Glucose activation and metabolism through UDP-glucose pyrophosphorylase in plants

0031-9422(94)00558-3

Pergamon

Phymhemutry, Vol. 37, No. 6, pp. 1507-1515, 1994 Copyright 0 1994 Elwvier Science Ltd Printed in Great Bntain. All rights -cd 0031~9422,FJ4 $24.00 + 0.00

REVIEW ARTICLE NUMBER 97 GLUCOSE

ACTIVATION AND METABOLISM THROUGH PYROPHOSPHORYLASE IN PLANTS

UDP-GLUCOSE

LESZEK A. KLECZKOWSKI Plant Physiology Department, Umd University, 901 87 Umd, Sweden (Receioed23 June 1994) Key Word Index-ADP-glucose pyrophosphorylase; sugars; pyrophosphate; starch, sucrose; UDP-glucose;

cellulose; cell wall biosynthesis; UDP-glucose pyrophosphorylase.

nucleotide

Abstract-Recent developments in studies on the characterization of properties and functions of UDP-glucose pyrophosphorylase (UGPase) in plant metabolism are presented. UGPase constitutes a reversible enzymatic step for interconversions between starch and sucrose metabolites, and is responsible for synthesis and metabolism of UDPglucose, a major form of nucleoside diphosphoglucose in plant cells. The enzyme, although considered not to have any regulatory function, has attracted considerable interest due to its ubiquitous distribution in plants, high activity, especially in sink tissues, and because of the key role of UDP-glucose as a direct or indirect precursor of sucrose, starch and structural polysaccharides. The enzyme has been the subject of biotechnological manipulations to engineer its kinetic properties and gene expression in relation to metabolic processes at the sucrose/starch interface. Depending on tissue type, the UGPase reaction may be channelled in oiuo, either toward UDP-glucose pyrophosphorolysis or synthesis, due to a metabolic coupling to other reactions of sugar pathways. Some strategies for future research on plant UGPase are discussed.

INTRODUCTION

UDP-glucose pyrophosphorylase (UGPAse) (EC 2.7.7.9) is an ubiquitous enzyme found both in plant and animal tissues, as well as in bacteria. It catalyses a reversible, magnesium-dependent, transfer of an uridylyl group from UDP-glucose to Mg-PP, (inorganic pyrophosphate) forming glucose-l-phosphate (glu-1-P) and Mg-UTP [l-3]. In photosynthetic tissues, UGPase activity is involved in the synthesis of sucrose by being ‘coupled’ to sucrose phosphate synthase (SPS) activity, which is a key, highly controlled, irreversible step in channelling photosynthetically derived carbon toward sucrose biosynthesis [4, 51. In tissues where sucrose degradation [catalysed by sucrose synthase (SuSy) and invertase] is metabolically important, e.g. developing seeds and tubers, UGPase is considered to have a role in the metabolism of UDPglucose formed by SuSy activity [6-81. The latter enzyme catalyses a readily reversible reaction in viuo [9], but in the presence of UGPase equate PP, levels UDP-glucose is driven away from SuSy, affecting the equilibrium toward sucrose degradation. Thus, depending on metabolic conditions, UGPase may be involved either in the synthesis or degradation of sucrose in uioo. Other than sucrose pathways, UGPase provides UDP-glucose for a variety of reactions leading to biosynthesis of cell wall components (see below).

In the present review, a concise account of the recent status of work on UGPase is presented along with the discussion of possible functions of the enzyme in processes of glucose activation and UDP-glucose metabolism in plant tissues. More comprehensive reviews on sugar activation through UGPase and other pyrophosphorylases in plants, containing older literature on the subject, can be found in refs [2, 3, lo].

FUNCTION

The discovery of UDP-glucose by Leloir and his coworkers [11] over 40 years ago provided a powerful stimulus for studies on carbohydrate biochemistry, particularly with respect to complex saccharide biosynthesis. Since then, a number of sugar nucleotides differing in sugar and base substitutions have been isolated from a variety of plant tissues [2, 3, 121. UDP-glucose is the major form of activated sugar and the major glucosyl donor for carbohydrates in higher plants. It represents a branchpoint in sugar metabolism, and is needed: (i) for sucrose synthesis in the cytosol via SPS and SPS phosphatase [S], (ii) as a direct or indirect (through conversion to other nucleotide sugars) precursor in the synthesis of cellulose, pectic substances, hemicelluloses, glycolipids and other assorted glycosylated molecules involved in cell

1507 PHYTO

37-6-C

OF UDP-GLUCOSE

L. A.

1508

KLECZKOWSKI

Sucrose -

Other nucleotide sugars :e g ADP-glucose, GDP-mannose)

I

I

Fig. 1. A simplified scheme for the involvement of UDP-glucose in metabolic processes of the plant. Details of biochemistry of UDP-glucose (UDPG) metabolic conversions are extensively covered in refs [2, 3,8, 10, 12-161.

wall formation [2, 13-211, and (iii) as a direct or indirect (through conversion to ADP-glucose) precursor of starch formation in the plastids [22-241 (Fig. 1). The metabolic pathways with UDP-glucose that are most important in terms of the magnitude of carbon fluxes are those concerned with sucrose synthesis and breakdown. Activities of enzymes involved in these processes, especially UGPase, are frequently orders of magnitude higher than those involved in other pathways requiring UDP-glucose [3, 191. Thus, levels of UDP-glucose are likely to be most affected by sucrose synthesis/metabolism. General metabolic pathways for biosynthesis of structural carbohydrates (e.g. cellulose, callose), involving nucleotide sugars as major precursors, have been extensively reviewed [2, 13, 15, 16-j. The processes involve formation of nucleotide sugars through activities of specific pyrophosphorylases, followed by a transfer of the monosaccharide moiety from a nucleotide sugar to the growing chain of a polysaccharide. The main routes of cell Wall polysaccharide synthesis appear to be localized in plant membranes: Golgi dictyosomes, endoplasmic reticulum and the plasmalemma [13, 15-J. It is unclear whether UDP-glucose can be formed in the above mentioned membrane structures or is imported there from the cytosol. In the latter case, activities of the cytosolic UGPase and/or SuSy would probably be responsible for production of virtually all the UDP-glucose utilized in biosynthesis of structural polysaccharides. In animal tissues, UDP-glucose is a direct precursor of glycogen (equivalent to plant starch). In plants, however, starch is believed to be derived entirely from ADPglucose which is formed by ADP-glucose pyrophosphorylase (AGPase). This belief is based mostly on genetic studies showing that mutants of AGPase are starch deficient (e.g. ref. [25]) [no mutants for plant UGPase have yet been reported], and on comparative enzymatic studies of the efficiency of starch formation from nucleotide sugars [26]. On the other hand, lysed amyloplasts from certain plants (e.g. corn kernels or sugar beet roots) are capable of synthesizing starch with the involvement of

uridine nucleoside phosphates, possibly through starch synthase reaction [23, 271. In maize developing kernels, synthesis of starch occurred at higher rates when lysed amyloplasts were incubated with gluconeogenic intermediates and UTP than with ATP [23]. Starch formation from UDP-glucose may possibly occur through the activity of a granule-bound starch synthase (but not soluble starch synthase), which can be highly specific toward UDP-glucose as a substrate [22]. The key, still unresolved, question with respect to these studies is whether the involvement of UDP-glucose in starch production is indeed occurring in viva If the answer is affirmative, then an immediate further problem would concern the origin of the plastidic pool of UDP-glucose: does it arise through the activity of some minor isozyme of UGPase (or some other UDP-glucose generating enzyme) or is it transported from the cytosol to the plastid stroma? Concerning the latter question, plastids are considered not to have any significant capability to uptake UDPglucose, even though they are capable of importing ADPglucose at high rates [28]. Moreover, UDP-glucose is thought to be compartmentalized entirely in the cytosol (e.g. ref. [29]). UDP-glucose was, at one time, implicated in the active transport of sucrose to vacuoles [30]. At present, however, the possibility of the UDP-glucose-dependent translocation at the tonoplast has been discounted, as discussed by Niemietz and Hawker [31]. Rather, sucrose is transported through the sucrose/proton antiport mechanism that is driven by a proton-translocating ATPase [31, 321. LOCALIZATION

OF UGPase

UGPase activity/protein has practically been found in every plant tissue investigated [2, lo]. For potato plants, e.g. immunoblots of proteins from various tissues have revealed the presence of UGPase in tubers, stolons, stems, leaves, roots, callus, as well as suspension cell cultures. The enzyme activity was markedly higher (per mg pro-

Glucose activation

and metabolism

through UDP-glucose

tein) in stolons, stems and tubers, when compared to roots and leaves [33]. In rice etiolated seedlings, UGPase is preferentially localized in the scutellum tissues. Based on the amount of UGPase protein, shoots, roots and dedifferentiated embryo cells contain ca a lo-fold lower level of the enzyme than scutellar tissues [34]. Concerning subcellular location in plant tissues, UGPase occurs almost exclusively in the cytosol (e.g. ref. [35]), but in some plants considerable activity was also found to be associated with membrane fractions [36,37]. Immunocytochemical and biochemical studies using antibodies against rice UGPase suggested that UGPase was localized at 90% in the cytosol of cultured rice cells, while the remaining 10% was distributed in the amyloplasts and Golgi membranes [34]. The latter organelles are involved in the production of cell wall polysaccharides, which either directly or indirectly are derived from UDPglucsoe [18]. In animal cells, ca 10% of all UGPase activity is also associated with Golgi membranes [38]. ENZYMOLOGY

OF UGl’ase

Structural properties

UGPase has been extensively studied from animal tissues, where its prime role is in the synthesis of UDPglucose, which acts as a precursor of other nucleotide sugars and oligo- and polysaccharides [39]. Studies on plant UGPase were relatively less detailed [2, 33, with most attention paid to the enzyme from non-photosynthetic tissues. UGPase has been highly purified from etiolated sorghum seedlings [40], Typka Zati$ofolia pollen 1411, soybean nodules [42], rice cells [34] and potato tubers [33, 433. Partially purified preparations of UGPase have been obtained from mung beans [44], Ldiurn longiforum pollen [45], Jerusalem artichoke tubers [46], and other plants (reviewed in ref.. [2,3, lo]). Purification procedures for UGPase usually included ammonium sulphate fractionation, gel filtration, as well as ion exchange and hydrophobic chromatography. Based on activity of the purified enzyme, the amount of UGPase in plant tissues can be estimated at ca 0.2-0.4% of total soluble proteins [33, 34,40,41,43]. Plant UGPase is an acidic protein [34], with a relatively low subunit M, value of ca 30-55 x 103. It is unclear whether the variation in the latter estimates reflects true differences in subunit structure or is the result of different experimental approaches. The potato tuber monomeric subunit M, of UGPase is 50-53 x lo3 [33, 431. This agrees with the reported values of 53-55 x lo3 for the enzyme from Typha latifolia [41], rice cell culture [34] and slime mould [39]. On the other hand, the enzyme from soybean nodules was reported to have M, values of 30 and 40 x 103, as determined by electrophoresis of denatured enzyme and by gel tiltration in native conditions, respectively [42]. Low M, values (possibly as low as 20 x 103’) were also reported for purified sorghum UGPase [40]. Preliminary evidence with non-denatured purified potato UGPase has suggested that the enzyme may have a dimeric structure in oitro [33], in contrast to

pyrophosphorylase

1509

an earlier report postulating a monomeric structure of the enzyme [43]. It has been suggested that potato tuber UGPase may exist in different oligomeric forms [33], but the mechanism of this isomerization phenomenon is not clear at present. The multimeric nature of plant UGPase would be consistent with the multiple subunit structure reported for UGPase from animal tissues [39]. Oligomerization of plant enzyme may possibly explain the ‘negative’ cooperativity phenomenon observed when glu-1-P or UTP were varied at a constant concentration of the second reactant [33]. Reversible oligomeric and/or conformational changes of protein structure have previously been implicated in hysteretic responses of UGPase from rat mammary glands [4fl. Substrate specijcity

Plant UGPase is specific for UDP-glucose as substrate. Numerous studies have demonstrated that the enzyme is specific both for the nucleotide and sugar part of UDPglucose [34,43-46]. Consistent with this observation is specificity of UGPase for UTP as the nucleotide donor [33,42,45]. UGPase has a requirement for divalent metal ion for activity. MgZ + is most effective, but Mn2 + could partially replace Mg* + ions at low concentration [42]. Kinetics of the enzyme at various Mg*+ concentrations suggest that the Mg-PP, and Mg-UTP complexes are the actual substrates for the reaction, and that free UTP acts as an inhibitor [40, 433. In the absence of Mg+, calf liver UGPase, which is an oligomer composed of eight identical subunits, could form enzyme-substrate complexes with UDP-glucose and UTP which were stable to Sephadex column chromatography even in the presence of 1 M EDTA [48]. Thus, the divalent metal ion is not required for substrate binding to the liver enzyme, even though enzyme activity totally depends on the presence of Mg*+ or on an appropriate substitute [48]. In contrast to the liver enzyme, potato tuber UGPase does not form any stable enzyme-substrate complex(es) [43]. Kinetic properties

The reaction catalysed by UGPase is freely reversible, at least under initial velocity assay conditions. Apparent equilibrium constant, defined as K,, = (glu-l-P)(UTP)/ (UDP-glucose)(PPi), experimentally determined for UGPase from artichoke tubers [46] and Typha pollen [41], was ca 6.2 and 2.1, respectively. For soybean nodule UGPase, the calculated K,, was 3.3 and 2.2, depending on assay pH [42]. The apparent K,, might also vary considerably (from 1.6 to 7.5) depending on the concentration of Mga+ in reaction mixtures for UGPase [40]. Similar K, values, retlecting mass action ratios slightly favouring glu-1-P formation, were reported for UGPase from non-plant tissues [39]. Plant UGPase has a relatively broad pH optimum both for the pyrophosphorolytic and UDP-glucose synthesis reactions, with the highest activity at ca pH 7.5-8.5 [33,34,40-42,45]. This pH range has usually been used

L. A. KLECZKOWSKI

1510

Table 1. Michaelis-Menten

constants (IQ) of UGPase from plant tissues & (mM) _. _. ._.__._

..___

..--.

Plant/tissue

UDP-glu

PP,

UTP

Glu-1-P

Reference

Potato tubers

0.12 0.14 0.44 0.06 0.33 NDt ND 0.13 0.07

0.11 0.13 0.23 0.05 0.36 0.03 ND 0.57 0.19

0.17 0.18* 0.30 0.03 0.42 ND 0.14 1.75 0.11

0.18 0.17* 0.94 0.05 0.61 ND 0.43 0.29 0.23

(43) (33) (46) (40) (34) (6) (45) (41) (42)

Artichoke tubers Sorghum etiolated seedlings Rice embryo cells Sycamore cells Loln4m pollen Typha pollen Soybean nodules3

*Determined at the concentration range of 0.05-5.0 mM. iNot determined. $Plant part of the nodule. Please note that UTP and PP, probably refer to magnesium complexes of these compounds.

in substrate kinetics and product inhibition studies of UGPase. The enzyme displays typical hyperbolic kinetics with all of its substrates. Only for potato tuber UGPase, a ‘negative cooperativity’ kinetics was found with respect both to UTP and glu-1-P as a varied substrate [33,49]. Plant UGPase usually has similar K,s of ca 0.1-0.4 mM for all its substrates (Table 1). However, with respect to the rypha pollen enzyme, a relatively high K, of 1.75 mM was reported for UTP [41], whereas UGPase from sycamore cells had a low K, of 0.03 mM for PP, [6]. Relatively low K,s for all substrates were determined for the sorghum enzyme [40]. The differences in the K,s for UGPase from different plants and tissues may partly be the result of distinct experimental approaches to measure substrate kinetics of the enzyme, but they may also reflect evolutionary changes in enzyme structure to adapt to various metabolic environments. The kinetic mechanism of UGPase from sorghum seedlings [40] and potato tubers [43], determined by substrate kinetics and product inhibition approaches, is consistent with the sequential binding of UTP and glu- lP, respectively, and the release of PP, followed by that of UDP-glucose. In other words, when considering both directions of the reaction, only UDP-glucose or UTP can bind to a free form of the enzyme, whereas PP, or glu- 1-P bind only to the enzyme form already associated with the corresponding substrate (Fig. 2). A similar mechanism was suggested for Typha pollen UGPase [41] and for the enzyme from non-plant tissues [l, SO]. The mechanism calls for a compulsory formation of an enzymesubstrates ternary complex before any product of the reaction can be released. The reaetion involves a transfer of the uridylyl moiety to the glu-1-P acceptor by processes involving cleavage of the pyrophosphate linkage in the UTP molecule. The uridylyl group transfer between UDP-glucose and UTP within the ternary complex does not involve any high energy enzyme-uridylyl intermediate [l]. It has been suggested [Sl] that the sequential ordered mechanism, with nucleoside triphosphate and

GIP

UTP

E

E-blP

PPI

(E-UT~-G’P~E-UDPG-PPI)

UDPG

E-UDPG

E

Fig. 2. The sequential ordered kinetic mechanism of UGPase. Please note that the reaction can proceed in both directions, with either UTP or UDP-glucose (UDPG) binding to the free form of the enzyme (E). GlP, glu-1-P. nucleoside

diphosphoglucose

binding

to the free form(s)

of the enzyme (Fig. 2), may represent an intrinsic feature of the nucleoside diphosphoglucose pyrophosphorylase family of enzymes. Regulation

The UGPase enzyme is strongly inhibited by UDPglucose (e.g. refs [3, 39, 40, 451). The inhibition (Ki of ca 0.1 mM) might be of physiological significance given the fact that some tissues have high content of UDP-glucose [3, 521. An obvious obstacle in such estimations is that the determination of (average) concentrations of metabolites in chloroplasts or cytosol might not reveal what the actual concentrations are at specific enzyme surfaces where localized differences and metabolic channelling might occur. UDP-glucose was a competitive inhibitor versus UTP for both soybean and potato UGPase [33, 421. For Typha pollen UGPase, however, UTP was a mixed inhibitor vs UDP-glucose, whereas UDP-glucose was a non-competitive inhibitor vs glu-1-P [41]. Lolium pollen UGPase showed non-competitive kinetics with UDP-glucose and other diphosphouridine sugars and acid derivatives vs glu-l-P, and had mixed competitive and non-competitive kinetic patterns with UTP [45]. The K, values varied from 0.13 mM for UDP-glucose to 9.6 mM for UDP-mannose. The inhibitors showed simple additive inhibition when provided at low concentrations in the presence of low substrate concentrations, suggesting that UGPase may be regulated through a

Glucose activation and metabolism through UDP-glucose pyrophosphorylase

cumulative feedback inhibition by precursors of polysaccharide biosynthesis [45]. Other inhibitors of UGPase include ATP and UDP [46]. On the other hand, the enzyme from soybean nodules was only weakly inhibited by UDP-glucose and the remaining products of UGPase reactions, and was not significantly at&ted by a number of other cell metabolites tested [42]. Activity of UGPase from the green alga Acetabuluria is controlled by blue light at the translational level [53]. Synthesis of the enzyme was accelerated ca four-fold during the period of biue-light-mediated activation. The increase in UGPase protein synthesis was caused by an overall blue light stimulation of cytosolic translation of the enzyme, whereas degradation of UGPase was unaffected by blue light [54]. To my knowledge, it is still unknown whether blue light can have any effect on activity of UGPase from higher plants. The enzyme is probably not regulated by any post-translational mechanism in uiuo (e.g. protein phosphorylation). For potato tuber UGPase, a linear relationship was found between the amount of protein and enzyme activity in transgenic plants expressing ‘antisense’ RNA for UGPase [55], which rather minimizes the possibility that the enzyme is regulated by some post-translation event. The oligomerization-induced changes in UGPase activity, as observed for the tuber enzyme [33], need yet to be proved to be of physiological significance. Overall, the accumulated evidence strongly indicates that the activity of UGPase in uivo is regulated mainly by substrate availability [42]. This is perhaps not surprising, given the fact that enzymes preceding/following the UGPase in metabolic pathways are thought to be prone to tine regulation by metabolite and/or post-translational mechanisms [S, 81. Substrate concentration, especially PP,, is the most likely factor affecting the direction and flux rate through this readily reversible reaction. PP, is probably provided by the reverse reaction of the PP,dependent phosphofructokinase [PFK(PP,)], which may regulate the concentration of PP, in the cytosol[7,8,56]. A removal of PP, may result in channelling of the reactants of UGPase toward UDP-glucose synthesis. This phenomenon is thought to be operating in source tissues, where UGPase is involved in sucrose biosynthesis. Expression of an Escherichia coli inorganic pyrophosphatase in the cytosol of tobacco and potato leaves resulted in a several-fold increase in sucrose and glucose. levels, and in drastic changes in growth and development [57J This occurred probably through making the UGPase reaction essentially unidirectional (by hydrolysing PP, produced by the enzyme), and thus channelling carbon exported from chloroplasts to UDP-glucose and then toward sucrose biosynthesis. On the other hand, in the sink tissue e.g. potato tubers, the effect of the heterologously expressed pyrophosphatase was to block sucrose metabolism, most probably due to the shortage of PP, for UGPase reaction [57]. Resides PFK(PP,), the PP, required for sucrose degradation can possibly also be supplied by a PP,/adenine nucleotide exchanger in the plastid envelope, or through a hypothetical synthesis of PP, at the tonoplast [58]. In barley seed endosperm, and

1511

UGPase

SUSY SUCROSE

-

ucpG7=-7

UTP

PPI

STARCH

+--

ADPG S-2,

AGPase

Fig. 3. A hypothetical ‘coupling’ of the UGPase and AGPase reactions in the cytosol of barley seed endosperm [59]. Please note that glu-1-P derived from sucrose might be directly incorp orated into ADP-glucose (ADPG) [star& precursor],-with the cytosolic AGPase acting as a PP,-recycling mechanism. UDPG, UDP-glucose.

perhaps in seeds of other cereal plants, at least part of the PP, may be generated through the reaction of a cytosolic form of AGPase (Fig. 3) [59]. The direction of carbon flow through the UGPase step may actually depend on a developmental stage of a tissue. In actively growing potato tubers, e.g., imported sucrose is most likely degraded by cytosolic SuSy to form fructose and UDP-glucose. The UDP-glucose molecule can then be utilized in the reaction of UGPase to form glu-l-P, the substrate for starch biosynthesis in the amyloplasts. In a sprouting tuber, however, where degradation of starch and sucrose formation occur, UGPase probably acts in the direction of UDP-glucose formation, which is then used in the reaction of SPS to make sucrose [7,49]. The ratio of SuSy to SPS activities is thought to be an important factor in the regulation of net direction of carbon flow through sucrose pathways. The ratio is usually high in carbon importing tissues and low in tissues which are net carbon exporters. However, there are exceptions to this rule (discussed in ref. [60]), which puts a note of caution in assigning definitive function to the two enzymes, and consequently, to UGPase, in predicting the direction of carbon flow through sucrose pathways. Site-directed affinity labelling

Recently, several photoafiity probes for studying nucleotide diphospho-sugar- and pyrophosphate-binding proteins have been developed (e.g. refs [Sl, 621). They have proved to be of value in probing protein structure of UGPase, thanks to the recent work by Fukui et al. (see ref. [63] for an overview). The active site of potato tuber UGPase was probed with several reactive analogues of UDP-glucose, e.g. uridine di- and triphosphopyridoxals, and pyridoxal(5’)diphospho-cc-D-glucose [64, 651. All these compounds similarly inactivated the enzyme in a stoichiometric manner and labelled as many as five different lysyl residues (Lys-263, Lys-329, Lys-367, Lys409 and Lys-410) [64]. Although unexpected, the finding could perhaps be rationalized by the fact that all the substrates of UGPase contain phosphate group(s) with negative charges, which may react with the positively charged lysyl residues. The results suggest that the UDPglucose binding site (or its close surroundings) contains a

1512

L. A. KLECZKOWSKI

high net positive charge due to the lysines present. Most labelled was Lys-367, regardless of the probe used. Based on comparison of the incorporation of analogues into each lysyl residue (comparative affinity labelling), Kazuta et al. [65], have presented a hypothetical model for the location of those residues around the substrate bound to the enzyme. A role for at least three lysine residues in substrate binding has also been implicated for UGPase from bovine liver [66]. In addition to analogues of UDP-glucose, an analogue of PP,, pyridoxal S-diphosphate, was used to probe the active site of potato tuber UGPase. This reagent bound effectively, in the presence of magnesium, to the enzyme-UDP-glucose complex, with Lys-329 being most labelled. Studies with mutant enzymes lacking Lys-263 and Lys-329 suggested the roles for these residues in binding of Mg-PP,, and in the UDP-glucose-induced conformational changes of the enzyme [67]. Interestingly, pyridoxal phosphate, a close analogue of pyridoxal diphosphate, is a functional analogue of 3-phosphoglycerate (PGA), but not of PP,, for spinach AGPase, which requires PGA for maximal activation [68].

MOLECULAR

BIOLOGY

OF UGPnse

Genes, mRNAs and regulation of gene expression

Little is known about gene and mRNA structure for plant UGPase. Based on biochemical studies implicating the presence of isozymes of UGPase in*plants [34, 421, there is probably more than one UGPase gene expressed in a given plant. In barley, the presence of at least two genes for UGPase has been indicated by means of genomic Southern analyses and RFLP mapping [Kilian, A., unpublished results]. At the transcript level, however, there is yet no evidence for the expression of more than one gene of UGPase in a given plant. Up until today nucleotide sequence of only one cDNA for plant UGPase, from potato tubers, has been published [69]. Partial amino acid sequence of the trypsindigested purified rice cell UGPase has high homology to the derived corresponding sequences of the tuber enzyme [34]. The derived amino acid sequence of potato UGPase [69] has significant homology (ca 50%) to those of the corresponding protein from slime mould and liver tissues, but very little or no homology is found between UGPase from eucaryotic and procaryotic species [66, 69-731. Positions of at least three lysine residues localized at the putative active site of the tuber UGPase [65] are conserved in the bovine liver enzyme [66]. Based on the coding sequence of the cDNA for potato UGPase, Katsube et al. [69] predicted the M, of 51800, which is consistent with estimates of ca 50-53 x lo3 for the denatured tuber enzyme [33, 433. Storage of mature potato tubers at low temperatures leads to an increase of the steady-state level of UGPase mRNA [SS], implicating a role of this enzyme in the process of ‘cold-sweetening’ [49]. Specific activity of the

enzyme from crude extracts of potato tubers also increases during this process 133,491. However, no details on mechanisms of gene expression of UGPase in plants have been published. For plant UGPase, regulation of gene expression occurs probably through translational rather than transcriptional control. As found by ‘antisense’ RNA inhibition of UGPase expression in transgenic potato tubers [55], only a fraction ofthe mRNA encoding UGPase might actually be translated into protein. Transgenie plants expressing ‘antisense’ RNA for UGPase showed up to a 95-96% reduction of UGPase activity, but no signs of changes in growth and development. Carbohydrate metabolism in tubers of these plants was not substantially affected, indicating that only 4% of the wild-type UGPase activity is sufficient for the enzyme to function in plant growth and development [SS]. Although certainly suggesting that UGPase is present in large excess in potato tubers, these data do not necessarily imply lack of any essential function for the enzyme. It should be emphasized that UGPAse activity, as measured in uitro, can be several orders of magnitude higher than those of other enzymes of carbohydrate synthesis/metabolism. For instance, even a 96% decrease in measurable activity of UGPAse would result in an enzyme whose activity is comparable to a fully-activated potato tuber AGPase [Kleczkowski, L. A., unpublished results]. Thus, it is conceivable that the remaining activity of UGPase in transgenic plants still fulfills its metabolic functions(s). Expression in heterologous systems

The full length cDNA for potato tuber UGPase was introduced into E. coli under the control of lac promotor [74]. Using this heterologous systems, the potato UGPase was expressed to up to 5% of total soluble proteins of the bacteria. The purified enzyme preparation was free of the E. coli host UGPase protein/ activity, and was structurally and catalytically identical with the enzyme purified from potato tubers, except for the absence of an N-terminal-blocking acetyl group [74]. These data have unequivocally proved the identity of the cDNA used for transformation as that coding for UGPase. The heterologous expression system was subsequently used to engineer kinetics and substrate binding properties of UGPase through site-directed mutagenesis of several critical lysine residues [63,67], which had been identified through affinity labelling approaches (see above). The lysines were substituted individually with glutamine to examine their functional roles [74]. Mutation of Lys-367 resulted in an almost completely inactive enzyme, supporting the critical role of this residue for UGPase activity. Kinetic properties of the mutant enzymes were consistent with the earlier proposed model of the configuration around and in the proximity of the active site of UGPase [65]. Random mutations resulting in substitutions for Pro-66 and Ile-153 led to a rapid inactivation of the recombinant enzyme during purification attempts [74], indicating importance of these amino acid residues in maintaining stability of the enzyme.

Glucose activation and metabolism through UDP-glucose pyrophosphorylase ARE THERE ISOZYMES OF IJGPase?

Very little is known about the possible presence of isozymes of UGPase in plants. UDP-glucose is required in all plants tissues, both at the very early stages of development (e.g. primary cell wall formation), throughout development (e.g. sucrose biosynthesis and breakdown in developing source and sink tissues, respectively) and at mature stages (e.g. secondary cell wall synthesis [75]). With so many cellular functions for UDP-glucose, it would be surprising, in my opinion, if only one gene of UGPase was expressed in all tissues during plant development. Presence of more than one gene for UGPase expressed in a given plant would be consistent with the apparent lack of reports on isolating mutant plants impaired in UGPase activity. More direct, although still unconclusive, evidence in favour of more than one UGPase enzyme was provided for purified soybean nodule enzyme, which could be resolved into three proteins bands that stained for UGPase activity on non-denaturing gels. The three bands were also observed in gels for crude proteins from the plant fraction of the nodule and in unnodulated roots [42]. Immunocytological analysis of rice cell culture using antibodies against UGPase revealed the label not only in the cytosolic compartment (90% of label), but also in the amyloplasts and Golgi membranes [34]. Since sorting of nuclei-encoded proteins to organelles and membranes requires presence of specific amino acid regions for intracellular targeting [763, the latter studies suggest the presence of more than one gene of UGPase expressed in rice cells.

PJ3RSPJXTIVES When reviewing the bulk of literature on plant UGPase, one is faced with a lot of reports on activities of the enzyme (usually in connection to sucrose pathways), but not with any comprehensive attempt to characterize. the properties of the enzyme from several tissues of a given plant species. In fact, no purihed UGPase from green leaves has been characterized. This is surprising, given the presumed involvement of UGPase in sucrose formation in source tissues. The question, of course, is whether it is worth the effort to study comprehensively leaf UGPase, if plants contain only one form of the enzyme that is ubiquitously distributed in all tissues. Moreover, leaves usually contain lower activity (per mg protein) of UGPase than other tissues [lo, 333, [Kleczkowski, L. A., unpublished results]. Possible approaches to tackle this problem should include a combination of biochemical and molecular biology techniques applied comprehensively to study enzymology and gene expression of UGPase, both in source and sink tissues of a given plant. The biochemical approach may involve search for selective inhibitors which could differentiate between activities of the supposedly distinct forms of UGPase. Inhibitors used at the enzymatic and physiological levels may eventually help elucidate the function of a given activity. Potential benefits of using isozyme-specific inhibitors have recently

1513

been reviewed in the context of photosynthetic metabolism [77]. Another strategy should involve screening of different tissues for the presence of UGPase protein and transcripts, using specific antibodies and cDNA probes. A side-by-side comparison of protein subunit or transcript size may eventually lead to the identification of isozymes of UGPase. Recent developments in molecular techniques, especially polymerase chain reaction (PCR) procedure, may make it possible to amplify full length or selected regions of cDNA(s) for UGPase from different tissues, using degenerate cDNA primers, for comparative restriction analysis. Similar approach with respect to AGPase has helped to identify several distinct cDNAs that are expressed in different tissues [78] and correspond to AGPase proteins with distinct regulatory properties [Sl]. Analyses of full-length cDNAs and derived amino acid sequences in search for ‘transit peptide’ and other ‘signal’ sequences necessary for intracellular targeting may represent the most convincing approach to identify possible minor non-cytosolic forms of plant UGPase. Studies with various affinity-labelling reagents and using recombinant wild-type and mutated forms of potato tuber UGPase have already made it possible to identify and modify the regions of UGPase involved in catalysis and substrate binding [63,74]. Further elucidation of the structure/function properties of UGPase should include studies on crystallization and X-ray analyses of purified wild-type and mutated UGPase to test the model for the three-dimensional location and the roles of critical lysine residues in UGPase structure [63, 651. Substrate labelling and site-directed mutagenesis studies on plant UGPase should bear direct consequences for research on the structure/function properties of AGPase, a related pyrophosphorylase which shares with UGPase two of the same substrates/ products (glu-lP and PP,), whereas the remaining substrates/ products (UTP/ATP and UDP-glucose/ADP-glucose) represent structurally closely related analogues. The two enzymes also share the same kinetic mechanism [So, 511, suggesting structural similarities in amino acid sequences that are responsible for substrate-binding and catalysis. When compared to AGPase, UGPase might be regarded as a model to study pyrophosphorylases at the protein/enzyme level, since (i) it is a single gene-encoded protein, (ii) it is very active both in source and sink organs, and (iii) it is stable both in crude and purified form. AGPase, in contrast, is a two-gene encoded protein, which needs activation (with the notable exception of the barley seed endosperm AGPase [51]), and is unstable during purification. Also of importance is further examination of subcellular location of UGPase in plant tissues. UDPglucose has, in some instances, been proposed to serve as a direct precursor for starch biosynthesis, being the substrate for a plastid granule-bound starch synthase(s) [21-23-J. Plastids may contain a minor isozyme of UGPase, in addition to the main cytosolic UGPase enzyme [34]. Alternatively, a pool of plastidic UDP-glucose could perhaps arise through the activity of a plastid membrane-bound UDP-glucose transporter [27]. Still

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L. A. KLECZKOWSKI

unclear is also the identity and possible roles of a membrane-bound UGPase activity found in some plants [33, 36, 373. Studies on the role of UGPase in metabolic conversions between starch and sucrose might be particularly important for cereal seeds, where AGPase, the first commi&d enzyme of starch biosynthesis, has recently been proposed to be localized both in the amyloplasts and cytosol, based on studies with barley enzyme [59]. Thus, the activity of the cytosolic seed UGPase, producing glu-1-P from sucrose, might be directly ‘coupled’ to that of cytosolic AGPase, utilizing glu-1-P for ADPglucose formation, providing a vital link between sucrose metabolism and starch formation (Fig. 3). The ADPglucose synthesized by cytoplasmic AGPase has been proposed [59] to be exported to the amyloplasts (where starch synthesis occurs) via the ADP-glucose/ adenylate carrier, which is present in a variety of plastids (e.g. ref. [ZS]). The AGPase reaction can recycle PP, used by UGPase. Assuming a close metabolic coupling between the two reactions, a net UDP-glucose to ADP-glucose conversion could be achieved without any net input of PP, (Fig. 3). Another feature of this scheme is that the extraplastidic AGPase may be an important generator of PPi in the cytosol of barley seed endosperm. Recent studies (carried out at the molecular level) on AGPase from maize seed endosperm also support the cytosolic location of the enzyme in this non-photosynthetic tissue L-791. Elucidating the properties of UGPase, which lies at the intersection of sucrose and starch metabolic pathways, and is the key activity responsible for maintaining intracellular levels of UDP-glucose in plants, should have an impact on a variety of biotechnological attempts to manipulate plant metabolism at the sucrose/starch interface [80,81]. Studies with heterologous pyrophosphatase in transgenic plants [57] have proved that the reversible reaction of UGPase can be made virtually unidirectional toward UDP-glucose synthesis, resulting in overproduction of sucrose in source tissues and inhibiting sucrose synthesis in sinks. Thus, if not by engineering properties of UGPase itself, there is still a potential to actually take advantage of this otherwise unregulated reversible step of sucrose pathways. With respect to prospects for affecting biosynthesis of cell wall polysaccharides in plants through manipulation of UGPase properties/ expression, the key question is whether the intracellular sites of structural polysaccharide synthesis (e.g. Golgi apparatus) contain their own membrane-bound UGPase, or whether UDP-glucose is provided entirely by the cytosolic enzyme. If UGPase is specifically targeted to the membranes, implying either distinct gene for this UGPase or some post-translational mechanism modifying structure of an otherwise soluble protein, this may have important biotechnological consequences. Genetic engineering of membrane-targeting of UGPase may affect the formation of structural polysaccharides, with UDP-glucose being the chief precursor. As more progress is being made in elucidating mechanisms of carbohydrate pathways, it is apparent that UGPase piays an important role in these

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