrease of affinity) afﬁnity) (for 1.95 mM IAA)IAA)

TheThe inhibition inhibition type type of of the the enzymatic enzymatic reaction reaction was was determined determined through through the the graphical graphical method using a Dixon plot. The inhibition constant (Ki) value was calculated using a method using a Dixon plot. The inhibition constant (Ki) value was calculated using a Dixon Dixon plot. The IC50 parameter defines an inhibitor concentration that causes 50% inhibi- plot. The IC50 parameter deﬁnes an inhibitor concentration that causes 50% inhibition of thetion enzyme of the enzyme activity andactivity was and calculated was calculat throughed thethrough graphical the graphical method. method. The resulting The result- data ing data were fit into the following equation:app KMapp = KM (1+ [I]/Ki). All the values were were ﬁt into the following equation: KM = KM (1 + [I]/Ki). All the values were expressed asexpressed the means as± theSD means (n = 3). ± SD (n = 3).

2.3.2.3. Sequence Sequence Analysis, Analysis,3D 3D S Structuretructure Prediction Prediction and and Molecular Molecular Docking Docking Studies Studies of of Maize Maize IAGlc IAGlc SynthaseSynthase UsingUsing Clustal Clustal Omega, Omega, we we aligned aligned IAGlc IAGlc synthase synthase with with other other IAA IAA glucosyltransferases glucosyltransfer- (Figureases (Figure 10). Clustal 10). Clustal Omega Omega analysis analysis revealed revealed that maize that IAGlc maize synthase IAGlc synthase (ZmIAGLU) (ZmIAGLU) shows ashows 69.35% a identity69.35% identity with OsIAGT1 with OsIAGT1 (Table4). (Table Both UGTs4). Both are UGTs from are monocotyledonous from monocotyledonous plants andplants utilize and IAA utilize and IAA IBA and as glucose IBA as acceptorsglucose acceptors [22], hence [22], high hence sequence high sequence identity. identity. Maize IAGlcMaize synthase IAGlc synthase shows a deﬁnitelyshows a definitely lower identity lower to dicotyledonousidentity to dicotyledonousA. thaliana UGTs. A. thaliana The lowestUGTs. sequence The lowest identity sequence (27.55%) identity is shared (27.55%) with is UGT76F1, shared with which UGT76F1, catalyzes which glucosylation catalyzes ofglucosylation IPyA, an IAA of precursor, IPyA, an but IAA does precursor, not utilize but IAA does or not IBA utilize as substrates IAA or [ 20IBA]. Maizeas substrates IAGlc synthase[20]. Maize catalyzes IAGlc glucosylation synthase catalyzes of IAA, glucosylation IBA and IPA with of IAA, a higher IBA preferenceand IPA with towards a higher the ﬁrstpreference auxin (Table towards1). Other the firstA. thalianaauxin (TableUGTs, 1). UGT74D1, Other A. UGT74E2,thaliana UGTs, UGT75D1 UGT74D1, and UGT84B1, UGT74E2, similarUGT75D1 to maize and UGT84B1, IAGlc synthase, similar utilizeto maize both IAGlc IAA synthase, and IBA utilize as substrates both IAA [17 and,19,29 IBA,32 ],as hence higher sequence identity, which is 39.55%, 40.41%, 35.27% and 34.10%, respectively substrates [17,19,29,32], hence higher sequence identity, which is 39.55%, 40.41%, 35.27% (Table4). and 34.10%, respectively (Table 4). The PSPG (plant secondary product glycosyltransferase) motif is the most conserved part of the GT sequence [4,34]. The identity of ZmIAGLU and OsIAGT1 PSPG motifs is 88.64% (Table 4). Even with the least similar UGT76F1, maize IAGlc synthase has a 59.09%

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Figure 10. Sequence alignment of IAA glucosyltransferases: maize IAGlc synthase (ZmIAGLU, Figure 10. Sequence alignment of IAA glucosyltransferases: maize IAGlc synthase (ZmIAGLU, NCBI Reference Sequence Database, accession number NP_001105326.1), rice OsIAGT1 (GenBank, NCBI Reference Sequence Database, accession number NP_001105326.1), rice OsIAGT1 (GenBank, accessionaccession number number BAS85861.1) BAS85861.1) and and A. thalianaA. thaliana glucosyltransferasesglucosyltransferases UGT74D1 UGT74D1 (UniProtKB/Swiss- (UniProtKB/Swiss- Prot,Prot, accession accession number number Q9SKC5.1), Q9SKC5.1), UGT74E2 UGT74E2 (GenBank, (GenBank, accession accession number number ABJ17124.1), ABJ17124.1), UGT75D1 UGT75D1 (UniProtKB/Swiss-Prot,(UniProtKB/Swiss-Prot, accession accession number number O23406.2) O23406.2),, UGT76F1 UGT76F1 (UniProtKB/Swiss-Prot, (UniProtKB/Swiss-Prot, accession accession numbernumber Q9M051.1) Q9M051.1) and and UGT84B1 UGT84B1 (GenBank, (GenBank, access accessionion number number AAO63432.1). AAO63432.1). The red The box red indicates box indicates thethe PSPG PSPG motif. motif. The The sequ sequenceence alignment alignment was wasgenerated generated using usingClustal Clustal Omega. Omega. ‘*’—positions with a single, fully conserved residue; ‘:’—conservation between groups of strongly similar properties, scoring > 0.5 in the Gonnet PAM (Percent Accepted Mutation) 250 matrix; ‘.’—conservation between groups of weakly similar properties, scoring ≤ 0.5 in the Gonnet PAM 250 matrix. Int. J. Mol. Sci. 2021, 22, 3355 13 of 23

Table 4. Sequence identities of IAA glucosyltransferases. The sequence identities were calculated using Clustal Omega.

Sequence Identity with PSPG Motif Identity with UGT Maize IAGlc Synthase Maize IAGlc Synthase UGT76F1 27.55% 59.09% UGT84B1 34.10% 61.36% UGT75D1 35.27% 65.91% UGT74D1 39.82% 65.91% UGT74E2 40.41% 75.00% OsIAGT1 69.35% 88.64%

The PSPG (plant secondary product glycosyltransferase) motif is the most conserved part of the GT sequence [4,34]. The identity of ZmIAGLU and OsIAGT1 PSPG motifs is 88.64% (Table4). Even with the least similar UGT76F1, maize IAGlc synthase has a 59.09% identity when only the PSPG motif is concerned. The PSPG motif consists of 44 amino acid residues that participate in binding of the sugar nucleotide substrate. The most conserved residues of the PSPG motif are Trp (W1 and 22), Gln (Q4 and 44), Leu (L8), His (H10 and 19), Cys (C20), Gly (G21), Asn (N23), Ser (S24), Glu (E27), Pro (P39) and Glu/Asp (E/D43) (numbers in the brackets indicate the amino acid position in the PSPG motif). Those residues are conserved in all analyzed IAA UGTs, with the exception of proline P39, which in maize IAGlc synthase is substituted with alanine. The last residue of the PSPG motif is crucial for sugar donor specificity of GTs. GTs which utilize glucose nucleotides have glutamine in this position (Q44), while activity required for recognition of the galactose moiety is determined by histidine (H44) [35]. Substitution of Q44 by the His residue caused Vitis vinifera UGT78A12 to switch activity form glucosyltransferase to galactosyltransferase. All IAA UGTs catalyze formation of the IAA–glucose conjugate; thus, they have glutamine as the last residue of the PSPG motif. Moreover, activity analysis of native maize IAGlc synthase revealed that the enzyme is strictly specific for UDP glucose and shows no catalytic activity towards UDP galactose or UDP xylose [13]. Homology modeling of IAGlc synthase was carried out on the iterative threading assembly refinement (I-TASSER) server (Figure 11). For the obtained model, the root-mean- square deviation (RMSD) and the TM-score (index for assessing the topological similarity of model and template) were 5.5 ± 3.5 Å and 0.82 ± 0.09, respectively. The C-score, which gives the confidence for the quality of the predicted models (C-score is typically in the range of (–5, 2)), was estimated to be 0.77. This 3D model of IAGlc synthase was used in molecular docking. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 14 of 24

Homology modeling of IAGlc synthase was carried out on the iterative threading assembly refinement (I-TASSER) server (Figure 11). For the obtained model, the root- mean-square deviation (RMSD) and the TM-score (index for assessing the topological similarity of model and template) were 5.5 ± 3.5 Å and 0.82 ± 0.09, respectively. The C-score,

Int. J. Mol. Sci. 2021, 22, 3355 which gives the confidence for the quality of the predicted models (C-score is 14typically of 23 in the range of (–5, 2)), was estimated to be 0.77. This 3D model of IAGlc synthase was used in molecular docking.

FigureFigure 11. 11. Three-dimensionalThree-dimensional structure structure of IAGlc of synthaseIAGlc wassyntha modeledse was with themodeled I-TASSER with server the (https://zhanglab. I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TAccmb.med.umich.edu/I-TASSER/, accessed onSSER/, 20 February accessed 2021) on 20 [36 February–39]. The sequence-based 2021) [36–39]. predictionThe sequence-based of the secondary prediction of thestructure secondary using structure I-TASSER/PSSpred. using I-TASSER/PSSpred. The helix is marked The in helix red, theis marked strand is in blue, red, and the the strand coil is is black blue, (a ).and Predicted the coil 3D is black (a). Predictedmodel ofIAGlc 3D model synthase of IAGlc in ribbon-style synthase representation in ribbon-style (b). representation (b).

InIn silico silico molecular molecular dockingdocking waswas performed performed with with two two different different computational computational pro- pro- grams,grams, SwissDock SwissDock [[40]40] andand AutoDock AutoDock Vina Vina [41], [41], to study to study the interaction the interaction of ATP andof ATP Glc-1-P and Glc- 1-Pwith with IAGlc IAGlc synthase. synthase. We We performed performed simple simple docking docking experiments experiments in order in order to predict to predict a a possible binding site of ATP and Glc-1-P to IAGlc synthase, with the use of the SwissDock possible binding site of ATP and Glc-1-P to IAGlc synthase, with the use of the SwissDock Server. Results indicated four different binding sites of these modulators to the protein Server.(Figure Results 12). This indicated may explain four thedifferent different binding modulation sites (inhibitionof these modulators or activation) to ofthe the protein (FigureIAGlc synthase12). This activitymay explain by these the two different compounds. modulation One of the (inhibition highest-ranked or activation) regions of of the IAGlcATP assynthase well as Glc-1-Pactivity binding by these to IAGlc two synthasecompou isnds. located One in of the the sugar highest-ranked donor pocket. regions This of ATPpocket as well is the as binding Glc-1-P site binding of UDPG, to therefore,IAGlc synthase we postulate is located that the in modulator the sugar binding donor atpocket. Thisthis pocket site decreases is the binding the activity site of of the UDPG, synthase. therefore, we postulate that the modulator binding at thisThe IAGlcsite decreases synthase possessesthe activity a highly of the conserved synthase. PSPG motif (plant secondary product glycosyltransferase), which is located on one side of the sugar donor pocket. Amino acids of the PSPG motif participate in binding interactions with the sugar donor (UDPG). There are 10 amino acids in this motif that play an important role in the binding interaction with the sugar donor substrate (W341, C342, P343, H359, W362, N363, S364, E367, D383 and Q384) [4]. Data on the structure of the substrate binding site of IAGlc synthase allowed for docking studies of UDPG and its modulators (ATP, Glc-1-P). The semiflexible molecular docking using AutoDock was performed. The protein molecule was kept rigid and the grid box was fixed around the sugar donor pocket of the enzyme which possessed the PSPG motif, whereas UDPG, ATP and Glc-1-P were kept as flexible molecules.

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FigureFigure 12. Results 12. Results of docking of docking of Glc-1-P of Glc-1-P (a ()a and) and ATP ATP ( (b) to IAGlcIAGlc synthase synthase performed performed with with the the SwissDock SwissDock server. server.

TheMolecular IAGlc synthase docking possesses results revealed a highly that cons the enzymeerved PSPG interacts motif with (plant UDPG, secondary ATP and prod- uctGlc-1-P glycosyltransferase), mainly through which residues is oflocated the PSPG on motifone side (Figure of the 13). sugar The uracil donor ring pocket. of UDP Amino forms interactions with the indole ring of W341 (Figure 13b). Similar interactions are acids of the PSPG motif participate in binding interactions with the sugar donor (UDPG). also observed in the complex structure of IAGlc synthase with ATP. The purine ring of ThereATP are is involved 10 amino in acids interactions in this with motif W341. that Moreover,play an important the hydrogen role bondsin the betweenbinding theinterac- tionnitrogen with the atoms sugar of adenine donor andsubstrate the oxygen (W341, atom C342, of S287 P343, and H359, E367 were W362, detected N363, (Figure S364, E367, D38313d). and The Q384) glucose [4]. moietyData on of the UDPG structure forms of hydrogen the substrate bonds binding with the site nitrogen of IAGlc atom synthase of allowedN363 andfor docking Q384 and studies the oxygen of UDPG atom and of D383. its modulators Furthermore, (ATP, contacts Glc-1-P). between The nitrogen semiflexible molecularof the W362 docking main using chain AutoDock and glucose was were performed. observed. TheThe αprotein-phosphate molecule of UDPG was forms kept rigid andhydrogen the grid bondsbox was through fixed around its oxygen the atom sugar and donor the N pocket atom ofof N363,the enzyme while β which-phosphate possessed theinteracts PSPG motif, through whereas its oxygen UDPG, atom ATP with theand N Glc-1-P atom of H359.were kept In the as case flexible of ATP molecules. binding, the several interactions between phosphates and the enzyme were also found. The hydrogen Molecular docking results revealed that the enzyme interacts with UDPG, ATP and bond between the oxygen atom of α-phosphate and nitrogen of the W362 main chain was Glc-1-Pobserved. mainly Moreover, throughβ -phosphateresidues of makes the PSPG contacts motif with (Figure the enzyme 13). The through uracil hydrogen ring of UDP formsbonds interactions between its with nitrogen the indole atoms ofring H359 of andW341 Q384. (Figure What 13b). is more, Similar thenitrogen interactions atom are of also observedH359 participates in the complex in the structure H-bond with of IAGlc the oxygen synthase atom with of the ATP. ribose The ring. purine Furthermore, ring of ATP is involvedthe oxygen in interactions atoms of the withγ-phosphate W341. Moreover form interactions, the hydrogen with the bonds nitrogen between atom of the W381 nitrogen atomsand of the adenine oxygen atomand the of D383. oxygen For atom Glc-1-P, of theS287 interactions and E367 betweenwere detected the glucose (Figure ring 13d). and The glucosefour residues moiety (W362,of UDPG N363, forms D383 hydrogen and H359) bond weres detectedwith the (Figurenitrogen 13 f).atom At the of N363 same time,and Q384 α and -phosphatethe oxygen forms atom interactionsof D383. Furthermore, with the N atom contacts of H358 between and the nitrogen main chain’s of the nitrogen W362 main atom. chain and glucose were observed. The α-phosphate of UDPG forms hydrogen bonds through its oxygen atom and the N atom of N363, while β-phosphate interacts through its oxygen atom with the N atom of H359. In the case of ATP binding, the several interactions between phosphates and the enzyme were also found. The hydrogen bond between the oxygen atom of α-phosphate and nitrogen of the W362 main chain was observed. Moreo- ver, β-phosphate makes contacts with the enzyme through hydrogen bonds between its nitrogen atoms of H359 and Q384. What is more, the nitrogen atom of H359 participates in the H-bond with the oxygen atom of the ribose ring. Furthermore, the oxygen atoms of the γ-phosphate form interactions with the nitrogen atom of W381 and the oxygen atom of D383. For Glc-1-P, the interactions between the glucose ring and four residues (W362, N363, D383 and H359) were detected (Figure 13f). At the same time, α-phosphate forms interactions with the N atom of H358 and the main chain’s nitrogen atom.

Int. J. Mol. Sci. 2021, 22, 3355 16 of 23 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 16 of 24

Figure 13. DockingFigure analysis13. Docking visualization analysis ofvisualization UDPG (a), ATPof UDPG (c) and (a Glc-1-P), ATP (ec) toand IAGlc Glc-1-P synthase (e) to performed IAGlc synthase with AutoDock. performed with AutoDock. Hydrogen bonding interactions of UDPG (b), ATP (d) and Glc-1-P (f) Hydrogen bonding interactions of UDPG (b), ATP (d) and Glc-1-P (f) with amino acid residues of the enzyme. Possible with amino acid residues of the enzyme. Possible hydrogen bonds are marked with yellow dashed hydrogen bonds are marked with yellow dashed lines. The PSPG motif is presented in the ribbon-style representation in lines. The PSPG motif is presented in the ribbon-style representation in green. green.

Int. J. Mol. Sci. 2021, 22, 3355 17 of 23

3. Materials and Methods 3.1. Expression of Zm-IAGLU in E. coli and Purification of Recombinant IAGlc Synthase We received the pET-15b-Zm-IAGLU vector used for the IAGlc synthase expression from Antoni Le´znickifrom the Department of Biochemistry of Nicolaus Copernicus Uni- versity who performed cloning of the gene into the expression vector based on Szerszen et al. [15]. The pET-15b-Zm-IAGLU bacterial expression construct was transformed into E. coli BL21-CodonPlus(DE3)-RIL. Bacteria cells were grown at 37 ◦C in an LB medium containing 50 µg/mL ampicillin and 0.5% (w/v) glucose until OD600 = 0.4. Protein synthesis was induced by addition of isopropyl-1-thio-β-D-galactopyranoside (IPTG) to a final concentration of 0.2 mM and the culture was continued for 20 h at 18 ◦C. The cells were pelleted by centrifugation (3500× g, 20 min, 4 ◦C, Sigma Sartorius Centrifuge, Goettingen, Germany) and resuspended in 25 mM HEPES buffer, pH 7.4, containing 0.5 M NaCl, 30 mM imidazole and 10% (v/v) glycerol (buffer A). The cells were lysed by sonication (4 × 20 s, Ultrasonic Disintegrator UD-20, Techpan, Warszawa, Poland). The lysate was centrifuged at 10,000× g for 10 min at 4 ◦C (Sigma Sartorius Centrifuge). Recombinant IAGlc synthase carrying N-terminal His-tag was purified from the supernatant using an ÄKTATM start system (GE Healthcare Bio-Sciences, Uppsala, Sweden). The supernatant was loaded into a His-TrapTM HP column pre-packed with the Ni Sepharose™ High Performance medium (GE Healthcare Bio-Sciences) previously equilibrated with buffer A. The unbound proteins were removed by washing the column with buffer A. IAGlc synthase was eluted with 77 mM and 170.5 mM imidazole in 25 mM HEPES buffer, pH 7.4, containing 0.5 M NaCl and 10% (v/v) glycerol. The preparation was concentrated using an Amicon-ultra 15 centrifugal filter unit (Millipore, Burlington, Massachusetts, USA) according to the manu- facturer’s instructions. Purity of the enzyme preparation was examined by SDS-PAGE [42] with silver nitrate staining [43]. The fraction of the highest purity was used for further analysis.

3.2. Enzyme Activity Assay 3.2.1. Determination of IAGlc Synthase Activity IAGlc synthase activity was determined in a total volume of 8 µL containing 25 mM HEPES buffer, pH 7.4, 7.5 mM UDPG, 4 mM IAA, 0.016 µCi [20-14C]IAA (55 mCi mmol−1, Hartmann Analytic GmBH, Braunschweig, Germany) and 2.5 mM MgCl2 with 3 µL of the enzyme preparation. The reaction was stopped after 15 min incubation at 30 ◦C by drying 4 µL of aliquots on a Silica Gel F260 TLC plate (Merck, Darmstadt, Germany). TLC was performed using ethyl acetate: n-butanone: ethanol: water (5/3/1/1, v/v/v/v) as a solvent. The indole ring compounds were visualized by staining the plate with the Van Urk–Salkowski reagent [44]. Bands identiﬁed as IAGlc were excised and placed in a vial with 2 mL EcoLite (+) scintillation ﬂuid (ICN). The radioactivity level was measured using a Wallac 1409 liquid scintillation counter (Turku, Finland).

3.2.2. Determination of UDPG Pyrophosphorylase Activity UDPG pyrophosphorylase (1) activity was analyzed using the spectrophotometric method using a coupled enzyme assay containing phosphoglucomutase (2) and glucose-6- phosphate dehydrogenase (3) according to the following equations:

UDPG + PPi ↔ UDP + glucose-1-phosphate (1)

glucose-1-phosphate ↔ glucose-6-phosphate (2) glucose-6-phosphate + NADP+ → 6-phosphogluconolactone + NADPH/H+ (3) The reaction mixture in the total volume of 1.0 mL contained 0.2 M HEPES–NaOH, pH 7.4, 6 mM MgCl2, 2 mM sodium pyrophosphate, 10 mM UDPG, 0.4 mM NADP +, 3 U glucose-6-phosphate dehydrogenase (Sigma-Aldrich, Saint Louis, MO, USA), 3 U phosphoglucomutase (Sigma-Aldrich) and 0.26 U/mg IAGlc synthase. The change Int. J. Mol. Sci. 2021, 22, 3355 18 of 23

of the NADPH/H+ absorbance was monitored at 340 nm using a Shimadzu UV-160A spectrophotometer (Shimadzu, Japan). The enzyme activity was calculated using the absorption coefﬁcient 6220 M−1 cm−1 for NADH/H+.

3.3. Kinetic Studies 3.3.1. The Effect of pH on IAGlc Synthase Activity The effect of pH on the catalytic activity of the recombinant IAGlc synthase was investigated in 25 mM HEPES buffer ranging from pH 5.8 to 8.8 (pH 5.8; 6.2; 6.6; 6.8; 7.0; 7.4; 7.6; 8.0; 8.2; 8.6; 8.8) using the radioactivity method in standard conditions. The reaction ◦ was stopped after 30 min incubation at 30 C by drying 4 µL of aliquots on a Silica Gel F260 TLC plate (Merck). The rest of the assay was performed as described above.

3.3.2. The Effect of Divalent Cations on IAGlc Synthase Activity The effect of divalent ions on the catalytic activity of recombinant IAGlc synthase was investigated in a total volume of 8 µL containing 25 mM HEPES buffer, pH 7.4, 7.5 mM UDPG, 4 mM IAA and 0.016 µCi [20-14C]IAA (55 mCi mmol−1, Hartmann Analytic GmBH, Braunschweig, Germany) with 3 µL of the enzyme preparation. The reaction mixture contained 5 mM MgCl2, 5 mM CaCl2, 5 mM MnCl2, 5 mM EDTA or no ions (control). The reaction was stopped after 15 min incubation at 30 ◦C by drying 4 µL of aliquots on a Silica Gel F260 TLC plate (Merck). The rest of the assay was performed as described above.

3.3.3. The Effect of Modulators on IAGlc Synthase Activity The effect of modulators on the catalytic activity of recombinant IAGlc synthase was investigated in a total volume of 8 µL containing 25 mM HEPES buffer, pH 7.4, 7.5 mM UDPG, 4 mM IAA, 0.016 µCi [20-14C]IAA (55 mCi mmol−1, Hartmann Analytic GmBH, Germany) and 2.5 mM MgCl2 with 3 µL of the enzyme preparation. The reaction mixture contained one of the potential modulators: 1 mM ATP; 10 mM oxidized glutathione (GSSG); 10 mM reduced glutathione (GSH); 4 mM glucose-1-phosphate (Glc-1-P); 4 mM pyrophosphate (PPi); 0.1 mM IAA–Asp or 1 mM IAA–Asp. The control did not contain any modulators. The reaction was stopped after 15 min incubation at 30 ◦C by drying 4 µL of aliquots on a Silica Gel F260 TLC plate (Merck). The rest of the assay was performed as described above.

3.3.4. The Effect of Phytohormones on IAGlc Synthase Activity The effect of phytohormones on the catalytic activity of recombinant IAGlc synthase was investigated in a total volume of 8 µL containing 25 mM HEPES buffer, pH 7.4, 7.5 mM UDPG, 4 mM IAA, 0.016 µCi [20-14C]IAA (55 mCi mmol−1, Hartmann Analytic GmBH, Germany) and 2.5 mM MgCl2 with 3 µL of the enzyme preparation. The reaction mixture contained one of the following phytohormones: 1 mM phenylacetic acid (PAA); 1 mM 2,4-dichlorophenoxyacetic acid (2,4-D); 1 mM Picloram; 1 mM Dicamba; 1 mM gibberellic acid (GA3) or 1 mM kinetin. The control did not contain any additional phytohormones. The reaction was stopped after 15 min incubation at 30 ◦C by drying 4 µL of aliquots on a Silica Gel F260 TLC plate (Merck). The rest of the assay was performed as described above.

3.3.5. Determination of Kinetic Parameters for UDPG The effect of UDPG concentration on the IAGlc synthase activity was analyzed in a total volume of 8 µL containing 25 mM HEPES buffer, pH 7.4, 3 mM IAA and 0.016 µCi [20-14C]IAA (55 mCi mmol−1, Hartmann Analytic GmBH, Germany) with 3 µL of the enzyme preparation with different UDPG concentrations (0.5–20 mM). The reaction was ◦ stopped after 15 min incubation at 30 C by drying 4 µL of aliquots on a Silica Gel F260 TLC plate (Merck). The rest of the assay was performed as described above. The Vmax and KM parameters were calculated using a Hanes–Woolf plot ([S]/v = f([S])) and veriﬁed by the Michaelis–Menten equation using SigmaPlot 11.0 (Systat Software Inc, San Jose, California, USA). Int. J. Mol. Sci. 2021, 22, 3355 19 of 23

3.3.6. Determination of Kinetic Parameters for IAA The effect of IAA concentration on the IAGlc synthase (4) activity was analyzed using the spectrophotometric method [16] as the release of UDP using a coupled enzyme assay containing pyruvate kinase (5) and lactate dehydrogenase (6) according to the following equations: IAA + UDPG ↔ IAGlc + UDP (4) UDP + phosphoenolpyruvate ↔ UTP + pyruvate (5) + + Pyruvate + NADH/H → L-lactate + NAD (6) The reaction mixture in the total volume of 1.0 mL contained 50 mM HEPES–NaOH, + pH 7.4, 2.5 mM MgCl2, 2 mM PEP, 4 mM UDPG, IAA (0.5–4 mM), 0.13 mM NADH/H , 3 U pyruvate kinase from rabbit muscles (Sigma-Aldrich), 4 U L-lactate dehydrogenase from rabbit muscles (Sigma-Aldrich) and 0.26 U/mg IAGlc synthase. The change of the NADH/H+ absorbance was monitored at 340 nm using a Shimadzu UV-160A spectrophotometer (Shimadzu, Japan). The enzyme activity was calculated using the absorption −1 −1 + coefﬁcient 6220 M cm for NADH/H . The Vmax and KM parameters were calculated using a Hanes–Woolf plot and veriﬁed by the Michaelis–Menten equation using SigmaPlot 11.0 (Systat Software). The effect of IAA–Asp on the KM and Vmax values was analyzed as described above using the reaction mixtures containing 0.5–4 mM IAA and additionally 0.1 mM IAA–Asp.

3.3.7. Determination of Substrate Speciﬁcity of IAGlc Synthase The substrate speciﬁcity of the IAGlc synthase (7) was analyzed using the spectrophotometric method [16] as the release of UDP using a coupled enzyme assay containing pyruvate kinase (8) and lactate dehydrogenase (9) according to the following equations:

substrate + UDPG ↔ substrate-Glc + UDP (7)

UDP + phosphoenolpyruvate ↔ UTP + pyruvate (8) + + Pyruvate + NADH/H → L-lactate + NAD (9) The reaction mixture in the total volume of 1.0 mL contained 50 mM HEPES–NaOH, + pH 7.4, 2.5 mM MgCl2, 2 mM PEP, 4 mM UDPG, 0.13 mM NADH/H , 3 U pyruvate kinase from rabbit muscles (Sigma-Aldrich), 4 U L-lactate dehydrogenase from rabbit muscles (Sigma-Aldrich), 4.3 µg ZmIAGlc synthase and 3 mM sugar acceptor (IAA, IBA, IPA, IPyA, PAA, 2,4-D, Dicamba, Picloram or tryptophan). The change of the NADH/H+ absorbance was monitored at 340 nm using a Shimadzu UV-160A spectrophotometer (Shimadzu, Japan). The enzyme activity was calculated using the absorption coefﬁcient 6220 M−1 cm−1 for NADH/H+.

3.3.8. Inhibition of IAGlc by ATP and Glucose-1-Phosphate The effect of ATP on the IAGlc synthase activity was studied in the standard reaction mixture with ATP at the ﬁnal concentration of 0–1 mM. The effect of glucose-1-phosphate (Glc-1-P) on the IAGlc synthase activity was studied in the standard reaction mixture with Glc-1-P at the ﬁnal concentration of 0–4 mM. The reaction was stopped after 15 min ◦ incubation at 30 C by drying 4 µL of aliquots on a Silica Gel F260 TLC plate (Merck). The rest of the assay was performed as described above.

3.3.9. Inhibition of IAGlc Synthase by 2,4-D The effect of 2,4-D on the IAGlc synthase activity was determined using the radioactivity assay in the standard reaction mixture with IAA concentrations of 1.95 mM and 3.0 mM in the presence of 0–625 µM 2,4-D. The inhibition kinetics were determined using a Dixon plot (1/v = f([I])). The inhibition constant (Ki) value was calculated using a Dixon plot. The IC50 value was calculated through graphical analysis. The resulting data were ﬁt into the Int. J. Mol. Sci. 2021, 22, 3355 20 of 23

app following equation: KM = Km (1 + [I]/Ki). Calculations were performed using SigmaPlot 11.0 (Systat Software).

3.4. Western Blot Analysis For Western blot analysis, the proteins were separated by SDS-PAGE according to the method of Ogita and Markert [42] and transferred onto a Protran BA 83 nitrocellulose membrane (Whatman, GmBH, Maidstone, UK) by the wet system (Bio-Rad) (200 mA, 90 V for 1 h at 4 ◦C) in 10 mM CAPS/NaOH buffer, pH 11.0, containing 10% (v/v) ethanol. The SpectraTM multicolor broad range protein ladder (10–260 kDa) (Thermo Scientiﬁc, Waltham, Massachusetts, USA) was used as a molecular mass standard. After blocking in TBS (Tris Buffered Saline) buffer containing 5% (w/v) nonfat dry milk, the membrane was incubated with monoclonal anti-polyHistidine antibodies produced in mice (1:12,000) (Sigma-Aldrich). The proteins were detected with goat anti-mouse IgG antibodies conjugated to alkaline phosphatase (1:1000) (Sigma-Aldrich) and visualized using NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate) tablets (Roche, Bazylea, Sweden).

3.5. Protein Determination Protein concentration was determined with the Bradford method [45] using bovine serum albumin as a standard.

3.6. In Silico Sequence Analysis Amino acid sequences of maize IAGlc synthase (ZmIAGLU, NCBI Reference Sequence Database, accession number NP_001105326.1), rice OsIAGT1 (GenBank, accession number BAS85861.1) and A. thaliana glucosyltransferases UGT74D1 (UniProtKB/Swiss-Prot, accession number Q9SKC5.1), UGT74E2 (GenBank, accession number ABJ17124.1), UGT75D1 (UniProtKB/Swiss-Prot, accession number O23406.2), UGT76F1 (UniProtKB/Swiss-Prot, accession number Q9M051.1) and UGT84B1 (GenBank, accession number AAO63432.1) were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/, access date: 1 February 2021). Identity of these sequences was calculated also using Clustal Omega. Prediction of disulﬁde bonds was performed using the DiANNA 1.1 web Server (http://clavius.bc.edu/~clotelab/DiANNA/, accessed on 2 February 2021).

3.7. Homology Modeling and Molecular Docking The secondary and three-dimensional structure of IAGlc synthase was predicted using the iterative threading assembly refinement (I-TASSER) server [36–39]. The five models were generated by the I-TASSER server and the best model was selected on the basis of sequence identity and the estimated global accuracy of the model (C-Score = 0.77, TM-score = 0.82 ± 0.09, and RMSD = 5.5 ± 3.5 Å). The obtained 3D structure of IAGlc synthase was used in the molecular docking process. The three-dimensional structure of UDPG, ATP and Glc-1-P were downloaded from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do, accessed on 20 Febru- ary 2021). Prediction of molecular interactions of IAGlc synthase with ligands was made using the SwissDock web service with default server values [40]. The results were visualized in UCFS Chimera [46]. The binding interactions between the substrate (UDPG) or modulators (ATP, Glc-1-P) and IAGlc synthase were simulated using the docking method implemented in AutoDock 4.2 along with Autodock Tools [41]. A semiflexible docking protocol was used. The protein molecule was kept rigid, whereas ligands were kept as flexible molecules. The grid box (72 × 70 × 62) with 0.375 Å was fixed around the active site of the protein (PSPG motif, TRP314, CYS342, PRO343, HIS359, TRP362, ASN363, SER364, GLU367, ASP383 and GLN384) [4]. The center of the grid box was set at points 70.199, 65.543, 63.479. Then, the grid maps were precalculated using Autogrid, one map for each atom type present in the docked ligand. The Lamarckian genetic algorithm (LGA) was used for searching the best conformation of complexes. The docking parameters were Int. J. Mol. Sci. 2021, 22, 3355 21 of 23

set to the default value. The position with the best binding afﬁnity was visualized using Pymol [47].

3.8. Statistical Analysis All data are presented as the means ± SD for three biological repetitions of each experiment (n = 3).

4. Conclusions Our research provides a biochemical characterization of IAGlc synthase—the enzyme modulating the IAA biological activity by its conjugation to glucose. The enzyme is highly speciﬁc towards IAA; however, it also utilizes other natural auxins (IBA, IPA and IPyA) as glucose acceptors, though with less efﬁciency. IAGlc synthase activity is modulated by a number of compounds. 2,4-D, a synthetic auxinic herbicide, acts as a competitive inhibitor of the synthase. We also report that IAA–Asp, another conjugated form of IAA, acts as an activator of IAGlc synthase. Such a role can suggest that auxin conjugate synthesis is a complex process regulated not only on the expression level, but also on the protein level by products of different conjugation pathways. We also report unique modulatory properties of ATP and glucose-1-phosphate whose action is concentration-dependent. At lower concentrations, both compounds act as activators of IAGlc synthase, however, a high concentration of these compounds strongly inhibits this enzyme. Molecular docking revealed that both ATP and glucose-1-phosphate, similar to the glucose donor—UDPG, can bind to the PSPG motif of IAGlc synthase. However, more potential binding sites for these two compounds are present in the enzyme structure. We suggest that IAGlc synthase may contain more than one binding site for these two modulatory compounds, one site being responsible for the activating effect of the modulator and another, possibly the PSPG motif, causing abolishment of synthase activity.

Author Contributions: Conceptualization, A.C. and M.O.; methodology, A.C., M.O. and A.K.; formal analysis, A.C., M.O. and A.K.; investigation, A.C., M.O. and A.K.; resources, A.C., M.O. and A.K.; writing—original draft preparation, A.C., M.O. and A.K.; writing—review and editing, A.C., M.O. and A.K.; supervision, M.O.; project administration, A.C.; funding acquisition, A.C. and A.K. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Nicolaus Copernicus University grant No. 1190-B. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in article. Acknowledgments: We are grateful to Antoni Le´znickifrom the Department of Biochemistry at Nicolaus Copernicus University for providing us with the pET-15b-Zm-IAGLU vector. Anna Koza- kiewicz is a member of the “Towards Personalized Medicine” Center of Excellence operating under the Excellence Initiative—Research University program. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

1-O-IAGlc 1-O-indole-3-acetyl-β-D-glucose 2,4-D 2,4-dichlorophenoxyacetic acid ATP adenosine triphosphate GA gibberellic acid GSH reduced glutathione GSSG oxidized glutathione GT glycosyltransferase IAA indole-3-acetic acid Int. J. Mol. Sci. 2021, 22, 3355 22 of 23

IBA indole-3-butyric acid IPA indole-3-propionic acid IPyA indole-3-pyruvic acid IPTG isopropyl-1-thio-β-D-galactopyranoside NAA naphthaleneacetic acid OxIAA oxindole-3-acetic acid PAA phenylacetic acid PEP phosphoenolopyruvate PPi pyrophosphate PSPG plant secondary product glycosyltransferase UDP uridine diphosphate UDPG uridine diphosphate glucose UGT uridine diphosphate glycosyltransferase Zm-IAGlc UDPG-dependent IAA glucosyltransferase gene

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