Andrzej Guranowski

Purification and characterization of adenosine nucleosidase from barley leaves

Adenosine nucleosidase (adenosine ribohydrolase, EC 3.2.2.7) has been purified to a nearly homogeneous state from barley leaves. The enzyme is soluble in concentrated salt solution while it aggregates and precipitates at low ionic strength, factors which enabled a simple purification procedure to be carried out. A molecular weight of 66 000 +/- 3000 was estimated for the native enzyme by gel filtration. In sodium dodecyl sulphate polyacrylamide gel electrophoresis of the most purified fraction a single major band of polypeptide chains, with molecular weight of 33 000, was observed. Thus, the native enzyme seems to be dimer of alpha2 type. The pH optima are 4.7 and 5.4 for citrate and (N-morpholino)ethanesulphonic acid buffers, respectively. Adenine and adenosine protect the enzyme against heat inactivation. The enzyme is resistant to -SH reagents, dithiothreitol inhibits it. The Km for adenosine varied from 0.8 to 2.3 micronM depending on temperature and buffer system. The Km for deoxyadenosine was 120 micronM. Besides adenosine, of several nucleosides tested only adenosine N1-oxide, deoxyadenosine and purine riboside acted as substrates. Adenine as well as its derivatives, including plant hormones (cytokinins), have an inhibitory effect on the enzyme. The Ki values of some modified nucleosides and free bases were determined. The physiological role of adenosine nucleosidase in plants is discussed.

Purine salvage in cotyledons of germinating lupin seeds

Purine metabolism is much less elucidated in plants [ 1,2] than in animal tissues. In contrast to animal tissues, in plants adenosine is hydrolyzed to adenine and ribose by adenosine nucleosidase (EC 3.2.2.7.) found in leaves of various species [3-71, as well as in the cotyledons of yellow lupin seedlings [8]. For the cotyledons, the highest adenosine nucleosidase activity has been observed on day 4-5 of seed germination [9]. However, as Brown found [ IO,1 l] in legume seeds, adenine does not accumulate in their cotyledons at any stage of germination. This could suggest that the activity of purine salvage pathways is relatively high. The aim of this work was to study changes in the activity of enzymes of the purine salvage pathways in cotyledons of germinating lupin seeds. In animals, two ways of purine salvage are postulated: (i) The one-step way catalyzed by purine phosphoribosyltransferases (EC 2.4.2.7. and EC 2.4.2.8.); (ii) The two-step way dependent on purine nucleoside phosphorylase (EC 2.4.2.1.) and nucleoside kinases (EC 2.7.1.20 and 2.7.1.73.) or nucleoside phosphotransferase (EC 2.7.1.77). Some of these enzymic activities have been studied in germinating wheat embryos, but only up to the second day of germination [ 121.

4-Coumarate:Coenzyme A Ligase Has the Catalytic Capacity to Synthesize and Reuse Various (Di)Adenosine Polyphosphates

4-Coumarate:coenzyme A ligase (4CL) is known to activate cinnamic acid derivatives to their corresponding coenzyme A esters. As a new type of 4CL-catalyzed reaction, we observed the synthesis of various mono- and diadenosine polyphosphates. Both the native 4CL2 isoform from Arabidopsis (At4CL2 wild type) and the At4CL2 gain of function mutant M293P/K320L, which exhibits the capacity to use a broader range of phenolic substrates, catalyzed the synthesis of adenosine 5′-tetraphosphate (p4A) and adenosine 5′-pentaphosphate when incubated with MgATP−2 and tripolyphosphate or tetrapolyphosphate (P4), respectively. Diadenosine 5′,5‴,-P1,P4-tetraphosphate represented the main product when the enzymes were supplied with only MgATP2−. The At4CL2 mutant M293P/K320L was studied in more detail and was also found to catalyze the synthesis of additional dinucleoside polyphosphates such as diadenosine 5′,5‴-P1,P5-pentaphosphate and dAp4dA from the appropriate substrates, p4A and dATP, respectively. Formation of Ap3A from ATP and ADP was not observed with either At4CL2 variant. In all cases analyzed, (di)adenosine polyphosphate synthesis was either strictly dependent on or strongly stimulated by the presence of a cognate cinnamic acid derivative. The At4CL2 mutant enzyme K540L carrying a point mutation in the catalytic center that is critical for adenylate intermediate formation was inactive in both p4A and diadenosine 5′,5‴,-P1,P4-tetraphosphate synthesis. These results indicate that the cinnamoyl-adenylate intermediate synthesized by At4CL2 not only functions as an intermediate in coenzyme A ester formation but can also act as a cocatalytic AMP-donor in (di)adenosine polyphosphate synthesis.

Fhit proteins can also recognize substrates other than dinucleoside polyphosphates

We show here that Fhit proteins, in addition to their function as dinucleoside triphosphate hydrolases, act similarly to adenylylsulfatases and nucleoside phosphoramidases, liberating nucleoside 5′‐monophosphates from such natural metabolites as adenosine 5′‐phosphosulfate and adenosine 5′‐phosphoramidate. Moreover, Fhits recognize synthetic nucleotides, such as adenosine 5′‐O‐phosphorofluoridate and adenosine 5′‐O‐(γ‐fluorotriphosphate), and release AMP from them. With respect to the former, Fhits behave like a phosphodiesterase I concomitant with cleavage of the P–F bond. Some kinetic parameters and implications of the novel reactions catalyzed by the human and plant (Arabidopsis thaliana) Fhit proteins are presented.

Recognition of different nucleotidyl-derivatives as substrates of reactions catalyzed by various HIT-proteins

Proteins that have a histidine triad in their active sites belong to the HIT-protein superfamily. They are ubiquitous, are involved in the metabolism of different nucleotides and catalyze their hydrolysis and/or phosphorolysis liberating either the corresponding 5′-NMP or 5′-NDP, respectively. We studied substrate specificity of nine recombinant HIT-proteins with adenosine 5′-phosphosulfate (1), adenosine 5′-phosphoramidate (2), adenosine 5′-phosphorothioate (3), adenosine 5′-phosphorofluoride (4), diadenosine 5′,5′′′-P1,P3-triphosphate (5), di(7-methylguanosine) 5′,5′′′-P1,P3-triphosphate (6) and adenosine 5′-hypophosphate (7). Preferences for the recognition of these compounds as substrates by individual proteins differed. All the proteins hydrolyzed (1) but the Arabidopsis thaliana Hint1 did it very slowly. None of the proteins cleaved (7). Only A. thaliana Hint1 and Escherichia coli HinT hydrolyzed (3). Three proteins known as dinucleoside triphosphatases, human and A. thaliana Fhit-proteins and Trypanosoma brucei HIT-45, cleaved (1), (2), (4), (5) and (6). Caenorhabditis elegans decapping protein DcpS degraded (1), (5), (6) and poorly (4). A. thaliana aprataxin-like protein and Hint4 hydrolyzed only (1), (2) and (4), in that order of efficiency. Velocities of those reactions and some Km values were determined. Applicability of this study to the metabolism of certain nucleotidyl-derivatives is discussed.

NEW TRIPODAL, SUPERCHARGED ANALOGUES OF ADENOSINE NUCLEOTIDES : INHIBITORS FOR THE FHIT AP3A HYDROLASE

Methanetrisphosphonic acids provide a branch point for synthetic nucleotide analogues which can be exploited either to generate novel tripodal nucleotides or to incorporate additional negative charge into linear analogues relative to the parent nucleotide, as exemplified in the picture for ATP and diadenosine tetraphosphate (Ap4 A). These compounds show valuable discriminatory behavior as competitive inhibitors for the tumor suppressor protein Fhit and a second Apn A pyrophosphohydrolase. X=H, Cl, F.

Adenosine 5′-Tetraphosphate Is a Highly Potent Purinergic Endothelium-Derived Vasoconstrictor

Besides serving as a mechanical barrier, the endothelium has important regulatory functions. The discovery of nitric oxide revolutionized our understanding of vasoregulation. In contrast, the identity of endothelium-derived vasoconstrictive factors still remains uncertain. The supernatant from mechanically stimulated human microvascular endothelial cells elicited a potent vasoconstrictive response in the isolated perfused rat kidney. Whereas a nonselective purinoceptor blocker blocked this vasoactivity most potently, the inhibition of the endothelin receptor by BQ123 weakly affected that vasoconstrictive response. As a compound responsible for that vasoconstrictive effect, we have isolated from HMECs and identified the mononucleotide adenosine 5′-tetraphosphate (AP4). This nucleotide proved to be the most potent vasoactive purinergic mediator identified to date, exerting the vasoconstriction predominantly through activation of the P2X1 receptor. The intraarterial application of AP4 in a Wistar–Kyoto rat induced a strong increase of the mean arterial pressure. The plasma concentration of AP4 is in the nanomolar range, which, in vivo, induces a significant change in the mean arterial pressure. To our knowledge, AP4, which exerts vasoactive effects, is the most potent endogenous mononucleotide identified to date in mammals. The effects of AP4, the plasma concentration of AP4, and its release suggest that this compound functions as an important vasoregulator.

Dual activity of certain HIT-proteins: A. thaliana Hint4 and C. elegans DcpS act on adenosine 5'-phosphosulfate as hydrolases (forming AMP) and as phosphorylases (forming ADP).

Histidine triad (HIT)‐family proteins interact with different mono‐ and dinucleotides and catalyze their hydrolysis. During a study of the substrate specificity of seven HIT‐family proteins, we have shown that each can act as a sulfohydrolase, catalyzing the liberation of AMP from adenosine 5′‐phosphosulfate (APS or SO4‐pA). However, in the presence of orthophosphate, Arabidopsis thaliana Hint4 and Caenorhabditis elegans DcpS also behaved as APS phosphorylases, forming ADP. Low pH promoted the phosphorolytic and high pH the hydrolytic activities. These proteins, and in particular Hint4, also catalyzed hydrolysis or phosphorolysis of some other adenylyl‐derivatives but at lower rates than those for APS cleavage. A mechanism for these activities is proposed and the possible role of some HIT‐proteins in APS metabolism is discussed.

Diadenosine polyphosphates (Ap3A and Ap4A) behave as alarmones triggering the synthesis of enzymes of the phenylpropanoid pathway in Arabidopsis thaliana

It is known that cells under stress accumulate various dinucleoside polyphosphates, compounds suggested to function as alarmones. In plants, the phenylpropanoid pathways yield metabolites protecting these organisms against various types of stress. Observations reported in this communication link these two phenomena and provide an example of a metabolic “addressee” for an “alarm” signaled by diadenosine triphosphate (Ap3A) or diadenosine tetraphosphate (Ap4A). In response to added Ap3A or Ap4A, seedlings of Arabidopsis thaliana incubated in full nutrition medium increased both the expression of the genes for and the specific activity of phenylalanine ammonia‐lyase and 4‐coumarate:coenzyme A ligase, enzymes that control the beginning of the phenylpropanoid pathway. Neither adenine mononucleotides (AMP, ADP or ATP) nor adenosine evoked such effects. Reactions catalyzed in vitro by these enzymes were not affected by Ap3A or Ap4A.

Substrate specificity of S-Adenosylhomocysteinase: Custeine is a substrate of the plant and mammalian enzymes

Substrate specificity of S-adenosylhomocysteinases (S-adenosyl-L-homocysteine hydrolase, EC 3.3.1.1) with respect to amino acid has been studied using homogeneous preparations of the enzymes from yellow lupin (Lupinus luteus) seeds and bovine liver. Both enzymes use cysteine, in addition to homocysteine, as a substrate. Homoserine, serine, pinicillamine, reduced glutathione and 2-mercaptoethanol are not substrates. In the presence of cysteine, the reaction of S-adenosylthio-amino acid synthesis is characterized by 20-40-fold lower kcat values (kcat = 0.23 s-1 or 0.11 s-1 in the presence of cysteine and either bovine or lupin enzyme) and 270-250-fold higher Km values (Km for cysteine is 15 mM and 35 mM with bovine and lupin enzyme, respectively) than the reaction in the presence of the normal substrate, homocysteine. In the reverse reaction, S-adenosylcysteine is hydrolyzed by the mammalian enzyme much faster than by the plant one. Specificity (kcat/Km) towards S-adenosylcysteine and S-adenosylhomocysteine is 0.9 M-1 . s-1 and 60 000 M-1 . s-1, respectively, with the plant enzyme and 15.3 M-1 . s-1 and 70 000 M-1 . s-1, respectively, with the mammalian enzyme. With plant enzyme, the reactions with cysteine and homocysteine are not competitive, i.e., cysteine does not inhibit the synthesis of S-adenosylhomocysteine, and homocysteine does not inhibit the synthesis of S-adenosylcysteine. This is consistent with independent binding of cysteine and homocysteine to both enzyme subunits. Using adenosine analogs and the mammalian S-adenosylhomocysteinase we were able to synthesize a number of novel S-adenosylcysteine analogs. These included: S-N6-hydroxyadenosyl-L-cysteine, S-2-aminoadenosyl-L-cysteine, S-nebularyl-L-cysteine, S-3-deazaadenosyl-L-cysteine, S-formycyl-L-cysteine, S-N6-methyladenosyl-L-cysteine and S-N1-oxideadenosyl-L-cysteine.