NAD (+) -dependent Formate Dehydrogenase from Plants.

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) widely occurs in nature. FDH consists of two identical subunits and contains neither prosthetic groups nor metal ions. This type of FDH was found in different microorganisms (including pathogenic ones), such as bacteria, yeasts, fungi, and plants. As opposed to microbiological FDHs functioning in cytoplasm, plant FDHs localize in mitochondria. Formate dehydrogenase activity was first discovered as early as in 1921 in plant; however, until the past decade FDHs from plants had been considerably less studied than the enzymes from microorganisms. This review summarizes the recent results on studying the physiological role, properties, structure, and protein engineering of plant formate dehydrogenases.

It is possible to distinguish two major FDH groups based on the differences in structure of these enzymes. the first group is comprised of formate dehydrogenases from anaerobic microorganisms and archae. FDHs in this group are heterooligomers with a complex quaternary structure and a high molecular weight. they are generally characterized by the presence of various prosthetic groups (iron-sulphur clusters, molybdenum and tungsten ions) in the active site and high sensitivity to oxygen [1,2]. the second group is comprised of nAD + -dependent formate dehydrogenases consisting of two identical subunits, both having two active sites and containing neither metal ions nor prosthetic groups in the protein globule. FDHs of this group belong to the superfamily of D-specific dehydrogenases of 2-oxyacids [3]. the reaction of formate oxidation catalyzed by a FDH from this group is the simplest example of dehydrogenation of carbonyl compounds, since there is neither the stage of proton transfer in the catalytic mechanism nor other stages of acid-base catalysis. the reaction rate is generally limited by the rate of hydride ion transfer from the substrate to the c4 atom of the nicotinamide ring [4]. thus, FDH can be used as a model enzyme for studying the mechanism of hydride ion transfer in the active site of dehydrogenases that belong to this superfamily. the active and systematic study of FDHs began in the early 1970s and was primarily devoted to enzymes from microorganisms. the physiological role of microbial FDHs is different. thus, in methanol-utilizing bacteria and yeast, this enzyme participates in the supply of energy to a cell, whereas in pathogenic bacteria and fungi FDH is a stress protein. the properties and protein engineering of FDH were thoroughly discussed in [5,6]. nAD + -dependent formate dehydrogenases from plants also belong to the second FDH group. recent studies have revealed that FDH also belongs to stress proteins in plants, similar to those found in pathogenic microorganisms. FDH synthesis strongly increases under the following conditions: drought, with abrupt changes in temperature, irradiation with hard ultravio-NAD + -dependent Formate Dehydrogenase from Plants let light, through the action of chemical agents [7][8][9], hypoxia [10], and action of pathogenic microorganisms [11]. the significance of the physiological role of this enzyme gives rise to the necessity of studying plant FDHs. till now, there are no publications in which the data on plant FDHs are systematized. the major features of plant formate dehydrogenases, as well as their kinetic properties and stability, are summarized in this review; a detailed description of the physiological role of FDH is also presented.

DISCOVERY HISTORY, LOCALIZATION, AND PHYSIOLOGICAL ROLE OF PLANT FDHS
Plant FDH was first found in beans (Phaseolus vulgaris) in 1921 [12].
the first attempt to provide a detailed description of FDH and to assess the role of this enzyme in plant metabolism was made by Davison in 1951 [13], by the example of formate dehydrogenases from pea and bean seeds. the role of FDH was believed to consist in the production of nADH, which was subsequently consumed for the formation of ethanol, succinate, and glutamate in coupled reactions. thus, the role of FDH as a "supplier" of nADH molecules to fit the various needs of a cell was first defined. An assumption concerning the mechanisms of the emergence of formate in a plant cell was made in the same study. According to the first hypothesis, formate could be formed along with ethanol and acetic acid as a result of anaerobic respiration. According to an alternative hypothesis, formate could be formed during the oxidation of glycolic acid; however, no unambiguous data that would corroborate a certain metabolic pathway, during which formate is formed, have been obtained thus far.
the first experiments for determining the localization of FDHs in plant cells were carried out in 1956. It was revealed that formate dehydrogenase activity was primarily present in mitochondria [14]. However, due to the fact that the samples under study were contaminated with other organelles, it couldn't be unequivocally proven that FDH is localized in mitochondria. In 1960, it was demonstrated that FDH was localized not only in seeds, but also in other plant parts. Formate dehydrogenase activity was revealed in cabbage and spinach leaves, roots of garden radish and turnip, cauliflower buds, and pumpkin fruits [15]. It was demonstrated using spinach leaves that there were at least two pathways for formate oxidation in a plant cell: by FDH in mitochondria and by peroxidase in peroxisomes [16]. It was ascertained in separate experiments that formate was oxidized by FDH in mitochondria at pH > 6, while peroxidase in peroxisomes plays the major role in formate oxidation at lower pH values. Later, it was shown that FDH in mitochondria was a component of a pro-tein complex with a molecular weight of approximately 200 kDa rather than being an individual molecule [17]. these complexes can potentially be formed by glycine decarboxylase and fumarase; their concentration increasing synchronically with rising FDH activity [9].
It was shown in systematic studies that formate dehydrogenase activity was strongly dependent both upon a plant and upon a particular plant organ containing the enzyme [18]. the dependence of enzymatic activity on the rate of oxygen consumption by a plant was also revealed. thus, in plants with high oxygen consumption (spinach, tobacco, etc.), formate dehydrogenase activity was higher than that in a plant with low oxygen consumption (the Leguminosae, lettuce, etc.) [18]. In this study, the hypothesis was postulated that in oxidation of nADH obtained via the formate dehydrogenase reaction, the accumulated energy was consumed for AtP formation via the electron transport chain, thus satisfying the energy demand of the cell [18]. unfortunately, high variation of FDH activity in different plants prevents the unambiguous answering of the question concerning the role of this enzyme in the metabolism. the relationship between formate metabolism and plant response to stress was first noted in 1978 [19], the increased formation of labelled carbon dioxide from formate was observed in barley, which was grown under overwatering conditions.
In 1992, research into the physiological role of formate dehydrogenase from plants was raised to a new level [20]. It was revealed that the mitochondria of non-photosynthesizing tissues of potato contained an unknown peptide with a molecular weight of approximately 40 kDa, which composed up to 9% of all mitochondrial proteins. cDnA of this polypeptide was cloned in 1993; the analysis of the amino acid sequence encoded by this cDnA demonstrated 55% homology with FDH from Pseudomonas sp. 101 [21]. comparison of the n-terminal sequences of natural FDH and polypeptide translated from cDnA revealed that the theoretical protein contained an additional signal peptide consisting of 23 amino acid residues, which provided the transport of pro-enzyme from cytoplasm inside mitochondria. Polypeptides of the same molecular mass were found in pea, tomato, and onion; the FDH content in the mitochondria of non-photosynthetic tissues (tubers and roots) was approximately eightfold higher than that in leaves [20]. Moreover, FDH concentration sharply increased in plants which were grown in the dark (pea stems, chicory leaves, carrot roots, sweet potato tubers, etc.) [20].
Actually, numerous data have been published supporting the fact that FDH is synthesized at a high concentration under conditions that are unfavourable to plant growth, e.g.: drought, low temperature, hard reVIeWS ultraviolet radiation, exposure chemical agents, deficiency of both light and iron, and low oxygen concentration. However, the response rate strongly depended on the type of interaction. thus, the fastest response of potato plants, which manifests itself by mrnA synthesis, was observed upon direct damage to plant tissue (~20 min), whereas the average response time for other types of impacts was equal to 8 h [7]. under conditions of iron deficiency, the amount of formate dehydrogenase mrnA in barley roots began to increase after 1 day, attaining the maximum value after 14 days [8,22], whereas the synthesis of formate dehydrogenase in leaves did not change. under anaerobic stress, the concentration of FDH mrnA in barley roots increased as early as after 12 h, attaining the maximum value by the 48th hour. In maritime pine, the biosynthesis of FDH is enhanced during a drought [23]. An increase in the level of FDH mrnA was also observed in Lotus japonicиs plants cultivated under conditions of hypoxia [10].
the gene expression in moss Physcomitrella patens responding to stress was studied in [24]. Moss plants were treated with abscisic acid (hormone inducing the transfer of plants to the rest period and being capable of decelerating stem growth, which is accumulated in seeds and buds in Autumn) followed by cooling to +4 о С. It was found that abscisic acid induced an increase in resistance of moss to low temperatures; it also altered the set of expressed genes. FDH is one of the enzymes whose gene is expressed under the action of abscisic acid. It turned out that the level of FDH gene expression increased during several hours following treatment with abscisic acid and when the plants were stored in cold temperature for 24 h. In the absence of abscisic acid, the response to the impact of low temperatures occurs much more slowly. treatment with sodium chloride at high concentrations (0.125 and 0.25 M) and mannitol (0.25 and 0.5 M) enhanced both the resistance of the moss to low temperatures and the expression of a number of genes, including the FDH gene. It was thus demonstrated that formate dehydrogenase was a stress protein both in higher plants and in mosses; the level of its biosynthesis could be regulated by hormones. Other plant hormones, such as auxin and cytokinin, also have an effect on FDH activity in higher plants [25]. the synthesis of FDH was also studied in Arabidopsis thaliana, being exposed to various factors. It was the first plant for which the complete nucleotide sequence of the genome was determined; therefore, in many cases A. thaliana is used as a model plant. the plants were sprayed with various c1-compounds (methanol, formaldehyde, and formate) followed by the northern blot analysis using FDH cDnA as a probe. the most intensive expression of the FDH gene was observed for treatment with formaldehyde or methanol. A lower level of expression was observed in the samples sprayed with formate and deionized water. An increase in the expression of the FDH gene was not recorded, neither in plants with their leaves pruned nor in the control sample. these data enabled one to reasonably conclude that the synthesis of FDH was induced to a larger extent not by the formate substrate, but by its reduced form (formaldehyde) [26]. It was also demonstrated [27] that one-carbon compounds (methanol, formaldehyde, and formate) induced the synthesis of FDH in plant leaves. Methanol has a direct effect on the synthesis of FDH transcripts, while its oxidized modifications (formaldehyde, formate) can act as signalling molecules. An analysis of the n-terminal region of the enzyme allowed one to assume that FDH can also be transported to chloroplasts. the dual localization of FDH, both in mitochondria and chloroplasts, was shown in transgenic A. thaliana and tobacco plants containing the AthFDH gene [28].
the origin of formate in cells of the plants exposed to stress remains unknown. the hypothesis has been put forward suggesting that formate may be synthesized during photorespiration, in the methanol metabolism, or from glyoxylate formed from different products of the Krebs cycle [7]. the formation of formate by the serine pathway as it takes place in bacteria [1] has been discussed, since the introduction of serine resulted in the increase in FDH concentration in potato plants. In further experiments [29], the transgenic potato with suppressed synthesis of FDH was obtained. It was revealed that formate that does not undergo further oxidation to carbon dioxide was accumulated in the tissues of transgenic plants. It was also shown that proline and its precursor glutamate were formed at a high concentration in transgenic potato under conditions of drought.
the metabolism of formate and its physiological role have been well studied [30]. In photosynthesizing potato tissues, formate is the major precursor of all other carbon-containing compounds; it is basically synthesized via ferredoxin-dependent fixation of carbon dioxide. In other tissues, formate is a side product of photorespiration and some enzymatic processes; its formation seems to result from the direct reduction of carbon dioxide in chloroplasts. In potato plants, the metabolism of formate is associated with the synthesis of serine.
A close relationship between the biosynthesis of formate and serine also exists in A. thaliana [31]. three lines of transgenic plants with enhanced expression of FDH were obtained. Formate concentration in transgenic plants was almost identical to that in wild-type A. thaliana. Following the introduction of labelled formate, the intensity of formation of radioactively labelled carbon dioxide in transgenic plants was much higher, whereas serine accumulation remained at the same level. transgenic A. thaliana plants with an enhanced level of FDH gene expression were also obtained in [32].
Phosphorylation is the most important method of metabolism regulation. 14 proteins of potato mitochondria, which can be presented in the phosphorylated form, have been discovered [33]; among them FDH can also be found. the amino acid residues of mitochondrial FDH of potato, which undergo phosphorylation (thr76 and thr333) have been identified [34]. An analysis of the FDH structure demonstrated that these two threonine residues were located on the surface of a protein globule and could be easily accessible for kinases catalyzing the phosphorylation process. A high phosphorylation level is observed in the Е1-α subunit of pyruvate dehydrogenase (PDH). the phosphorylation of both FDH and pyruvate dehydrogenase is regulated by the variation of concentrations of nAD + , formate, and pyruvate, which attests to the similarity in the mechanisms of regulation of the function of these enzymes. the level of phosphorylation of the enzyme is considerably reduced with increasing concentrations of nAD + , formate, and pyruvate. It is assumed that pyruvate can be converted into formate in the reaction catalyzed by pyruvate formate lyase (PFL) followed by the oxidation of formate with the participation of FDH.
Formate ion takes part in a great number of metabolic processes with complicated regulation, as can clearly be seen from the data provided. the most complete scheme of participation of formate in plant metabolism can be found in [11]. recent studies have attested to the fact that FDH content in plant mitochondria increases in response not only to physical and chemical factors, but also as a result of a "biological attack". the activation of biosynthesis of FDH was observed following infection of the english oak with the pathogenic fungus, Piloderma croceum [35]; wheat, with fungus Blumeria graminis f. sp. tritici [36]; and the common bean, with fungus Colletotrichum lindemuthianum [11]. the common bean genome contains three FDH genes; their expression is regulated by the type of exposure factor. It is assumed that synthesis of FDH in wheat is induced by methanol as a result of the impact of pectin methylesterase on pectin. In plants of the common tobacco Nicotiana attenuate damaged by Manduca sexta caterpillars, fatty acid conjugates initiating the synthesis of a number of proteins, including FDH, are released [37].
Summarizing this section, let us note that formate dehydrogenase is a universal enzyme involved in the cell stress response caused by both exogenic (negative ambient impact) and endogenic (deficiency of essential microelements, exposure to pathogens) processes. this fact attests to the key role of FDH in metabolism processes of higher plants. the production of mutant forms of FDH characterized by an enhanced catalytic activity and the insertion of their genes into the plant genome instead of wild-type enzyme genes represents a fundamentally new approach to the design of plants with an enhanced resistance to stress.

THE FEATURES OF THE PRIMARY STRUCTURE OF PLANT FDH
Due to the active development of mega-sequencing methods, a new genome structure of various organisms, including plants, is published almost every day. Searching in GenBank (GB), eMBL, and KeGG (http://www. genome.jp/) databases enabled us to find nucleotide sequences of genes (complete or as cDnA) of plant FDH from over 70 sources. Moreover, study [11] presents a number of sequences that are not present in the databases. the presence of a signal peptide, which is responsible for FDH transport from the cytoplasm to mitochondria, at the n-terminus of the synthesized proenzyme is the distinctive feature of plant FDHs [21]. Bacterial and yeast FDHs contain no signal peptides. the genes of FDHs from a number of pathogenic fungi also contain the nucleotide sequence encoding the signal peptide. However, depending on the condition of a host cell, the rnA synthesized from the FDH gene undergoes alternative splicing, resulting in the formation of different mrnAs encoding proteins both with and without the signal peptide [49]. Figure 1 shows the signal sequences of formate dehydrogenases from various sources. the potential specific sequences providing the transport of an enzyme to mitochondria are underlined. the residue, after which the cleavage of the signal peptide occurs, is shown in green italics. In the majority of formate dehydrogenases, it is the arginine residue. Serine residue (FDH from sorghum SbiFDH1, castor bean tree rcoFDH1), lysine (grape VviFDH1), proline (FDHs from soybean SoyFDH1 and SoyFDH2, isoforms 1 and 2) can also be found in this position. the signal sequence of FDH is enriched in amino acid residues containing hydroxyl or positively charged groups and is capable of form-reVIeWS Sinorhizobium meliloti nitrogen-fixing Sinorhizobium meliloti SmeFDH Ae006469, [52] reVIeWS ing an amphiphilic α-helix. the signal sequence is highly conserved. thus, the deletion of only two n-terminal amino acids blocks the transport of the enzyme to the mitochondria [53]. It was ascertained that the n-terminal MAM motif enabled swift transport of the enzyme to mitochondria to be performed.  Fig. 1 (the names are highlighted in pink). two enzyme isoforms from Aj. capsulatus (AjcFDH1 and AjcFDH3) are also given, which result from the alternative splicing of mrnA [49]. Some sequences also contain an arginine residue, at which the cleavage of the signal peptide may occur (the residue is highlighted in pink italics). A similar mechanism of the FDH transport to different cell organelles seems to exist in fungi. the Lys and Val residues that are totally conserved in all formate dehydrogenases are shown in red in Fig. 1. the n-terminal sequence of formate dehydrogenase from Pseudomonas sp. 101 that is highly homologous to the nterminal region AjcFDH3 without the signal peptide is shown for comparative purposes. As can be seen in Fig. 1, signal peptide sequences in enzymes from the plants belonging to one family (the family Solanaceae: tomatoes, potato; the family Gramineae: rice, barley, rye, etc.) have a high degree of homology.
the differences in sequences of signal peptides are more strongly
Plant enzymes are highly homologous (approximately 80%); the similarity between the bacterial and plant FDHs is ~50%.

OBTAINMENT OF PLANT FORMATE DEHYDROGENASES
In plants, FDHs are localized in mitochondria; they therefore constitute a small part of all soluble proteins within a cell, and the isolation of an enzyme directly from plants is a labour-intensive and time-consuming procedure. Plant FDHs are mostly not very stable, which results in an appreciably significant inactivation of the enzyme during the extraction. therefore, the specific activity of the resulting FDH specimens is much lower than one may expect ( Table 2). the purified plant FDH was first obtained in 1951 from pea and French bean [13]. In 1983, FDH was extracted from soybean G.max in appreciably large amounts; this enabled the determination of the amino acid composition of plant formate dehydrogenase [57].
Since 1993 several cDnA of plant FDH have been cloned: in 1993 -from potato [21], in 1998 -from barley [8], in 2000 -from rice [41] and A. thaliana [26,27]. transgenic A. thaliana and tobacco plants expressing AthFDH were developed [28]; however, the yield of the enzyme was not very high. the expression of full size cDnA of potato FDH in Escherichia coli cells yielded insoluble inclusion bodies [21].
the active and soluble recombinant formate dehydrogenase from plants was first obtained in E. coli cells, [41]; however, the protein content was very low, approximately 0.01% of all soluble proteins within the cell. In our laboratory, we obtained E. coli strains, which are superproducers of active FDHs from A. thaliana (Ath-FDH) and soybean G. max (isoenzyme SoyFDH2) with an enzyme content attaining 40% of all soluble proteins in the cell [58] (the gene of soybean FDH was kindly provided by Professor n. Labrou from the Agricultural university of Athens (Greece); the FDH gene from A. thaliana, by Professor J. Markwell from the university of nebraska (Lincoln, uSA)). there is no system of transport to mitochondria in E. coli cells; therefore, in order to obtain the "natural enzyme", we deleted the sequences encoding the signal peptide from the cDnA [58]. After the optimization of the cultivation conditions, the yield of recombinant FDH from A. thaliana and soybean G. max reached 500-600 mg/l of the medium [6]. A procedure was developed which enabled the obtaining of several hundred milligrams of homogenous FDH specimen via a single chromatographic stage per extraction run. thus, all necessary conditions for the performance of systematic studies of FDHs from A. thaliana and soybean G. max were provided, including genetic engineering experiments and X-ray diffraction determination of the structure. the experiments were successfully carried out for the expression of FDH from L. japonicas in E. coli cells [10].  * NA -Data not available.

KINETIC PROPERTIES OF PLANT FORMATE DEHYDROGENASE
reVIeWS most thoroughly studied bacterial and yeast enzymes are provided for comparative purposes. Several major conclusions can be drawn from the data in Table 2: 1. It is clearly visible that in case of FDH from A. thaliana the multistage extraction of the natural enzyme is accompanied by a considerable loss in activity. the specific activity of the specimens, even those obtained from transgenic plants, is several times lower than the activity of recombinant AthFDH expressed in E. coli cells. It is noteworthy that AthFDH belongs to stable FDHs. In terms of thermal stability, it is even superior to FDH from Moraxella sp. c2 and yeast C. boidinii (Table 3). It is obvious that the degree of inactivation of less stable, FDHs (in particular, in the case of SoyFDH) being extracted from natural sources will be higher. this fact should be taken into consideration when analysing formate dehydrogenase activity in plants.
2. the specific activity of recombinant AthFDH and SoyFDH is comparable with that of formate dehydrogenases from microorganisms; although it is inferior to FDHs from methylotrophic bacteria and baker's yeast. As previously mentioned, a partial inactivation of enzymes may occur during the extraction; therefore, calculation of the catalytic constant based on the values of specific activity and molecular weight may give underestimated k cat values. this fact is of particular significance for SoyFDH, the thermal stability of which is much lower than that of other FDHs (see the thermal stability section below). therefore, we developed a procedure for determining the concentration of active sites of recombinant SoyFDH based on the quenching of the intrinsic fluorescence of the enzyme as it is titrated with azide ion in the presence of coenzyme nAD + [64]. the azide ion is a strong competitive inhibitor of Soy-FDH (K I = 3.6 × 10 -7 M). therefore, a linear dependence of quenching of FDH fluorescence on the azide concentration is observed under the conditions ensuring the equimolar binding of enzyme and inhibitor. the k cat value determined from these experiments actually coincided with that calculated using the specific activity and molecular weight. the obtained data attest to the fact that despite the low thermal stability, SoyFDH extracted using the designed procedure is not inactivated. today, this procedure is being actively used for determining the k cat values of the mutant forms of SoyFDH.
3. Plant FDHs are characterized by significantly lower values of the Michaelis constants with respect to both formate and coenzyme nAD + in comparison with the bacterial and yeast enzymes. It is very important for the practical application of FDH. today, recombinant FDHs from Pseudomonas sp. 101 and C. boidinii, whose chemical and thermal stability have been enhanced by protein engineering methods, are used for the regeneration of the reduced coenzyme (nADH or nADPH) in the synthesis of chiral compounds using dehydrogenases [6]. 4. Almost all FDH that have been studied are highly specific to the coenzyme nAD + . their catalytic activity in the reaction with nAD + is higher than that in the reaction with nADP + by a factor of 2500 (PseFDH) to 3 × 10 9 . the exception is the recently described FDH from pathogenic bacteria Burkholderia stabilis, which is twenty-six-fold more efficient in the reaction with nADP + in comparison with nAD + [51]. It should be noted that the mutant PseFDH, whose coenzyme specificity changed from nAD + to nADP + , was obtained as early as in 1993 [6,68]; this enzyme has been successfully used for nADPH regeneration [69,70]. Having the lowest К m value with respect to nADP + among all nAD + -specific wild-type FDHs, SoyFDH has a huge potential for obtaining the nADP + -specific enzyme [58] ( Table 2).

THERMAL STABILITY OF PLANT FORMATE DEHYDROGENASES
Prior to obtaining recombinant AthFDH and SoyFDH in E. coli cells, no systematic studies on the thermal stability of plant FDHs were performed. According to [59], the values of К M with respect to formate and nAD + decrease after the incubation of transgenic AthFDH for 5 min at 60°С; however, the specific activity value decreased by 13 times. We performed systematic studies of the thermal stability of recombinant AthFDH and SoyFDH using two approaches: determining the kinetics of thermal inactivation and the differential scanning calorimetry [71,72]. It was revealed that the thermal inactivation of plant FDHs occurred via a monomolecular mechanism, similar to the FDHs from bacteria and yeasts. time dependences of decrease in the activity of AthFDH and SoyFDH are described by the kinetics of the first-order reaction, the observed constant of inactivation rate was independent of enzyme concentration. the thermal stabilities of AthFDH and Soy-FDH strongly differ. AthFDH lost 50% of its activity after 20 min at 59.5°С, whereas SoyFDH lost 50% of its activity at 52.8°С. the difference by almost 7°С corresponds to a difference in the inactivation rate constants of more than 1000 times. AthFDH is actually inferior in terms of its thermal stability only to FDH from Pseudomonas sp. 101 (63.0°С, the most stable FDH among the known ones) [6] and Staphylococcus aureus (62.0°С) and is superior to all known microbial formate dehydrogenases. In contrast, SoyFDH is inferior in terms of stability to all known FDHs, with the exception of the enzyme from baker's yeast that is characterized by another inactivation mechanism. the temperature dependences of the rate constants of inactivation of Ath-FDH and SoyFDH are described by the transition state theory. the calculated values of activation enthalpy ΔH ≠ and activation entropy ΔS ≠ are listed in Table 3. It can be noticed that thermal stability of formate dehydrogenases correlates well with the ΔH ≠ and ΔS ≠ values. the highest values are typical of the most stable Pse-FDH, whereas the lowest ones refer to SoyFDH.
the results of experiments studying the thermal stability of plant FDHs using differential scanning calorimetry are in close agreement with the data of inactivation kinetics. In Table 3 the values of the temperature and heat of the phase transition are shown. All investigated FDHs are characterized by their high cooperativity of the denaturation process. PseFDH is characterized by the highest melting point, whereas this value is lower by a factor of 2.5 for SoyFDH. the melting point of Ath-FDH is higher than that of cboFDH and MorFDH. experiments on enhancing the thermal stability of recombinant SoyFDH using genetic engineering techniques are currently being performed.

CHEMICAL STABILITY OF PLANT FORMATE DEHYDROGENASES
FDH has been actively used in the process of synthesizing optically active compounds catalyzed by dehydrogenases. In these processes, the operational stability (the operating time of an enzyme) plays the greatest role. under conditions of biocatalytic process, the inactivation of FDH is associated with either oxidation with oxygen or chemical modification of sulfhydryl groups of the enzyme. the operational stability of PseFDH and cboFDH was enhanced by site-directed mutagenesis of two cys residues [6,65]. As previously mentioned, ) is likely to increase in a cell under the same conditions. It should be expected that in order to ensure high activity under stress, plant FDHs should be far more resistant to the action of these agents than formate dehydrogenases functioning under non-stress conditions. In order to verify this hypothesis, the kinetics of inactivation of recombinant AthFDH, SoyFDH, PseFDH, FDH from wild-type S. aureus (SauFDH) and three mutant PseFDH, where one or two cys residues were replaced, under the action of hydrogen peroxide was studied. FDH from S. aureus is a stress protein as well, since the biosynthesis of this enzyme increases by 20 times when staphylococci pass from planktonic growth to biofilm formation [73]. It was revealed that the inactivation rate of AthFDH and SoyFDH under the action of Н 2 О 2 is virtually equal and is 18 times lower than that in wild-type PseFDH. under the same conditions, the stability of SauFDH was sixfold higher than that in plant enzymes [74]. PseFDH with the stability identical to that of SoyFDH was successfully obtained only after double cys145Ser/cys255/Ala replacement. It was thus demonstrated that plant and bacterial FDHs synthesized under stress impact indeed possess a higher chemical stability than formate dehydrogenases, whose synthesis is induced by other factors. Moreover, studying the thermal inactivation of formate dehydrogenase in the presence of hydrogen peroxide can be used to comparatively assess the in vivo chemical stability of FDHs.

STRUCTURAL STUDIES OF PLANT FORMATE DEHYDROGENASES
FDH has not been well studied from a structural perspective. until recently, only the FDH structures from three sources, namely, bacteria Pseudomonas sp. 101 and Moraxella sp. c2 (unbound enzyme, and the enzyme-nAD + -azide ternary complex) and C. boidinii yeast (structures of two mutant forms of an apoenzyme) were deposited into the protein data bank (PDB). In case of formate dehydrohenases, free enzyme is in its «open» conformation. When the FDH-nAD + -azide ternary complex is formed (its structure being considered similar to that of the enzyme in the transition state), a considerable compacting of the protein globule occurs, and FDH is transformed into the «closed» state. the development of a highly efficient system of expression of AthFDH and SoyFDH in E. coli cells enabled the transition to their crystallization and identification of their structure., nowadays AthFDH structures both in open and closed conformations (3nAQ and 3n7u, with resolution of 1.7 and 2.0 Å, respectively) have been identified. enzyme crystals were produced in space [75] in order to obtain AthFDH structure in open conformation with higher resolution (3JtM, 1.3 Å), Fig. 4 shows the structures of apo-and holoFDHs (the open and closed conformations, respectively). A more detailed analysis of these structures will be provided in individual articles. the crystals of the SoyFDH-nAD + -azide ternary complex of FDH from soybean G. max have been obtained both on the earth and in space. the structures of these complexes were identified with resolution of 1.9 Å; the deposition of these structures into the PDB data bank is in progress.

CONCLUSIONS
Both our own and the published data attest to the fact that plant FDHs are extremely significant, in particular when responding to stress factors. Biosynthesis regulation and the physiological role of FDHs are diverse and complex; they have not been completely elucidated thus far. the systematic investigation of the structure and function of these enzymes is still in its infancy. the results of these studies are of significant interest to fundamental science and are of great practical importance. Production of mutant forms of FDH with a high activity opens a new approach for the design of plants with an enhanced resistance to unfavourable factors. More active mutant enzymes will supply the cell with the energy required to more efficiently overcome unfavourable effects of stress with the same expression level of FDH. Soybean FDH is also considered to be an exceptionally promising FDH for the design of a highly efficient biocatalyst for nAD(P)H regeneration in the synthesis of optically active compounds using dehydrogenases. the natural enzyme is notable for its high operational stability and has one of the lowest values of the Michaelis constant with respect both to nAD + and formate among all of the known FDHs. However, in order to practically implement SoyFDH, its catalytic activity and thermal stability need to be enhanced. We are currently performing active research in this direction.