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NAD+-dependent Formate Dehydrogenase from Plants

A.A. Alekseeva1,2,3, S.S. Savin2,3, V.I. Tishkov1,2,3,* 1Chemistry Department, Lomonosov Moscow State University 2Innovations and High Technologies MSU Ltd 3Bach Institute of Biochemistry, Russian Academy of Sciences *E-mail: [email protected] Received 05.08.2011 Copyright © 2011 Park-media, Ltd. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT 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 , , 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 from microorganisms. This review summarizes the recent results on studying the physiological role, properties, structure, and protein engineering of plant formate dehy- drogenases. KEYWORDS plant formate dehydrogenase; physiological role; properties; structure; expression; ; protein engineering. ABBREVIATIONS FDH – formate dehydrogenase; PseFDH, CboFDH – formate dehydrogenases from bacteria Pseudomonas sp. 101 and Candida boidinii, respectively; SoyFDH, AthFDH – plant formate dehydrogenases from soybean Glycine max and Arabidopsis thaliana, respectively.

INTRODUCTION this group is the simplest example of dehydrogenation NAD+-dependent formate dehydrogenases (FDHs) of carbonyl compounds, since there is neither the stage [EC 1.2.1.2] belong to the family of enzymes catalyzing of proton transfer in the catalytic mechanism nor other the oxidation of the formate ion to , cou- stages of acid–base catalysis. The reaction rate is gener- pled with NAD+ reduction to NADH: ally limited by the rate of hydride ion transfer from the substrate to the C4 atom of the nicotinamide ring [4]. - + HCOO + NAD → CO2↑ + NADH. Thus, FDH can be used as a model for studying the mechanism of hydride ion transfer in the It is possible to distinguish two major FDH groups of dehydrogenases that belong to this superfamily. based on the differences in structure of these enzymes. The active and systematic study of FDHs began in The first group is comprised of formate dehydrogenas- the early 1970s and was primarily devoted to enzymes es from anaerobic microorganisms and archae. FDHs from microorganisms. The physiological role of micro- in this group are heterooligomers with a complex qua- bial FDHs is different. Thus, in -utilizing bac- ternary structure and a high molecular weight. They teria and yeast, this enzyme participates in the supply are generally characterized by the presence of various of energy to a cell, whereas in pathogenic bacteria and prosthetic groups (iron–sulphur clusters, molybdenum fungi FDH is a stress protein. The properties and pro- and ions) in the active site and high sensitivity tein engineering of FDH were thoroughly discussed in to [1, 2]. [5, 6]. The second group is comprised of NAD+-dependent NAD+-dependent formate dehydrogenases from formate dehydrogenases consisting of two identical plants also belong to the second FDH group. Recent subunits, both having two active sites and containing studies have revealed that FDH also belongs to stress neither metal ions nor prosthetic groups in the protein proteins in plants, similar to those found in pathogen- globule. FDHs of this group belong to the superfamily ic microorganisms. FDH synthesis strongly increases of D-specific dehydrogenases of 2-oxyacids [3]. The re- under the following conditions: drought, with abrupt action of formate oxidation catalyzed by a FDH from changes in temperature, irradiation with hard ultravio-

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let light, through the action of chemical agents [7–9], tein complex with a molecular weight of approximately hypoxia [10], and action of pathogenic microorganisms 200 kDa rather than being an individual molecule [17]. [11]. The significance of the physiological role of this en- These complexes can potentially be formed by glycine zyme gives rise to the necessity of studying plant FDHs. decarboxylase and ; their concentration in- Till now, there are no publications in which the data creasing synchronically with rising FDH activity [9]. on plant FDHs are systematized. The major features of It was shown in systematic studies that formate plant formate dehydrogenases, as well as their kinetic dehydrogenase activity was strongly dependent both properties and stability, are summarized in this review; upon a plant and upon a particular plant organ con- a detailed description of the physiological role of FDH taining the enzyme [18]. The dependence of enzymatic is also presented. activity on the rate of oxygen consumption by a plant was also revealed. Thus, in plants with high oxygen DISCOVERY HISTORY, LOCALIZATION, consumption (spinach, tobacco, etc.), formate dehydro- AND PHYSIOLOGICAL ROLE OF PLANT FDHs genase activity was higher than that in a plant with Plant FDH was first found in beans (Phaseolus vulgaris) low oxygen consumption (the Leguminosae, lettuce, in 1921 [12]. etc.) [18]. In this study, the hypothesis was postulated The first attempt to provide a detailed description that in oxidation of NADH obtained via the formate of FDH and to assess the role of this enzyme in plant dehydrogenase reaction, the accumulated energy was was made by Davison in 1951 [13], by the consumed for ATP formation via the electron trans- example of formate dehydrogenases from pea and port chain, thus satisfying the energy demand of the bean seeds. The role of FDH was believed to consist cell [18]. Unfortunately, high variation of FDH activity in the production of NADH, which was subsequently in different plants prevents the unambiguous answer- consumed for the formation of ethanol, succinate, and ing of the question concerning the role of this enzyme glutamate in coupled reactions. Thus, the role of FDH as in the metabolism. The relationship between formate a “supplier” of NADH molecules to fit the various needs metabolism and plant response to stress was first noted of a cell was first defined. An assumption concerning in 1978 [19], the increased formation of labelled carbon the mechanisms of the emergence of formate in a plant dioxide from formate was observed in barley, which cell was made in the same study. According to the first was grown under overwatering conditions. hypothesis, formate could be formed along with etha- In 1992, research into the physiological role of for- nol and acetic acid as a result of . mate dehydrogenase from plants was raised to a new According to an alternative hypothesis, formate could level [20]. It was revealed that the mitochondria of be formed during the oxidation of glycolic acid; how- non-photosynthesizing tissues of potato contained ever, no unambiguous data that would corroborate a an unknown peptide with a molecular weight of ap- certain , during which formate is proximately 40 kDa, which composed up to 9% of all formed, have been obtained thus far. mitochondrial proteins. cDNA of this polypeptide was The first experiments for determining the localiza- cloned in 1993; the analysis of the sequence tion of FDHs in plant cells were carried out in 1956. It encoded by this cDNA demonstrated 55% homology was revealed that formate dehydrogenase activity was with FDH from Pseudomonas sp. 101 [21]. Compari- primarily present in mitochondria [14]. However, due son of the N-terminal sequences of natural FDH and to the fact that the samples under study were contami- polypeptide translated from cDNA revealed that the nated with other organelles, it couldn’t be unequivocal- theoretical protein contained an additional signal pep- ly proven that FDH is localized in mitochondria. In 1960, tide consisting of 23 amino acid residues, which provid- it was demonstrated that FDH was localized not only in ed the transport of pro-enzyme from cytoplasm inside seeds, but also in other plant parts. Formate dehydro- mitochondria. Polypeptides of the same molecular mass genase activity was revealed in cabbage and spinach were found in pea, tomato, and onion; the FDH con- leaves, roots of garden radish and turnip, cauliflower tent in the mitochondria of non-photosynthetic tissues buds, and pumpkin fruits [15]. It was demonstrated us- (tubers and roots) was approximately eightfold higher ing spinach leaves that there were at least two path- than that in leaves [20]. Moreover, FDH concentration ways for formate oxidation in a plant cell: by FDH in sharply increased in plants which were grown in the mitochondria and by peroxidase in peroxisomes [16]. It dark (pea stems, chicory leaves, carrot roots, sweet po- was ascertained in separate experiments that formate tato tubers, etc.) [20]. was oxidized by FDH in mitochondria at pH > 6, while Actually, numerous data have been published sup- peroxidase in peroxisomes plays the major role in for- porting the fact that FDH is synthesized at a high con- mate oxidation at lower pH values. Later, it was shown centration under conditions that are unfavourable to that FDH in mitochondria was a component of a pro- plant growth, e.g.: drought, low temperature, hard

VOL. 3 № 4 (11) 2011 | Acta naturae | 39 REVIEWS ultraviolet radiation, exposure chemical agents, defi- level of expression was observed in the samples sprayed ciency of both light and iron, and low oxygen concen- with formate and deionized water. An increase in the tration. However, the response rate strongly depended expression of the FDH gene was not recorded, neither on the type of interaction. Thus, the fastest response of in plants with their leaves pruned nor in the control potato plants, which manifests itself by mRNA synthe- sample. These data enabled one to reasonably conclude sis, was observed upon direct damage to plant tissue that the synthesis of FDH was induced to a larger ex- (~20 min), whereas the average response time for other tent not by the formate substrate, but by its reduced types of impacts was equal to 8 h [7]. Under conditions form (formaldehyde) [26]. It was also demonstrated [27] of iron deficiency, the amount of formate dehydro- that one-carbon compounds (methanol, formaldehyde, genase mRNA in barley roots began to increase after and formate) induced the synthesis of FDH in plant 1 day, attaining the maximum value after 14 days [8, leaves. Methanol has a direct effect on the synthesis of 22], whereas the synthesis of formate dehydrogenase in FDH transcripts, while its oxidized modifications (for- leaves did not change. Under anaerobic stress, the con- maldehyde, formate) can act as signalling molecules. centration of FDH mRNA in barley roots increased as An analysis of the N-terminal region of the enzyme al- early as after 12 h, attaining the maximum value by the lowed one to assume that FDH can also be transported 48th hour. In maritime pine, the biosynthesis of FDH is to chloroplasts. The dual localization of FDH, both in enhanced during a drought [23]. An increase in the lev- mitochondria and chloroplasts, was shown in transgenic el of FDH mRNA was also observed in Lotus japonicиs A. thaliana and tobacco plants containing the AthFDH plants cultivated under conditions of hypoxia [10]. gene [28]. The gene expression in moss Physcomitrella patens The origin of formate in cells of the plants exposed to responding to stress was studied in [24]. Moss plants stress remains unknown. The hypothesis has been put were treated with abscisic acid (hormone inducing the forward suggesting that formate may be synthesized transfer of plants to the rest period and being capable during photorespiration, in the methanol metabolism, of decelerating stem growth, which is accumulated in or from glyoxylate formed from different products of seeds and buds in Autumn) followed by cooling to +4оС. the Krebs cycle [7]. The formation of formate by the It was found that abscisic acid induced an increase in serine pathway as it takes place in bacteria [1] has been resistance of moss to low temperatures; it also altered discussed, since the introduction of serine resulted in the set of expressed genes. FDH is one of the enzymes the increase in FDH concentration in potato plants. In whose gene is expressed under the action of abscisic further experiments [29], the transgenic potato with acid. It turned out that the level of FDH gene expres- suppressed synthesis of FDH was obtained. It was re- sion increased during several hours following treatment vealed that formate that does not undergo further with abscisic acid and when the plants were stored in oxidation to carbon dioxide was accumulated in the tis- cold temperature for 24 h. In the absence of abscisic sues of transgenic plants. It was also shown that pro- acid, the response to the impact of low temperatures line and its precursor glutamate were formed at a high occurs much more slowly. Treatment with sodium chlo- concentration in transgenic potato under conditions of ride at high concentrations (0.125 and 0.25 M) and man- drought. nitol (0.25 and 0.5 M) enhanced both the resistance of The metabolism of formate and its physiological role the moss to low temperatures and the expression of a have been well studied [30]. In photosynthesizing potato number of genes, including the FDH gene. It was thus tissues, formate is the major precursor of all other car- demonstrated that formate dehydrogenase was a stress bon-containing compounds; it is basically synthesized protein both in higher plants and in mosses; the level of via ferredoxin-dependent fixation of carbon dioxide. In its biosynthesis could be regulated by hormones. Other other tissues, formate is a side product of photorespira- plant hormones, such as auxin and cytokinin, also have tion and some enzymatic processes; its formation seems an effect on FDH activity in higher plants [25]. to result from the direct reduction of carbon dioxide in The synthesis of FDH was also studied in Arabidop- chloroplasts. In potato plants, the metabolism of for- sis thaliana, being exposed to various factors. It was the mate is associated with the synthesis of serine. first plant for which the complete nucleotide sequence A close relationship between the biosynthesis of for- of the genome was determined; therefore, in many mate and serine also exists in A. thaliana [31]. Three cases A. thaliana is used as a model plant. The plants lines of transgenic plants with enhanced expression of were sprayed with various C1-compounds (methanol, FDH were obtained. Formate concentration in trans- formaldehyde, and formate) followed by the Northern genic plants was almost identical to that in wild-type blot analysis using FDH cDNA as a probe. The most in- A. thaliana. Following the introduction of labelled for- tensive expression of the FDH gene was observed for mate, the intensity of formation of radioactively la- treatment with formaldehyde or methanol. A lower belled carbon dioxide in transgenic plants was much

40 | Acta naturae | VOL. 3 № 4 (11) 2011 REVIEWS higher, whereas serine accumulation remained at the fact attests to the key role of FDH in metabolism proc- same level. Transgenic A. thaliana plants with an en- esses of higher plants. The production of mutant forms hanced level of FDH gene expression were also ob- of FDH characterized by an enhanced catalytic activity tained in [32]. and the insertion of their genes into the plant genome Phosphorylation is the most important method of instead of wild-type enzyme genes represents a funda- metabolism regulation. 14 proteins of potato mitochon- mentally new approach to the design of plants with an dria, which can be presented in the phosphorylated enhanced resistance to stress. form, have been discovered [33]; among them FDH can also be found. The amino acid residues of mitochondrial THE FEATURES OF THE PRIMARY FDH of potato, which undergo phosphorylation (Thr76 STRUCTURE OF PLANT FDH and Thr333) have been identified [34]. An analysis of the Due to the active development of mega-sequencing FDH structure demonstrated that these two threonine methods, a new genome structure of various organisms, residues were located on the surface of a protein globule including plants, is published almost every day. Search- and could be easily accessible for kinases catalyzing the ing in GenBank (GB), EMBL, and KEGG (http://www. phosphorylation process. A high phosphorylation level genome.jp/) databases enabled us to find nucleotide is observed in the Е1-α subunit of pyruvate dehydro- sequences of genes (complete or as cDNA) of plant FDH genase (PDH). The phosphorylation of both FDH and from over 70 sources. Moreover, study [11] presents a is regulated by the variation of number of sequences that are not present in the da- concentrations of NAD+, formate, and pyruvate, which tabases. Table 1 lists the names of the plants and the attests to the similarity in the mechanisms of regula- contracted notations of FDHs. FDHs that are character- tion of the function of these enzymes. The level of phos- istic of various microorganisms, such as enzymes from phorylation of the enzyme is considerably reduced with methylotrophic bacteria Pseudomonas sp. 101 (the most increasing concentrations of NAD+, formate, and pyru- well studied FDH to this moment), Moraxella sp. C2, vate. It is assumed that pyruvate can be converted into pathogenic bacteria Burkholderia stabilis and Borde- formate in the reaction catalyzed by pyruvate formate tella bronchiseptica RB50 (Alcaligenes bronchisepticus), (PFL) followed by the oxidation of formate with uncultured marine alpha proteobacteria and nitrogen- the participation of FDH. fixing bacteria Sinorhizobium meliloti, yeasts Saccha- Formate ion takes part in a great number of meta- romyces cerevisiae and Candida boidinii, were used for bolic processes with complicated regulation, as can comparison. clearly be seen from the data provided. The most com- The presence of a signal peptide, which is responsi- plete scheme of participation of formate in plant me- ble for FDH transport from the cytoplasm to mitochon- tabolism can be found in [11]. dria, at the N-terminus of the synthesized proenzyme Recent studies have attested to the fact that FDH is the distinctive feature of plant FDHs [21]. Bacterial content in plant mitochondria increases in response and yeast FDHs contain no signal peptides. The genes of not only to physical and chemical factors, but also as FDHs from a number of pathogenic fungi also contain a result of a “biological attack”. The activation of bio- the nucleotide sequence encoding the signal peptide. synthesis of FDH was observed following infection of However, depending on the condition of a host cell, the the English oak with the pathogenic fungus, Piloderma RNA synthesized from the FDH gene undergoes alter- croceum [35]; wheat, with fungus Blumeria graminis native splicing, resulting in the formation of different f. sp. tritici [36]; and the common bean, with fungus mRNAs encoding proteins both with and without the Colletotrichum lindemuthianum [11]. The common bean signal peptide [49]. genome contains three FDH genes; their expression is Figure 1 shows the signal sequences of formate de- regulated by the type of exposure factor. It is assumed hydrogenases from various sources. The potential spe- that synthesis of FDH in wheat is induced by methanol cific sequences providing the transport of an enzyme to as a result of the impact of pectin methylesterase on mitochondria are underlined. The residue, after which pectin. In plants of the common tobacco Nicotiana at- the cleavage of the signal peptide occurs, is shown in tenuate damaged by Manduca sexta caterpillars, fatty green italics. In the majority of formate dehydroge- acid conjugates initiating the synthesis of a number of nases, it is the arginine residue. Serine residue (FDH proteins, including FDH, are released [37]. from sorghum SbiFDH1, castor bean tree RcoFDH1), Summarizing this section, let us note that formate lysine (grape VviFDH1), proline (FDHs from soybean dehydrogenase is a universal enzyme involved in the SoyFDH1 and SoyFDH2, isoforms 1 and 2) can also be cell stress response caused by both exogenic (negative found in this position. The signal sequence of FDH is ambient impact) and endogenic (deficiency of essential enriched in amino acid residues containing hydroxyl microelements, exposure to pathogens) processes. This or positively charged groups and is capable of form-

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Table 1. The sources and contracted notations of formate dehydrogenases considered in the present study

Organism FDH Reference Latin name English name

PLANTS

Antirrhinum majus Common Snapdragon AmaFDH1 KEGG: EST 2545 Aquilegia formosa x Aquilegia ApuFDH1 KEGG: EST 273 Buttercup pubescens ApuFDH2 [11] Arabidopsis thaliana Mouse-ear cress AthFDH EMBL AF208029 Brachypodium distachyon Purple false brome BdiFDH1 [11] BnaFDH1 [11] Brassica napus Rapeseed BnaFDH2 KEGG: EST 21261 Brassica oleracea Cabbage BolFDH1 [11] Cryptomeria japonica Japanese cedar CjaFDH1 KEGG: EST 5066 Carica papaya Papaya CpaFDH1 KEGG: EST 3924 Citrus reticulata Tangerine CreFDH3 KEGG: EST 11052 Citrus sinensis Sweet orange CsiFDH1 [11] Coffea canephora Coffea canephora CcaFDH1 KEGG: EST 1007 Festuca arundinacea Tall fescue FarFDH1 KEGG: EST 5855 SoyFDH1 GB AK244764, [38] SoyFDH2 GB Bt094321 Glycine max Soybean SoyFDH3 GB AK243932, [38] SoyFDH4 GB BT095613 SoyFDH5 KEGG: EST 19520 Gossypium arboreum Tree cotton GarFDH1 KEGG: EST 1085 Gossypium hirsutum Tree cotton GhiFDH1 KEGG: EST 19680 Gossypium raimondii Tree cotton GraFDH1 KEGG: EST 213 Helianthus annuus Common sunflower HanFDH1 [11] Hordeum vulgare Barley HvuFDH1 GB D88272, [8] Ipomoea batatas Sweet potato IbaFDH EMBL BM878811 Lactuca saligna Willowleaf lettuce LsaFDH1 KEGG: EST 1616 Lotus japonicus – LjaFDH1 GB FM865900, [10] Lycopersicon esculentum Tomato LesFDH1 GB AJ849378 Malus domestica Apple MdoFDH EMBL CN496368 Manihot esculenta Cassava MesFDH1 KEGG: EST 2788 Medicago truncatula Barrel Medic MtrFDH1 KEGG: EST 1503 Mesembryanthemum crystallinum Common ice plant McrFDH GB BE035085 Nicotiana tabacum Common tobacco NtaFDH1 [11] Oryza sativa Japonica group Japanese rice OsaFDH_Ja GB AK065872, [39] Oryza sativa indica cultivar-group Indian rice OsaFDH_In GB CT832868, [40] Oryza sativa Rice OsaFDH1 AB019533, [41] Panicum virgatum Switchgrass PviFDH1 KEGG: EST 8602 Phaseolus vulgaris Common bean PvuFDH1 GB ACZ74695, [42] Phyllostachys edulis Moso bamboo PedFDH GB FP093692 Physcomitrella patens Moss Physcomitrella patens PpaFDH GB XM001768721, [43] Picea glauca White spruce PglFDH1 KEGG: EST 2327 Picea sitchensis Sitka spruce PsiFDH GB EF085163, [44] Pinus pinaster Maritime pine PpiFDH1 KEGG: EST 174

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PtaFDH1 [11] Pinus taeda Loblolly pine PtaFDH2 KEGG: EST 2972 PtaFDH3 KEGG: EST 15504 Populus nigra Lombardy poplar PniFDH2 KEGG: EST 7989 Populus tremula Aspen PtmFDH1 KEGG: EST 4757 Populus trichocarpa Western balsam poplar PtrFDH1 PtrFDH1 GB XM002320465, [45] Prunus persica Peach tree PpeFDH1 KEGG: EST 4281 Quercus robur English oak QroFDH1 GB AJ577266.2, [35] Raphanus raphanistrum subsp. Wild radish RraFDH1 KEGG: EST 15157 raphanistrum Ricinus communis Castor bean tree RcoFDH1 GB XM_002517292 Saccharum officinarum Sugarcane SofFDH1 KEGG: EST 18227 Solanum tuberosum Potato StuFDH1 GB Z21493, [21] SbiFDH1 GB XM002438363, [46] Sorghum bicolor Sorghum SbiFDH2 GB XM002454363, [46] Taraxacum officinale Common dandelion TofFDH1 [11] Theobroma cacao Cacao tree TcaFDH1 KEGG: EST 10274 Triphysaria pusilla Dwarf owl’s-clover TpuFDH1 KEGG: EST 5550 Triticum aestivum Common wheat TaeFDH1 TaeFDH1 GB AK332605, [47] Vigna unguiculata Cowpea VunFDH1 KEGG: EST 6491 Vitis vinifera Grape VviFDH1 GB XM002278408 Yucca filamentosa Bear grass YfiFDH1 YfiFDH1 [11] Zea mays Maize ZmaFDH GB EU967680, [48] Zingiber officinale Ginger ZofFDH1 KEGG: EST 5316

fungi

Aspergillus oryzae Fungus Aspergillus oryzae AorFDH1 NCBI XM001827498 Mycosphaerella graminicola Fungus Mycosphaerella graminicola MgrFDH GB AW180713 180985 Penicillium marneffei Fungus Penicillium marneffei PmaFDH1 GB XM002153251 AjcFDH1 Ajellomyces capsulatus Darling’s disease fungus [49] AjcFDH3

YEASTS

Methylotrophic yeast Candida Candida boidinii CboFDH EMBL AF004096 boidinii Saccharomyces cerevisiae Baker’s yeast SceFDH EMBL Z75296

BACTERIA

Methylotrophic bacterium Pseudomonas sp. 101 PseFDH [50] Pseudomonas sp. 101 Methylotrophic bacterium Moraxella sp. С2 MorFDH EMBL Y13245 Moraxella sp. Bacterium Burkholderia stabilis BstFDH [51] Burkholderia stabilis Bacterium Bordetella bronchiseptica RB50 Bordetella bronchiseptica RB50 BbrFDH EMBL BX640441 (Alcaligenes bronchisepticus) (Alcaligenes bronchisepticus) Uncultured marine alpha proteo- Uncultured marine alpha proteobac- BbrFDH EMBL BX640441 bacterium terium Nitrogen-fixing Sinorhizobium Sinorhizobium meliloti SmeFDH Ae006469, [52] meliloti

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BdiFDH1------MAMWRAAARQLVDRALVGSRAAHTSAG-SKKIVGVF PedFDH------MAMWRAAARQLVDRALGSRAAHTSAG-SKKIVGVF ing an amphiphilic α-helix. The signal FarFDH1------MWRAAARHLVDRALGSRAAHTSAG-SKKIVGVF sequence is highly conserved. Thus, the HvuFDH1------MAAMWRAAARQLVDRAVGSRAAHTSAG-SKKIVGVF TaeFDH1------MAAMCRAAARQLVDRAVGSRAAHTSAG-SKKIVGVF deletion of only two N-terminal amino SbiFDH1------MAMWRAAARQLVDRALGSSAAHTSAG-SKKIVGVF acids blocks the transport of the enzyme SofFDH1------MAMWRAAARKLVDRALGSRAAHTSAG-SKKIVGVF ZmaFDH------MAMWRAAARQLVDRALGSRAAHTSTG-SKKIVGVF to the mitochondria [53]. It was ascer- PviFDH1------MAMWRAAARQLVDRALGARAAHTSAG-SKKIVGVF tained that the N-terminal MAM motif OsaFDH1------MAMWRAAAGHLLGRALGSRAAHTSAG-SKKIVGVF SbiFDH2------MAMRRAAQQAARFAMGPHVPHTAPAARSLHASAG-SKKIVGVF enabled swift transport of the enzyme LjaFDH1------MAMKRAASSAVRSLLTAPTPNPSSSIFSRNLHASGG-KKKIVGVF to mitochondria to be performed. Fig- MtrFDH1------MAMKRAASTLITASSKISSLSSPSSIITRDLHASGG-KKKIVGVF PvuFDH1------MAMKRAAASSAFRSLLSSTFSRNLH------KKIVGVF ure 1 shows the N-terminal sequences VunFDH1------MAMRRAAGSSAIRSLFSSTFSRNLHVSGE-KKKIVGVF of 63 plant FDHs; 35 enzymes of those SoyFDH3------MAMMKRAASSSVRSLLSSSSTFTRNLHASGE-KKKIVGVF SoyFDH4------MAMMKRAASSALRSLIASSSTFTRNLHASGE-KKKIVGVF have the MAM motif at position 1–3. SoyFDH1--MSNFTLKMSDPTLAQQHLVKVHTTTHETVVTTHNHNQTPSINASGE-KKKIVGVF A number of plant FDHs have similar SoyFDH2--MLNFTLKMSDPTLAQPHLVKVHTT-LETVVTTHNHNHRPSINASGE-KKKIVGVF SoyFDH5------MAMKRAVQSLLASSSTLTRNLHASGE-KKKIVGVF motifs at their N-terminal fragments: TofFDH1------MAIAMKRAAAAAATRAISSANSGSIFTRHLHASSG-KKKIVGVF MAAM (in two plant FDHs) and MAS LsaFDH1------MAIAMKSDSGSILTRHLHASSG-KKKIVGVF HanFDH1------MAMSMAMKRSAAAATRALSSATSSSILTRDLHSSSG-KKKIVGVF (in eight plant FDHs). The N-terminal AmaFDH1------MAMKRAAVTAVRALTSSAPSSVLTRGLHASPG-SKKIVGVF amino acid sequences of isoenzymes 1 TpuFDH1------MAMKRAVASTVGAITSSGNPASSVLARYLHASPG-SKKIVGVF LesFDH1------MAMRRVASTAARAIASPSSLVFTRELQASPG-PKKIVGVF and 2 of soybean FDHs are significantly StuFDH1------MAMSRVASTAARAITSPSSLVFTRELQASPG-PKKIVGVF different from those of other plant for- NtaFDH1------MAMRRVASTAARAFASSSPSPSSLVFTRELQASPG-SKKIVGVF VviFDH1------MAMMKRVAESAVRAFALGSTSGALTKHLHASAG-SKKIVGVF mate dehydrogenases, both in terms of MesFDH1------MKRAATSAIRAFPSSFGISGSSALGRHLHASAG-SKKIVGVF their composition (it starts with MSN GraFDH1------MKQVANSAIKAIANSGSSSLLTRQLHASPG-SKKIVGVF GarFDH1------MKQVANSAIKAIANSGSSSLLTRQLHASPG-SKKIVGVF an MLN) and size, which attests to the GhiFDH1------MKQVANSAIKAIANSGSSSLLTRQLHASPG-SKKIVGVF possible specific function of these FDH TcaFDH1------MKQVASSAIKALANSGSSSVLTRQLHASPG-SKKIVGVF CcaFDH1------MAMKRVAASALRAFTSSGNSTSSLLTRRLHASPG-SKKIVGVF isoforms. The N-terminal sequences of IbaFDH------MAMRRVAASGLRAFASYGNPS--LLTRQLHASPG-SKKIVGVF FDHs from fungi Aspergillus oryzae, ApuFDH1------MATRKAVVLGAQSLLRSSSTSSPSIRNLHASSE-SKKIVGVF ApuFDH2------MKKAALSTVQSVLSSSSFSTRLVRHSHTSPG-SKKIVGVF As. flavus, Penicillium marneffei, My- YfiFDH1------MAMLRAAKQAIQTLGSRIPSSSTFSRHLHASPG-SKKIVGVF cosphaerella graminicola, and Ajel- ZofFDH1------MAMLRAAKHAMRALGSRAPDASPFARMLHASTG-SKKIVGVF QroFDH1------MAGAATSAIKSVLTRHLHASPG-SKKIVGVF lomyces capsulatus are also shown in RcoFDH1------MKSYSKRIALWLQRIEDGASDVTEELGVSINSASAG-SKKIVGVF Fig. 1 (the names are highlighted in BnaFDH2------MAMRRVTRAAIRASCVSSSSSGYFARKFNASSGDSKKIVGVF BolFDH1------MAMRRVIRASCVSSSSTGYLARKFHASSGDSKKIVGVF pink). Two enzyme isoforms from Aj. CsiFDH1------MAMKRVASSAINAFASSGYLRSSSRFSRHY-ASSG-RKKIVGVF capsulatus (AjcFDH1 and AjcFDH3) MdoFDH------MASKGVIASAVRALASSGSSASSTTFTRHLHASGG-SKKIVGVF PpeFDH1------MKGVIASAVRTLASSGSSASSTTFTRHLHASAG-SKKIVGVF are also given, which result from the al- CpaFDH1------MKRAATSAIKAFASSQTSFSGLSTNFARNLHASPG-SKKIVGVF ternative splicing of mRNA [49]. Some CreFDH3------MKRVASSAINAFASSGYLRSSSRFSRHY-ASSG-SKKIVGVF BnaFDH1------MAMRRITGAIRASCVSSSSSGYFARQFHASSGDSKKIVGVF sequences also contain an arginine resi- AthFDH------MAMRQAAKATIRACSSSSSSGYFARRQFNASSGDSKKIVGVF due, at which the cleavage of the signal RraFDH1------MAMQAAIRACVSSNSSGFLSRHLHASSGDSKKIVGVF PpiFDH1------MASRRAVISAFRAASRRPICSPVSSIASSVRELHAPAG-SNKIVGVF peptide may occur (the residue is high- PtaFDH2------MASRRSVISAFRAASRRPICSPVSSVRELHAPAG-SNKIVGVF lighted in pink italics). A similar mecha- PtaFDH3------MASKRAVISAFRAASRRPICSPVSSIASSVRELHAPAG-SNKIVGVF PtaFDH1------MASRRSVISAFRAASRRPICSPVS----SVRELHAPAG-SNKIVGVF nism of the FDH transport to different PsiFDH------MASKRAVISTFRAASRKPIFSSVSPLASSVRELHAPAG-SNKIVGVF cell organelles seems to exist in fungi. PglFDH1------MASKRAVISTFRAASRRPICSSVSPLASSVRKLHAPAG-SNKIVGVF CjaFDH1------MASKRAVKS---AAQ------AFSPL-SSIRALHAPAG-PNKIVGVF The Lys and Val residues that are to- PtrFDH1------MAMKRAATSAIRAFSSSSPASSVSSGSSTRLLHASAE-SKKIVGVF tally conserved in all formate dehydro- PtmFDH1------MAMKRAATSAIRAFSSSSPSSSLSSGSSTRLLHASAE-SKKIVGVF PniFDH2------MAMKRAATSAIRAFSSASPASSVSSGSSTRLLHASAE-SKKIVGVF genases are shown in red in Fig. 1. The McrFDH------MKRATASAIRAMVASSTNSSTILSRNLHASSD-SKKIVGVF N-terminal sequence of formate dehy- AorFDH1------MTFARSITRAALKASPLSRASRTFSSSSSAQSKVLMVL MgrFDH--MVFARSSLRMARPASSLLSQRATASFTQRGANLARAGGVRTLTSTSSRQGKVLLVL drogenase from Pseudomonas sp. 101 PmaFDH1------MVFSRSIPRALQRPATSLLAIPARQWRAPVFSGVRTLTASAPRQGKVLMVL that is highly homologous to the N- AjcFDH3------MGRGLPRSSSAPFPGYNTQSYGPLPRLPSLTRVITLTASPKLQGKVLLVL AjcFDH1------MGKVLLVL terminal region AjcFDH3 without the PseFDH------MAKVLCVL signal peptide is shown for comparative purposes. As can be seen in Fig. 1, sig- nal peptide sequences in enzymes from Fig. 1. Signal sequences of plant formate dehydrogenases. Here and in Figs. 2,3, abbreviations of enzymes are those from Table 1. Plant en- the plants belonging to one family (the zymes are highlighted in green; fungi enzymes are highlighted in ma- family Solanaceae: tomatoes, potato; genta; FDHs from bacteria are highlighted in blue. Specific sequences the family Gramineae: rice, barley, rye, that are responsible for the transport of the enzyme to mitohondria are etc.) have a high degree of homology. underlined. The residue, after which the signal peptide is eliminated, are In most plants, FDH is located in the highlighted in green italics. mitochondria; however, the thorough

44 | Acta naturae | VOL. 3 № 4 (11) 2011 REVIEWS

0.1 SoyFDH2 SoyFDH1 PtmFDH1 PtrFDH1 PniFDH2 MtrFDH1 PtaFDH3 PtaFDH2 PtaFDH1 PpiFDH1 PsiFDH PglFDH1 CjaFDH1 VviFDH1 MesFDH1 PpeFDH1 MdoFDH CsiFDH1 CreFDH3 TcaFDH1 GraFDH1 GhiFDH1 GarFDH1 CpaFDH1 IbaFDH CcaFDH1 TpuFDH1 AmaFDH1 YfiFDH1 ZofFDH1 SbiFDH2 NtaFDH1 StuFDH1 LesFDH1 McrFDH SoyFDH3 SoyFDH4 VunFDH1 PvuFDH1 LjaFDH1 TofFDH1 LsaFDH1 HanFDH1 QroFDH1 ApuFDH2 ApuFDH1 SoyFDH5 PpaFDH RraFDH1 BolFDH1 BnaFDH2 BnaFDH1 AthFDH RcoFDH1 OsaFDH_Ja OsaFDH_In OsaFDH1 TaeFDH1 HvuFDH1 SofFDH1 FarFDH1 PviFDH1 SbiFDH1 ZmaFDH PedFDH BdiFDH1

Fig. 2. Phylogenetic tree of N-terminal sequences for plant formate dehydrogenases.

VOL. 3 № 4 (11) 2011 | Acta naturae | 45 REVIEWS study of the signal peptide of the enzyme from A. thaliana has shown that the enzyme can also be trans- ported to chloroplasts. The N-termi- nal fragment of this enzyme strongly HVL I T PF H P A Y -VSAER HVL I T PF H P A Y -VTAEK HVL I T PF H P A Y -VTAER HVL I T PF H P A Y -VTAER HVL I T PF H P A Y -VSAER HVL I T PF H P A Y -VTADR HVL I S T PF H P A Y -VTAER HVI I S T PF H P A Y -VTAER HVI I S T PF H P A Y -VTAER HVL I T PF H P A Y -VTAER differs from the signal sequences of HVL I S T PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I T PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I T PF H P A Y -VTEER HVL I T PF H P A Y -VTAER HVL I T PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I T S PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I S T PF H P A Y -VTAER HVL I S T PF H P A Y -MTAER HVL I T PF H P A Y -VTAER D A EV I T PF H P G Y -LTADR D AD I T PF H P A Y -ITKER D AE I V T PF F P A Y -ISRNR FDHs from potato, barley, and rice. D A EV I T PF H P G Y -LTAER E KE L V D AD I S Q PF F P Y -LTKER E RE L V D AE I S Q PF W P A Y -LTAER E RE L V D AD I S Q PF W P A Y -LTPER There is a hypothesis that under cer- E KH L H D AE V I S Q PF W P A Y -LTAER E RR L P D AD V I S Q PF W A Y -LTAER E RE L P D AD V I S Q PF W A Y -LTAAR tain conditions AthFDH localizing in chloroplasts is capable of catalyzing

the reverse reaction, i.e., the conver- ble for transport of the enzyme sion of carbon dioxide into formate [27]. It was shown by using another T LVV N D K DA-PGCVA G H T LVV S D K DG-PDSVF G H T LVV S D K DG-PDSVF algorithm to compare the signal pep- G YE LVT T I D K DPEPTSTVDRELK G H E LVV T SS K DG-PDSEL G H T LIV S D K DG-PDSEF G H T LVV A D K DG-PGSVF G H tides of FDHs that all the enzymes, with the exception of FDH from to- mato plants, were capable of being transported both to mitochondria T able 1. Plant enzymes are highlighted in green; and chloroplasts [28]. An analysis of signal sequences carried out using E K L Y G CT NK LG IAN W LKDQ H LIT T S D EGETSELDKHIP- E K L G CI NE LG IRNFIEEQ P G L ATENE LG R K W LEEK H T LVT S D DGA-NSKFDQELV P R L G TT E NE LG K W IEDQ H T LVT S D EGE-NSKFDQELV NP N F V G CV E GA LG IRN W LESK H YIV T D K EG-PNSELEKHIEDM NP N F V G CV E GA LG IRD W LESK H YIV T D K EG-FNSELEKHIEDM NP N F V G CV E GA LG IRD W LESK H YIV T D K EG-LNSELEKHIEDM NP N F V G CV E GA LG IRG W LESQ H Q YIV T D K EG-PNCELEKHIEDM NP N F V G CV E GA LG IRE W LESK H YIV T D K EG-LNSELEKHIEDM NP N F V G CA E HA LG IRG W LESQ H Q YIV T D K DG-PNCELEKHIADA NP N F V G CV E GA LG IRE W LEAQ H YIV T D K EG-LDSELEKHIPDL NP N F V G CV E GA LG IRE W LESQ H Q YIV T D K EG-PDSELEKHIPDA NP N F V G CV E GA LG IRE W LESQ H Q YIV T D K EG-PDSELEKHIPDA NP N F L G CV E NS LG IRD W FYSE H Q YIV T P D K DA-PDCELEKHIPEM NP D F V G CA E NA LG IRE W LESK H YIV T P K DG-PDCELEKHIPDL NP N F L G CA E NA LG IRE W LESK H Q YIV T P D K EG-PDCELEKHIPDL NP N F L G CA E NA LG IRE W LESK H Q YIV T P D K EG-PDCELEKHIPDL NP N F V G CV E GA LG IRD W LESQ H Q YIV T D K EG-PDCELEKHIPDL NP N F V G CV E GA LG LRP W LESQ H Q YIV T D K EG-PDCELEKHIPDL NP N F V G CV E GA LG LRE W LESQ H Q YIV T D K EG-PDCELEKHIPDL NP N F L G CT E NA LG IRQ W LESQ H Q YIV T S D K EG-PHCELEKHIPDL NP N F V G CA E GA LG IRD W LESQ H Q YIV T D K DG-PNSELEKHIPDL NP K F V G CV E GS LG IRD W LESQ H Q YIV T D EG-PNCELEKHIPDM NP N F V G CV E GS LG IRD W LESQ H Q YIV T D K EG-PNSELEKHIPDL NP N F S G CA E GA LG IRD W LESQ H Q YIV T D K EG-PHCELEKHIPDL NP N F L G CV E NA LG IRN W LESQ H YIV T D K EG-PNCELEKHIPDL NP N F L G CV E NA LG IRN W LESQ H YIV T D K EG-PDCELEKHIPDL NP N F L G SQ E RA LG IRD W LESQ H YIV T D K EG-PNCELEKHIEDL NP N F L G CE E RA LG IKD W LESQ H K YIV T D DG-PDCELDKHIQDL NP N F V G CV E NA LG IRN W LESQ H Q YIV T D K EG-PNCELEKHIPDL NP N F L G CV E NA LG IRD W LESQ H Q YIV T D K EG-PDCELEKHIPDL NP N F L G CV E NA LG IRE W LESN H Q YIV T D K EG-PDCELEKHIPDV NP N F L G CV E NA LG IRE W LESN H Q YIV T D K EG-PDCELEKHIPDA NP N F L G CV E NA LG IRE W LESK H Q YIV T D K EG-PDCELEKHIPDL NP N F V G SL E GA LG IRD W LESQ H Q YIV T D K EG-LDSELEKHIPDL PG A L G SV S GE LG LRKFLEGQ PG Q L G SV S GE LG LRKYLESN the Predotar, TargetP, and Mitoprot PG A L G SV S GE LG LRKYLESQ PG E L V G SV S GA LG LRGYLEAH PG H L G CV S GE LG LRPFLQAR PG E L G CV S GE LG LRKFLEDA

software [11] confirmed the fact that Pseudomonas sp. 101 (PseFDH). The asterisk and red font FDH is basically localized in mito- P T KAIDF-E P T KAIDF-T chondria. P T KAIDF-T P T AGPPGFR P T LALDF-T P T KGIDF-N In plants, formate dehydrogenase is often represented by several iso- forms, also known as isoenzymes, P TLERYPGGQTL P KIDHYPGGQTL whose synthesis is determined by P RIDKYPDGQTL P VITRYADGQTA P AIARYPGGATL P KIEKYPDGQTL the condition of a plant. The differ- ences in the isoenzyme composition of FDHs in healthy and affected palms Pericopsis mooniana are used to select trees when selective cutting Y KAG E HA------DK Y QAG E YA------DK Y QAG E YA------DK Y KAG E YA------DK Y KGG E YA------DK Y KGG E YA------DR Y KAN E YA------AL Y KGN E YA------KL Y KGN E YA------KL Y KAN E YA------SM Y KAN E YA------SM Y KAN E YA------EM Y KAN E YA------EM Y KAN E YA------AM Y KAN E YF------TK Y KAN E YY------EK Y DAK E YA------AK Y KAN E YA------SM Y KAN E YA------SM Y KAN E NA------AL Y KAN E YA------SM Y KAN E YA------SK Y KAN E YA------SK Y KAN E YA------EL Y KAN E YA------EL Y KAN E YA------TE Y KAN E YA------TK Y KAN E YA------SL Y KAN E YA------SL Y KAN E YA------SL Y KAN E YA------SL is performed [54]. Polymorphism is CVL Y PD P VDGY PHYVRDTI also typical for FDH from the almond K VA CVL Y DD P VDGY TAYARDGL K VL CVL Y DD P VDGY KTYARDDL KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF K IV LVL Y DAG HA------ADE K VL LVL Y EGG HA------EEQ K VV CVL Y DD P INGY TSYARDDL K IL CVL Y DD P VGGM ATYARDSL K VL C I L Y DD P KGGM SSYPVETL K VLL VL Y DGGRHA------KNQ KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF KIVGVF K VL LVL Y DGH E HA------QQE tree Prunus dulcis [55] or P. amy- gdalus [56]. Based on the data of the analysis of isoforms of FDHs and sev- R AAHTSAG-SK R AAHTSAG-SK R AAHTSAG-SK R AAHTSTG-SK R AAHTSAG-SK R SLHASAG-SK R NLHASGG-KK R NLHASGE-KK P SINASGE-KK R GLHASPG-SK eral other dehydrogenases, a method R YLHASPG-SK R ELQASPG-PK R ELQASPG-PK K HLHASAG-SK R QLHASPG-SK R QLHASPG-SK R RLHASPG-SK R NLHASSE-SK R MLHASTG-SK R HLHASPG-SK S INSASAG-SK R KFNASSGDSK R KFHASSGDSK R HLHASGG-SK R HLHASAG-SK R NLHASPG-SK R QFNASSGDSK R ELHAPAG-SN R ELHAPAG-SN R ELHAPAG-SN R LLHASAE-SK R TLTSTSSRQG for identifying plant genotype was proposed. As previously mentioned above, phosphorylation may be the reason for the formation of different FDH isoforms [34]. Depending on the modification degree, the formation of numerous forms of the enzyme with pI varying from 6.75 to 7.19 was observed. Moreover, it was ascer- tained that the additional isoforms of potato FDH emerged as a result of post-translational deamidation of ------EMA ------A ------MWRAAARHLVDRALGS ------MAAMWRAAARQLVDRAVGS ------MAAMCRAAARQLVDRAVGS ------MAMWRAAARQLVDRALGS ------MAMWRAAAGHLLGRALGS ------MAMRRAAQQAARFAMGPHVPHTAPAA ------MAMKRAASSAVRSLLTAPTPNPSSSIFS ------MAMMKRAASSSVRSLLSSSSTFT -MLNFTLKMSDPTLAQPHLVKVHTT-LETVVTTHNHNHR ------MAMKRAAVTAVRALTSSAPSSVLT ------SKG ------A ------ATVL ------A ------MAMRRVASTAARAIASPSSLVFT ------MAMSRVASTAARAITSPSSLVFT ------MAMMKRVAESAVRAFALGSTSGALT ------MKQVANSAIKAIANSGSSSLLT ------MKQVASSAIKALANSGSSSVLT ------MAMKRVAASALRAFTSSGNSTSSLLT ------MATRKAVVLGAQSLLRSSSTSSPSI ------MAMLRAAKHAMRALGSRAPDASPFA ------MAGAATSAIKSVLT ------MAMRRVTRAAIRASCVSSSSSGYFA ------MAMRRVIRASCVSSSSTGYLA ------MASKGVIASAVRALASSGSSASSTTFT ------MKGVIASAVRTLASSGSSASSTTFT ------MKRAATSAIKAFASSQTSFSGLSTNFA ------MAMRQAAKATIRACSSSSSSGYFAR ------MASRRAVISAFRAASRRPICSPVSSIASSV ------MASRRSVISAFRAASRRPICSPVSSV ------MASKRAVISTFRAASRKPIFSSVSPLASSV ------MAMKRAVASTVGAITSSGNPASSVLA ------MKSYSKRIALWLQRIEDGASDVTEELGV ------MAMKRAATSAIRAFSSSSPSSSLSSGSST ------G

Asn329 and Gln330 residues [34]. -VFARSSLRMARPASSLLSQRATASFTQRGANLARAGGV The differences in sequences of SmeFDH PseFDH 1 * ** 107 Fig. 3. Alignment of formate dehyfrogenases from different sources. Abbreviations enzymes see in FarFDH1 fungi enzymes are highlighted in magenta; FDHs from bacteria blue. Specific sequences that responsi to mitochondria are underlined. The numeration of residues is the same as for FDH from mark the conserved amino acid residue. HvuFDH1 TaeFDH1 ZmaFDH OsaFDH1 SbiFDH2 LjaFDH1 SoyFDH3 SoyFDH2 AmaFDH1 CboFDH SceFDH MorFDH BstFDH BbrFDH UmaFDH LesFDH1 StuFDH1 VviFDH1 GhiFDH1 TcaFDH1 CcaFDH1 ApuFDH1 ZofFDH1 QroFDH1 BnaFDH2 BolFDH1 MdoFDH PpeFDH1 CpaFDH1 AthFDH PpiFDH1 PtaFDH2 PsiFDH MgrFDH AjcFDH1 signal peptides are more strongly TpuFDH1 RcoFDH1 PtmFDH1

46 | Acta naturae | VOL. 3 № 4 (11) 2011 REVIEWS D VVT D IVN D VIS D VVT D VVT D VVT V A Q D IV T V A Q S D VV T L P KC D VV V L P KC D VV V L P RC D VI V L P KC D VI V L P KC D VI V L P KC D VI V L P KC D VI V L P KC D II V L P KC D VV V L P KC D II V L P KC D VI V L P KC D VI V L P KC D VI V L P KC D II V L P KC D VI V L P KC D IV V L A QC D VV T L P KC D VV V L P KC D VL V L P KC D VV V L S KC D VV V L A QC D VV T L P KC D VV V L P KC D VI V L P KC D VI V L P KC D VI V L P KC D II V L P KC D II V L S KC D IV V L S KC D IV V L P KC D II V L P KC D II V L P KC D II V A KYVESLDEM A KYVESLDEM A KFEDDLDAM A KFEEDLDAM A KFEEDLDKM A KFVEDLNEM A KFEEDLDAM A KFEEDLDKM A KFEEDLDAM A KYEEDLDAM A KFEEDLDAM A KFEEDLDKM A KFEEDLDAM A NFEEDLDAM A KYEEDLDAM A KYEEDLDAM C RRVDTLEEM A TLETKLDEM A TLETNLDEM A TLETNLDDM A KFEEDLDSL C RRVDTLEEM A QFEADLDAM A KFEEDLDAM A KFEEDLDAM A KFEEDLDAM A QFQEDPDAM A KFEEDLDAM A KFEEDLDKM A KFEEDLDKM A KFEEDVDVM A KFEEDLDAM A KFEEDLDAM Y GQRK Y GQRNLIDKK A Y GQKK------A Y GKHDKK------ARRVENIEEL G------G------G------G------G------G------G------G------G------G------G------G------G------G------G------G------G ------G------G------G------G------G ------G------G------G------G------G------G------G------G------G------G------G------AENY I V- KE G EL A S---- Q Y K------R NEY-L I VDG- G TL A GTG QS Y RLT------R DEY-L I VQG- G GL A GVG HS Y SKGNATGGSEEAAKYEKLDA R DEY-L I VQG- G AL A GTG HS Y SKGNATGGSEEAAKFKKAV- R EEY-L I VSG- G KL A GAG HS Y SAGDATRGSEEAAKYKK--- R TEY-T I VKD- G AL A GTG HS Y SEGSATSGSEEAAKYERK-- R TEY-L I VDQ- G RL A GAG HA Y TPGDTTAGSEDAARFHP--- H D R LKMEPELENEI LL Y H D R LQMGPEMEKET LL Y H D R LQMGPEMEKET LL Y H D R VKIDPEVEQHT LL Y H D R FKIDPELEQQI LL Y H D R VKMDPELESQI LL Y H D R LQMAPELEKET LL Y H D R LQINPELEKEI LL Y H D R IKMDPELENQT LL Y H D R LQINPELEKEI LL Y H D R VKMDSDLESQT LL Y H D R LQINPELEKEI LL Y H D R LKMEPDLEKEI LL Y H D R LQIDPELEKEI LL Y H D R LKMDPELENQI LL Y H D R LKIDPELEKEI LL Y H D R IKMDPELENQT LL Y H D R LSIGPELEKET LL Y H D R LSIGPELEKET LL Y H D R LSIGPELEKET LL Y H D R LKMDPELEKQT LL Y H D R LRIDPALEAET LL Y H D R LKMEPELEKEI LL Y F D R LRIDPELEKEI LL Y F D R LRIDPELEKEI LL Y H D R LKMDPELESQI LL Y LL Y H D R LKMDSELENQI LL Y H D R LKMDSELENQI LL Y H D R IKMDPELENQI LL Y H D R VKIDPELEKQT LL Y H D R VKIDPELEKQT T KDM L ERY F KGE-E FP SQNY I V- KG G KL A S---- Q Y Q------KT V GT GA GRIG KL L QR KPFGC-N GMTP H I S G TS L SA Q A RYA TLE QCWFDGRPI AMTP H I S G TTID A Q L RYA AMTP H I S G TTID A Q L RYA V KDM DRH F KGE-D FP EQNY V- KE QL S---- Y R------AMTP H I S G TTID A Q L RYA V KDM DRY F KGE-D FP AQHY V- KE QL S---- Y Q------AMTP H I S G TTID Q L RYA A V KDM DRY F KGE-E FP PQNY V- KE KL S---- Y L------AMTP H I S G TTID A Q L RYA V KDM DRY F KGE-D FP AQNY V- KD KI S---- Y Q------GMTP H I S G TS L SA Q T RYA A TRE ECYFEGRPI AMTP H I S G TTID Q L RYA A T KDM DRY F KGQ-D FP AQNY V- KE KL S---- Y ------AMTP H I S G TTID A Q L RYA V KDM ERH F KGE-D FP EQNY V- KE QL S---- Y R------AMTP H I S G TTID A Q L RYA T KDM DRY F KGE-D FP AENY V- KD EL P---- Y R------AMTP H I S G TTID A Q RYA T KDM L DRY F RGE-D FP PQHY V- KE KL S---- Y L------AMTP H I S G TTID A Q L RYA T KDM DRY F KGE-E FP AQNY V- KD KL S---- Y Q------GMTP H I S G TT L TA Q A RYA TRE ECFFEGRPI AMT V H I S G T LD A Q K RYA N L NSY F SKKFD Y RPQ-D IVQN SYAT---R AMTP H I S G TTID A Q L RYA V KDM DKY F KGE-D FP AQNY V- KE EL S---- Y R------AMTP H I S G TTID Q L RYA A V KDM ERY F KGQ-D FP VQNY V- KE NL G---- Y Q------AMTP H I S G TTID A Q L RYA T KDM DRY F KGE-D FP AENY V- KD EL P---- Y R------AMTP H I S G TTID Q RYA A T KDM L ERY F KGE-D FP SQNY V- KE KL S---- Y L------AMTP H I S G TTID A Q L RYA V KDM DRY F KGE-D FP AMTP H I S G TTID A Q L RYA T KDM EKY F KGE-D FP AQNY V- KD EL P---- Y R------GMTP H I S G SS L SA Q A RYA TRE ECWFEGRPI AMTP H Y S G T LD A Q RYA E K N I L ESF F TGKFD RPQ-D ILLN EYVT---K AMTP H I S G TTID A Q RYA V KDM L DRY F KGE-D FP PQHY V- KD EL S---- Y R------AMTP H I S G TTID A Q L RYA V KDM DRY F KGE-D FP VQNY V- KE QL S---- Y Q------AMTP H I F G TTID A Q L RYA V KDM ERY KGE-D FP PQHY V- KD EL S---- Y R------AMTP H I S G TTID A Q RYA T KDM L DRY F KGE-D FP SQNY V- KE KL S---- Y L------AMTP H I S G TTID A Q L RYA V KDM DRY F KGE-E FP VENY V- KE EL S---- Y K------AMTP H I S G TTID A Q L RYA T KDM EKY F KGE-D FP AQNY V- KD EL P---- Y R------GMTP H T S G TS L SA Q A RYA VRE I ECFFAGEVQ AM V P H M S G SS ID A Q RYA T K AI L ESY F SGKYD Y RPEDL I V-HA DYAT---KS AMTP H I S G TTID A Q L RYA V KDM DRY F KGE-E FP AQNY V- KE EL P---- Y R------AMTP H I S G TTID A Q L RYA D V RDM NRY F KGE-D FP VQNY V- KE QL S---- Y Q------AMTP H I S G TTID A Q L RYA P V KDM ENY F KGE-D FP PQNY V- KD EL S---- Y R------AMTP H T S G TTID A Q L RYA KDM ERY F KGE-D FP TENY I V- KD EL P---- Y R------AMTP H I S G TTID A Q L RYA V KDM DRY F KGE-D FP AENY V- KE EL S---- Y K------AMTP H I S G TTID A Q L RYA V KDM DRY F KGE-E FP LQNY V- KE KL S---- Y Q------GMTP H I S G SS L PA Q A RYA TRE ECWLDGRAI AM V P H M S G T ID A Q RYA K AI L DSY F SGRED Y RPEDL I V-HK DYAT---KA AMTP H I S G TTID A Q L RYA V KDM ERY F KGE-D FP EQNY V- KA EL P---- Y R------AMTP H I S G TTID A Q L RYA V KDM DRH F KGE-D FP EQNY V- KE QL S---- Y R------AMTP H I S G TTID A Q L RYA V KDM DRY F KGE-D FP AENY V- KD QL S---- Y R------YD IEG K TIA T I GA GRIG YRV L ER LP F NPKE LL Y D QA PKEAEEKVG YD LED K IIS T V GA GRIG YRV L ER VA F NPKK LL Y D QE PAEAINRLNEASKLFNGRGDIVQRVEKLEDM YD VEG M HV G T V A GRIG LRV L RL AP F DMH-L H Y D R HRL PEAVEKELN------LTWHATREDMYGAC YD VEG M HF G T V A GRIG LAV L RR KP F GLH-L H Y TQR HRL DAAIEQELG------LTYHADPASLAAAV YD LEG M QV G V A GRIG SAV L RR KP F DVG-L H Y T D Q HRL PAATEQELG------ARYHPDAAALAGAC YD LEG KT V GT GA GRIG RL L QR KPFNC-N YD LEG M HV G T V A GRIG LDA L RK KH F DVH-M H Y D R HRL PESVEKELN------LTFHDSVESMVAVC YD IEG M DI G T V A GRIG TAV L RR KP F DVK-L H Y D R HRL PDEVAKELG------VTFHQTAAEMVPVC YD LEA M HV G T V A GRIG LAV L RR AP F DVH-L H Y D R HRL PESVEKELN------LTWHATREDMYPVC YD LEG KT V GT GA GRIG RL L QR KPFNC-N YD LEG KT V GT GA GRIG KL L QR KPFNC-N YD LEG KT V GT GA GRIG KL L QR KPFNC-N YD LEG KT V GT GA GRIG RL L QR KPFNC-N YD LEG YD IEH KV V GT VGV GRIG ERV L RR KP F D C KE LL Y QPLPPAVEQEI YD LEG KT I GT GA GRIG KE L KR KPFNC-K YD LEG KT I GT GA GRIG KE L KR KPFNC-K YD LEG KT I GT GA GRIG KE L KR KPFNC-K YD LEG KT V GT GA GRIG KL L QR KPFNC-N YD L E G KV V GT VAV GRIG ERV RR KP F D C KE LL Y QALAPEVEKEI YD LEG KT V GT GA GRIG RL L QR KPFNC-N YD LEG KT V GT GA GRIG RL L QR KPFNC-N YD LEG KT V GT GA GRIG RL L QR KPFNC-N YD LEG KT V GT I GA GRIG KL L QR KPFNC-N YD LEG KT V GT I GA GRIG RL L QR KPFNC-N G W NI A DCVARS G W NI A DCVSRS G W NI A DCVARS G W NI A DAVQRS G W NV A DCVARS G W NI A DCVSHA G D W V A AVAKNE G D W N V A AVAKNE DH P W R Y MPN ------H Q IINHD W EV A AIAKDA Q AINGE W DI A GVAKNE MR N FL PG HH Q AIS G E W D V A GVAHRA YD LEG KT GT GA GRIG RL L QR RPFNC-K SG DVW NP QP A P K DH W R Y MPN ------Q G H--VA GY SG DVW FP QP A P K G Q--IG GY G H--LA GY GG DVW FP QP AP A DH P W R MP F------N G H--VA GY SG DVW FP QP A P K DH W R Y MPN ------H G H--VA GY SG DVW FP QP A P K DH W R Y MPN ------H G H--IA GY SG DVW YP QP A P K DH W R Y MPN ------Q G Q--IG GY SG DVW NP QP A P K DH W R Y MPN ------Q G H--IA GY SG DVW NP QP A P K DH W R Y MPN------H G R--LA GY AG DVW FP QP AP N DH P W R T MP H------N G H--IA GY SG DVW NP QP A P K DH W R Y MPN ------H G H--IA GY SG DVW FP QP A P K DH W R Y MPN ------H G H--IA GY SG DVW YP QP A P K DH W R Y MPN ------Q G H--IG GY SG DVW FP QP A P K DH W R S MPN ------H G H--IA GY SG DVW NP QP A P K DH W R Y MPN ------H G R--LA GY AG DVW FP QP AP K DH P W R T MP Y------N G K--LA GY GG DVW DK QP APK DH P W R T M D N K-DHVGN G H--IA GY SG DVW FP QP A P K DH W R Y MPN ------Q G H--IA GY GG DVW HP QP A P K DH W R Y MPN ------N G H--IA GY SG DVW YP QP A P K DH L W R Y MPN ------Q G H--IG GY SG DVW SA QP A P K DH W R S MPN ------H G H--IA GY GG DVW FP QP A P K DH W R Y MPN ------H G H--IG GY SG DVW DP QP A P K DH W R Y MPN ------Q G Q--LA GY AG DVW FP QP AP K DH P W R S MP H------H G Q--LR GY GG DVW FP QP APK DH P W R D M N K-YGAGN G Q--IG GY SG DVW NP QP A P K DH W R Y MPN ------Q G Q--VA GY GG DVW FP QP A P K GP W R Y MPN ------H G H--IA GY SG DVW YP QP A P K DH W R Y MPN ------Q G Q--IG GY SG DVW FP QP A P K DH W R S MPN ------H G H--IA GY GG DVW FP QP A P K DH W R Y MPN ------H G H--IG GY SG DVW DP QP A P K DH W R Y MPN ------Q G Q--LS GY AG DVW FP QP AP N DH V W R T MP N------H G HGHLR GY GG DVW FP QP A P K DH PL R YAQGPW-GGGN G H--IA GY SG DVW YP QP A P K DH W R F MPN ------Q G H--IA GY GG DVW FP QP A P K DH W R Y MPN ------H G H--IA GY SA DVW YP QP A P K DH W R Y MPN ------Q G H--IG GY SG DVW DP QP A P K DH W R Y MPN ------Q G H--IA GY GG DVW FP QP A P K DH W R Y MPN ------H G H--IG GY SG DVW YP QP A S K DH P W R Y MPN ------Q G Q--LA GY AG DVW FP QP AP R DH P W S MP H------H G Q--LR GY GG DVW FP QP V P A DH PF R TASYSTWGGGN G H--IA GY SG DVW YP QP A P K DH W R Y MPN ------Q VV SV A E H VL M TI L V VR N FV PA HEQVVG A VVRAVTS A IVRALES A VARALES A VARALES A VADACSS A IARALES A VADACSS A VVQALAS V L EV T G S N VV SV A E H VVMTM VR FVPAHE V T EV G S N VV SV A E H VMATI L IR YNGGHQ V A EV T YC N SN SV E H VM MV L G VRNYIPSHDWARN V A EV T GS N SI SV E H VM TT L VRNYLPSHAIAQQ V A EV T YS N SI SV S E H VM MV L VRNYLPSYQCVLD VAEV T G S N VV SV A E D ELMRV L I VR FV PG HH Q VIS W V GIAYRA V M EV T FC N SR SV A E H I VM MI L S VRDYHNQYRIINE V A EV T YC N SI SV S E H VM MI L ARNYIPSYQWVVK V A EV T YC N SI SV E H VM MI L S VRNYLPSHEWARK T AR GKICDRD K R G AYLI N K R G AYLI N T AR AKLVDRD K R G AYLV N T AR GKLCDRD K R G AYIV N T AR GKLCDRD K D G AYL VN T AR A I CVAE VAEAVKS K R G AYLV N T AR GKICNRD K G AWL VN T AR A I CVAE D VAAALES K G AYIV N T AR GKICNRD I AKC K G VLI V N AR A ADTQ IADACSS I AKM K G VRI V N AR A MDTQ VVDACNS I AKC K G VLI V N AR A MDIQ VADACSS I AKC K G VLI V N AR A ADTQ IADACSS I AKL K G VLI V N AR A MDTQ VADACSS I AKM K G VLI V N AR A MDTQ VVDACSS I AKC K G VLI V N AR A MDTQ VVDASSS I GKL K G VLI V N AR A MDTQ VADACSS I AKL K G VLI V N AR A MDTQ VADACSS I AKL K G VLI V N AR A MDTQ VVDACNS I SKM K G VLI V N AR A MDAQ VADASAS I AKC K G VLI V N AR A MDTQ VVDACSS I AKL K G VLI V N AR A MDTQ VADACSS I ARM K G VII V N AR A MDTQ VADACAT I AKL K G VLI V N AR A MDTQ VVDACNS I SKL K G VLI V N AR A MDAQ VADASAS I AKM K G VII V N AR A MDTQ VADACSS I GKM K G VLI V N AR A MDRQ VVEAVES I ARL K G VLI V N AR A MDTQ VVDGCSS I AKM K G VII V N AR A MDTQ VADACSS I AKL K G VLI V N AR A MDTQ VVDACSS I SKM K G VLI V N AR A MDAQ VADASAS I AKM K G VII V N AR AIMDTQ I GKM K G VLI V N AR A MDRQ VVEAVES I AKM K G SWL VN T AR A VVKEDVADAIKS I AKL K G VLI V N AR A MDTQ VADACSS I AKM K G VIV V N AR A MDAQ VADACSS I AKL K N G VLI V T AR A MDTQ VVDACSS I GKL K G VLI V N AR A MERQ VVDAVES I AKM K G VIV V N AR GIMDAQ I AKL K G VLI V N AR A MDTQ VADACSS I SKM K G SWL VN TAR A VVKEEVAAALKF I AKM K G VLI V N AR A MDTQ VADACSS LLLT AG I GS D H DL Q AA AA-- L T VAEV G S N VV SV A E ELMRI MR FV PG YN VVN W V GIAYRA YD LEG KT GT GA GRIG KL QR KPFGC-N LLLT AG I GS D H DL K AA PA-- L T VAEV G S N VG SV A E ELMKI VP FV PG YQ Q IVT W V GIAHRA YD LER KT GT GA GRIG RL LT NPVHC-D LLLT AG V GS D H I DL K AA AA-- L T VAEV G S N VV SV A E ELMRI VR FV PG YT Q IVN W GIAHRA YD LEG KT GT GA GRIG KL QR KPFNC-H LLLT AG I GS D H DL K AA AA-- L T VAEV G S N VV SV A E EVMRV MR FV PG YQ Q VVN W V AIACRS YD LEG KT GT GA GRIG RL QR KPFNC-N LLLT AG I GS D H DL Q AA AA-- L T VAEV G S N VV SV A E ELMRI MR FV PG YN VVK W V GIAYRA YD LEG KT GT GA GRIG KL QR KPFGC-N LLLT AG I GS D H DL P AA AA-- L T VAEV G S N TV SV A E ELMRI VR FL PG YQ Q VVQ W V GIAHRA YD LEG KT GT GA GRIG RL QR KPFNC-N LLLT AG I GS D H DL P AA AA-- L T VARV G S N TV SV A E ELMRI LR FL PG YQ Q VVK W V GIAHRA YD LEG KT GT GA GRYG RL QR KPFNC-N LLLT AG I GS D H DL K AA AA-- L T VAEV G S N VV SV A E ELMRI VR FV PG YK Q VIT W V AIAHRA LLLT AG I GS D H DL P AA AA-- L T VAEV G S N TV SV A E ELMRI LR FL PG YQ Q VVK W V GIAHRA YD LEG KT GT GA GRIG RL QR KPFNC-N LLLT AG I GS D H DL K AA AA-- L T VAEV G S N TV SV A E ELMRI VR FL PG YH Q VIN W V AIAYRA LLLT AG I GS D H DL P AA AA-- L T VAEV G S N TV SV A E ELLRI LR FL PG YQ Q VVQ W V GIAHRA YD LEG KT GT GA GRIG RL QR KPFNC-N LLLT AG I GS D H DL P AA AA-- L T VAEV G S N VV SV A E ELMRI VR FL PG YH Q AIS W V AISHRA LLLT AG I GS D H DL P AA AA-- L T VAEV G S N TV SV A E ELMRI LR FL PG YQ Q VVH W V GIAYRA YD LEG KT GT GA GRIG RL QR KPFNC-N LLLT AG I GS D H DL K AA AE-- L T VAEV G S N VV SV A E ELMRI VR FL PG YH Q VIS W V GIAYRA LLLT AG I GS D H V DL P AA AA-- L T VAEV G S N TV SV A E QLMRV LLLT AG I GS D H DL Q AA AA-- L T VAEV G S N VV SV A E ELMRI MR FV PG YN VVN W V GIAYRA LLLT AG I GS D H DL N AA AA-- V T VAEV G S VV SV A E ELMRI L MR FV PG YK Q IVE W K AISYRS LLLT AG I GS D H DL N AA AA-- V T VAEV A S VV SV E ELMRI L MR FV PG YK Q IVE G W K AISYRS LLLT AG I GS D H DL N AA AA-- V T VSEV G S VV SV A E ELMRI L VR FV PG YK Q IVN W K AISYRS LLLT AG I GS D H DL K AA AA-- L T VAEV G S N VV SV A E ELMRI VR FL PG YH Q VIN W V AIAYRA LLLT AG I GS D H DL N AA AA-- L T VAEV G S TV SV A E ELMRI VR FL PG YH Q AIT W V GIAHRA YD LEG KT GT GA GRIG KL QR KPFNC-N LLLT AG I GS D H V DL K AA AA-- L T VAEV G S N VV SV A E ELMRI MR FL PG YH Q AVK W GIAHRA YD LEG KT GT GA GRIG KL QR KPFNC-N LLLT AG I GS D H V DL K AA AA-- L T VAEV G S N VV SV A E ELMRI MR FL PG YH Q AVK W GIAHRA YD LEG KT GT GA GRIG KL QR KPFNC-N LLLT AG I GS E H DL K AA AD-- L T GAEG G S N VV SV A D ELMRI VR FL PG HH Q VIN W V AIAYRS YD LEG KT GT GA GRIG RL QR KPFNC-N LLLT AG I GS D H DL Q AA AD-- L T VAEV G S N VV SV A E ELMRI VR FL PG HH VIN W V AIAHRA YD LEG KT GT GA GRIG RL QR KPFNC-N LLLT AG I GS D H V DL K AA AA-- L T VAEV G S N TV SV A E ELMRI VR FL PG HH Q VIN W AIAHRA LLLT AG I GS D H V DL K AA AA-- L T VAEV G S N TV SV A E ELMRI VR FL PG HH Q VIN W AIAHRA LLLT AG I GS D H DL K AA AA-- L T VAEV G S N VV SV A E ELMRI VR FL PG HH Q VIS W V GIAYRA LLLT AG I GS D H V DL K AA AE-- L T VAEV G S N VV SV A E ELMRI VR FV PG YH Q VIT W GIAYRA LLLT AG I GS D H V DL K AA AE-- L T VAEV G S N VV SV A E ELMRI VR FL PG HH Q VIT W GIAYRA LLLT AG I GS D H V DL K AA AD-- L T L K V AG GS D H I DL DYINQTGKKIS AEAPN L K C V T AG GS D H DL E AA NE--RKIT AKAPK L K LA T AG I GS D H V DL Q AA ID--NNIT ARAPK L R LA T AG I GS D H V DL AA AR--AHIT AKAPR L K LA I T AG GS D H V DL Q AA AQ--HGLT KKAKNLQ KKAKNLE KKAKNLQ KKAKNLQ KKAKNLK QEGKELK KKAKNLE AMAKN L K MA I T AG GS D H V DL Q AA MD--NKID VKAAR L K LA I T AG GS D H V DL Q AA ID--RGIT AKAKN L K LA T AG I GS D H V DL QS A ID--RNVT KKAKTPE KKAKNLQ KKAKNLE KKAKNLQ KNAKNLE TKAKNLQ KKAKNLE KKAKNLQ ARAKNLE KKAKNLQ QEGKELK KKAKNLK KRAKNLQ KKAKNLE KKAQKLE KKAQKLE NKAKNLQ KKAKNLQ KKAKNLQ KKAKNLQ KKAKNLQ KKAKNLQ KKAKNLQ KKAKNLQ LHC PL HPG T EHLFDAAMLARM I N T PL TEQ RGLFDKNR I N M PL TEK T RGMFDKDR V N T PL TEK RGMFNKER LQI PL YPS T EHLFDAAMIARM I N T PL TDK RGLFDKDR I N T PL TEQ RGLFDKNR I N M PL TEK T KGMFNKER I N T PL TEK RGMFDKER I N T PL TEK RGLFDKER LNC PL HPE T EHMINDETLKLF I N T PL TEK RGLFNKER I N T PL TDK RGLFDKNR I N T PL TEK KGMFDKER I N M PL SDK T RGMFNKEK V N T PL TEK RGLFDKER LNC PL HPE T EHMINDETLKLF I N C PL HKDSRGLFNKKLISHM I N M PL TEK T AGMFNKEK L N M PL TEK T RGMFDKER I N T PL TEK KGMFDKER I N M PL SDK T RGMFNKEK I N T PL TEK RGMFNKEK V N T PL TEK RGMFNKEM INA PL HPE T ENLFNEAMIGKM V N A PL HAG T KGLINKELLSKF I N M PL TEK T RGMFDKDR I N T PL TEK RGMFNKER I N T PL TEK KGMFDKDK I N M PL SDK T RGMFNKEK I N T PL TEK RGMFNKEK V N T PL TEK RGMFNKEM INC PL HPE T ENLFDDEMIGKM I N C PL HEK T RGLFNKDL I N M PL TEK T RGMFDKDR I N T PL TEK RGMFNKER I N T PL TEK KGMSDKDR I N M PL TEK T RGMFNKEL I N T PL TEK RGMFNKEK I N T PL TEK RGLFNKDR LDKAKN L AKAKH KL AVT AG V GS D H DL AA NKTN GG I TV A EV T GC N I N C PL HEK T RGLFNKEL L AKAKK KI AVT AG I GS D H V DL N AA NKTN GG TV A EV T VV SV E M TM LVL R FV PA HEQIAA BbrFDH SoyFDH2 GhiFDH1 MgrFDH CpaFDH1 BstFDH QroFDH1 SoyFDH3 VviFDH1 PtmFDH1 PpeFDH1 253 ** * *** 400 MorFDH ZofFDH1 LjaFDH1 StuFDH1 PsiFDH MdoFDH PseFDH SceFDH ApuFDH1 SbiFDH2 LesFDH1 PtaFDH2 TaeFDH1 BolFDH1 SmeFDH CboFDH CcaFDH1 OsaFDH1 TpuFDH1 PpiFDH1 HvuFDH1 BnaFDH2 SceFDH I MorFDH I BstFDH I BbrFDH I UmaFDH AjcFDH1 TcaFDH1 ZmaFDH AmaFDH1 BolFDH1 I MdoFDH I PpeFDH1 I CpaFDH1 I AthFDH I PpiFDH1 I AthFDH FarFDH1 I UmaFDH I SmeFDH I PseFDH I 108 ** * **** 252 FarFDH1 HvuFDH1 I ApuFDH1 I TaeFDH1 I ZofFDH1 I ZmaFDH I QroFDH1 I OsaFDH1 I RcoFDH1 I SbiFDH2 I BnaFDH2 I RcoFDH1 CboFDH PtaFDH2 I PsiFDH I PtmFDH1 I MgrFDH AjcFDH1 LjaFDH1 I SoyFDH3 I SoyFDH2 I AmaFDH1 I TpuFDH1 I LesFDH1 I StuFDH1 I VviFDH1 I GhiFDH1 I TcaFDH1 I CcaFDH1 I

VOL. 3 № 4 (11) 2011 | Acta naturae | 47 REVIEWS

pronounced when compared with those in plant FDHs; within a cell, and the isolation of an enzyme directly the homology level between which is ~80%. It is most from plants is a labour-intensive and time-consuming clearly demonstrated in the case of isoenzymes of soy- procedure. Plant FDHs are mostly not very stable, bean FDH. Homology of the sequences of isoenzymes is which results in an appreciably significant inactiva- 98%, whereas for the signal sequences, it is lower than tion of the enzyme during the extraction. Therefore, 40%. Figure 2 shows a phylogenetic tree of the signal the specific activity of the resulting FDH specimens peptides of plant FDHs. As can be seen, two isoenzymes is much lower than one may expect (Table 2). The pu- of FDH from soybean Glycine max, SoyFDH1 and rified plant FDH was first obtained in 1951 from pea SoyFDH2, form an individual group. The N-terminal and French bean [13]. In 1983, FDH was extracted from fragment of these enzymes is much longer than that soybean G.max in appreciably large amounts; this ena- in FDHs from other plants (Fig. 1) and strongly differs bled the determination of the amino acid composition of in terms of its amino acid composition. Next, there is a plant formate dehydrogenase [57]. large branch of the family Gramineae, which includes Since 1993 several cDNA of plant FDH have been the enzymes of rice (OsaFDH), wheat (TaeFDH), bar- cloned: in 1993 – from potato [21], in 1998 – from bar- ley (HvuFDH), sugarcane, moso bamboo, etc. A large ley [8], in 2000 – from rice [41] and A. thaliana [26, 27]. group is represented by the enzymes of plants belong- Transgenic A. thaliana and tobacco plants expressing ing to the family Brassicaceae (Cruciferae), namely, AthFDH were developed [28]; however, the yield of the wild radish (RraFDH), cabbage (BolFDH), rapeseed enzyme was not very high. The expression of full size (BnaFDH), and A. thaliana (AthFDH). The other groups cDNA of potato FDH in Escherichia coli cells yielded are formed of proteins from Asteroideae, Leguminosae, insoluble inclusion bodies [21]. Solanaceae, Malvaceae, Pinaceae, and Salicaceae. As The active and soluble recombinant formate dehy- previously mentioned, five isoenzymes of soybean FDH drogenase from plants was first obtained in E. coli cells, do not form a separate group. Since the signal peptide [41]; however, the protein content was very low, ap- is basically responsible for the transport of the enzyme proximately 0.01% of all soluble proteins within the cell. inside the cell, an assumption can be made that differ- In our laboratory, we obtained E. coli strains, which are ent isoenzymes of soybean FDH are transported into superproducers of active FDHs from A. thaliana (Ath- different organelles. FDH) and soybean G. max (isoenzyme SoyFDH2) with Figure 3 shows the alignment of some complete se- an enzyme content attaining 40% of all soluble proteins quences of plant FDHs that are known at the present in the cell [58] (the gene of soybean FDH was kindly time; the sequences of a number of similar enzymes provided by Professor N. Labrou from the Agricultur- from microorganisms are provided for the purposes al University of Athens (Greece); the FDH gene from of comparison. The absolutely conserved regions are A. thaliana, by Professor J. Markwell from the Univer- highlighted in red; the residues repeating only in plant sity of Nebraska (Lincoln, USA)). There is no system of FDHs are highlighted in green. A significant differ- transport to mitochondria in E. coli cells; therefore, in ence of bacterial enzymes from the plant ones is an N- order to obtain the “natural enzyme”, we deleted the terminal rigid loop. This region embraces a consider- sequences encoding the signal peptide from the cDNA able part of the enzyme subunit. Its interaction with [58]. After the optimization of the cultivation condi- other amino acid residues is likely to be accounted for tions, the yield of recombinant FDH from A. thaliana by a higher thermal stability of bacterial enzymes as and soybean G. max reached 500–600 mg/l of the me- compared with the plant ones. In addition, in FDHs of dium [6]. A procedure was developed which enabled microbial origin, the C-terminal region is longer than the obtaining of several hundred milligrams of ho- that in plant enzymes. Meanwhile, the differentiation mogenous FDH specimen via a single chromatographic of FDH into two groups on the basis of its homology is stage per extraction run. Thus, all necessary conditions clearly observed in the remaining part of the amino for the performance of systematic studies of FDHs acid sequence. The first group contains bacterial and from A. thaliana and soybean G. max were provided, plant enzyme, whereas the second group consists of the including genetic engineering experiments and X-ray enzymes from yeasts and fungi. diffraction determination of the structure. The experi- Plant enzymes are highly homologous (approximate- ments were successfully carried out for the expression ly 80%); the similarity between the bacterial and plant of FDH from L. japonicas in E. coli cells [10]. FDHs is ~50%. KINETIC PROPERTIES OF PLANT OBTAINMENT OF PLANT FORMATE DEHYDROGENASES FORMATE DEHYDROGENASE In plants, FDHs are localized in mitochondria; they Table 2 summarizes the kinetic properties of natural therefore constitute a small part of all soluble proteins and recombinant plant FDHs. The characteristics of the

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Table 2. Kinetic parameters of formate dehydrogenases from different sources

NADP+ kcat Specific K NADP+ + M + K kcat, KM (NAD ), KM (NADP ), M FDH specimen activity, (formate), + Reference s–1 µM mM NAD U/mg mM kcat NAD+ KM Arabidopsis thaliana, native, after affinity NA* NA 65 10 NA NA [27] chromatography A. thaliana, native, from NA NA 76 11 NA NA [59] mitochondria A. thaliana, recombinant 1.3 0.87 78 11 NA NA [59] from transgenic tobacco A. thaliana, recombinant from transgenic tobacco 0.1 0.07 35 3.3 ND ND [59] + thermal treatment for 5 min at 60oC A. thaliana, from 1.9 1.27 34 1.4 NA NA [60] mitochondria from leaves A. thaliana, recombinant 6.5 3.8 20 2.8 10 5.0 10-5 [58] from E. coli × Pea (seeds) 2 [61] NA NA 22 NA NA [61, 62] Pisum sativum 1.67; 6.25 [62] Mung bean, Phaseolus NA NA 7.2 1.6 NA NA [63] aureus, native

Soybean, G. max, native NA NA 5.7 0.6 NA NA [57]

Soybean, G. max, 4.0 2.83 [64] 13.2 1.5 1 8.7 10-4 [58, 64, 76] recombinant × 1.2 (NAD+) Lotus japonicus NA 29.5 6.1 29.5 3.7 10-6 [10] 0.005 (NADP+) × Spinach, Spinacia NA NA NA 1.7 NA NA [16] oleracea L., from leaves Potato NA NA 19 0.54 NA NA [29] Solanum tuberosum

C. boidinii,wild-type 6.3 3.7 37 5.9 NA NA [65]

C. metillica, wild-type 2.1 1.4 55 NA NA < 4 × 10-6 [66]

Saccharomyces cerevisiae, 10 6.5 36 5.5 NA < 3.3 10-10 [67, 68] recombinant × 1.66 (NAD+) Burkholderia stabilis NA 1430 55.5 0.16 25.0 [51] 4.75 (NADP+) Moraxella sp. C2, 10.0 7.3 80 7.5 NA NA [6] recombinant

Pseudomonas sp. 101 10.0 7.3 60 7 > 200 4.2 × 10-4 [6]

* NA – Data not available.

VOL. 3 № 4 (11) 2011 | Acta naturae | 49 REVIEWS most thoroughly studied bacterial and yeast enzymes NADPH) in the synthesis of chiral compounds using are provided for comparative purposes. Several major dehydrogenases [6]. conclusions can be drawn from the data in Table 2: 4. Almost all FDH that have been studied are highly 1. It is clearly visible that in case of FDH from A. thal- specific to the coenzyme NAD+. Their catalytic activ- iana the multistage extraction of the natural enzyme ity in the reaction with NAD+ is higher than that in the is accompanied by a considerable loss in activity. The reaction with NADP+ by a factor of 2500 (PseFDH) to specific activity of the specimens, even those obtained 3 × 109. The exception is the recently described FDH from transgenic plants, is several times lower than the from pathogenic bacteria Burkholderia stabilis, which activity of recombinant AthFDH expressed in E. coli is twenty-six-fold more efficient in the reaction with cells. It is noteworthy that AthFDH belongs to stable NADP+ in comparison with NAD+ [51]. It should be FDHs. In terms of thermal stability, it is even superior noted that the mutant PseFDH, whose coenzyme spe- to FDH from Moraxella sp. C2 and yeast C. boidinii (Ta- cificity changed from NAD+ to NADP+, was obtained ble 3). It is obvious that the degree of inactivation of less as early as in 1993 [6, 68]; this enzyme has been suc- stable, FDHs (in particular, in the case of SoyFDH) be- cessfully used for NADPH regeneration [69, 70]. Having + ing extracted from natural sources will be higher. This the lowest Кm value with respect to NADP among all fact should be taken into consideration when analysing NAD+-specific wild-type FDHs, SoyFDH has a huge formate dehydrogenase activity in plants. potential for obtaining the NADP+-specific enzyme [58] 2. The specific activity of recombinant AthFDH and (Table 2). SoyFDH is comparable with that of formate dehydro- genases from microorganisms; although it is inferior to THERMAL STABILITY OF PLANT FDHs from methylotrophic bacteria and baker’s yeast. FORMATE DEHYDROGENASES As previously mentioned, a partial inactivation of en- Prior to obtaining recombinant AthFDH and SoyFDH zymes may occur during the extraction; therefore, cal- in E. coli cells, no systematic studies on the thermal sta- culation of the catalytic constant based on the values bility of plant FDHs were performed. According to [59], + of specific activity and molecular weight may give un- the values of КM with respect to formate and NAD de- derestimated kcat values. This fact is of particular sig- crease after the incubation of transgenic AthFDH for nificance for SoyFDH, the thermal stability of which is 5 min at 60°С; however, the specific activity value de- much lower than that of other FDHs (see the thermal creased by 13 times. We performed systematic studies stability section below). Therefore, we developed a pro- of the thermal stability of recombinant AthFDH and cedure for determining the concentration of active sites SoyFDH using two approaches: determining the kinet- of recombinant SoyFDH based on the quenching of the ics of thermal inactivation and the differential scanning intrinsic fluorescence of the enzyme as it is titrated calorimetry [71, 72]. It was revealed that the thermal with azide ion in the presence of coenzyme NAD+ [64]. inactivation of plant FDHs occurred via a monomolecu- The azide ion is a strong competitive inhibitor of Soy- lar mechanism, similar to the FDHs from bacteria and -7 FDH (KI = 3.6 × 10 M). Therefore, a linear dependence yeasts. Time dependences of decrease in the activity of quenching of FDH fluorescence on the azide concen- of AthFDH and SoyFDH are described by the kinet- tration is observed under the conditions ensuring the ics of the first-order reaction, the observed constant of equimolar binding of enzyme and inhibitor. The kcat inactivation rate was independent of enzyme concen- value determined from these experiments actually co- tration. The thermal stabilities of AthFDH and Soy- incided with that calculated using the specific activity FDH strongly differ. AthFDH lost 50% of its activity and molecular weight. The obtained data attest to the after 20 min at 59.5°С, whereas SoyFDH lost 50% of its fact that despite the low thermal stability, SoyFDH ex- activity at 52.8°С. The difference by almost 7°С corre- tracted using the designed procedure is not inactivated. sponds to a difference in the inactivation rate constants Today, this procedure is being actively used for deter- of more than 1000 times. AthFDH is actually inferior in mining the kcat values of the mutant forms of SoyFDH. terms of its thermal stability only to FDH from Pseu- 3. Plant FDHs are characterized by significantly domonas sp. 101 (63.0°С, the most stable FDH among lower values of the Michaelis constants with respect to the known ones) [6] and Staphylococcus aureus (62.0°С) both formate and coenzyme NAD+ in comparison with and is superior to all known microbial formate dehy- the bacterial and yeast enzymes. It is very important drogenases. In contrast, SoyFDH is inferior in terms for the practical application of FDH. Today, recom- of stability to all known FDHs, with the exception of binant FDHs from Pseudomonas sp. 101 and C. boidi- the enzyme from baker’s yeast that is characterized by nii, whose chemical and thermal stability have been another inactivation mechanism. The temperature de- enhanced by protein engineering methods, are used for pendences of the rate constants of inactivation of Ath- the regeneration of the reduced coenzyme (NADH or FDH and SoyFDH are described by the transition state

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Table 3. Parameters of thermal inactivation of formate dehydrogenases from different sources

Kinetics of thermal inactivation* Differential scanning calorimetry* [72] FDH source ≠ ≠ o o ΔH kJ/mol ΔS , J/(mol·K) Cp, kJ/mol Tm, C T1/2, C

Pseudomonas sp. 101 540 [6] 1320 [6] 2060 67.6 5.4

Moraxella sp. C2 NA** NA** 1830 63.4 4.9

Candida boidinii 480 [77] 1250 [77] 1730 64.4 5.3

Saccharomyces cerevisiae 420 [67] NA 820 46.4 3.2

Arabidopsis thaliana 490 [6] 1200 [6] 1330 64.9 5.9

Glycine max 370 [76] 860 [76] 820 57.1 7.5

* All measurements were carried out in the 0.1 M phosphate buffer, pH 7.0 ** Data not available

theory. The calculated values of activation enthalpy the synthesis of FDHs in plants increased under vari- ΔH≠ and activation entropy ΔS≠ are listed in Table 3. It ous stress factors. The concentration of active forms can be noticed that thermal stability of formate dehy- of oxygen (superoxide radicals, hydrogen peroxide, drogenases correlates well with the ΔH≠ and ΔS≠ values. etc.) is likely to increase in a cell under the same condi- The highest values are typical of the most stable Pse- tions. It should be expected that in order to ensure high FDH, whereas the lowest ones refer to SoyFDH. activity under stress, plant FDHs should be far more The results of experiments studying the thermal sta- resistant to the action of these agents than formate de- bility of plant FDHs using differential scanning calorim- hydrogenases functioning under non-stress conditions. etry are in close agreement with the data of inactivation In order to verify this hypothesis, the kinetics of inac- kinetics. In Table 3 the values of the temperature and tivation of recombinant AthFDH, SoyFDH, PseFDH, heat of the phase transition are shown. All investigated FDH from wild-type S. aureus (SauFDH) and three FDHs are characterized by their high of mutant PseFDH, where one or two Cys residues were the denaturation process. PseFDH is characterized by replaced, under the action of hydrogen peroxide was the highest melting point, whereas this value is lower studied. FDH from S. aureus is a stress protein as well, by a factor of 2.5 for SoyFDH. The melting point of Ath- since the biosynthesis of this enzyme increases by 20 FDH is higher than that of CboFDH and MorFDH. times when staphylococci pass from planktonic growth Experiments on enhancing the thermal stability of to biofilm formation [73]. It was revealed that the in- recombinant SoyFDH using genetic engineering tech- activation rate of AthFDH and SoyFDH under the ac- niques are currently being performed. tion of Н2О2 is virtually equal and is 18 times lower than that in wild-type PseFDH. Under the same conditions, CHEMICAL STABILITY OF PLANT the stability of SauFDH was sixfold higher than that in FORMATE DEHYDROGENASES plant enzymes [74]. PseFDH with the stability identical FDH has been actively used in the process of synthe- to that of SoyFDH was successfully obtained only af- sizing optically active compounds catalyzed by dehy- ter double Cys145Ser/Cys255/Ala replacement. It was drogenases. In these processes, the operational stability thus demonstrated that plant and bacterial FDHs syn- (the operating time of an enzyme) plays the greatest thesized under stress impact indeed possess a higher role. Under conditions of biocatalytic process, the inac- chemical stability than formate dehydrogenases, whose tivation of FDH is associated with either oxidation with synthesis is induced by other factors. Moreover, study- oxygen or chemical modification of sulfhydryl groups ing the thermal inactivation of formate dehydrogenase of the enzyme. The operational stability of PseFDH and in the presence of hydrogen peroxide can be used to CboFDH was enhanced by site-directed mutagenesis comparatively assess the in vivo chemical stability of of two Cys residues [6, 65]. As previously mentioned, FDHs.

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А B

Fig. 4. The structures of apo-(A) and holo-(B) forms of FDH from A. thaliana. Figures were obtained using PDB structures 3JTM and 3N7U, respectively. In the structure of the holo-form, the molecules of NAD+ and azide ion are highlighted in magenta and red, respectively.

STRUCTURAL STUDIES OF PLANT CONCLUSIONS FORMATE DEHYDROGENASES Both our own and the published data attest to the fact FDH has not been well studied from a structural per- that plant FDHs are extremely significant, in particular spective. Until recently, only the FDH structures from when responding to stress factors. Biosynthesis regu- three sources, namely, bacteria Pseudomonas sp. 101 lation and the physiological role of FDHs are diverse and Moraxella sp. C2 (unbound enzyme, and the en- and complex; they have not been completely elucidated zyme–NAD+–azide ternary complex) and C. boidinii thus far. The systematic investigation of the structure yeast (structures of two mutant forms of an apoen- and function of these enzymes is still in its infancy. The zyme) were deposited into the protein data bank (PDB). results of these studies are of significant interest to In case of formate dehydrohenases, free enzyme is in its fundamental science and are of great practical impor- «open» conformation. When the FDH–NAD+–azide ter- tance. Production of mutant forms of FDH with a high nary complex is formed (its structure being considered activity opens a new approach for the design of plants similar to that of the enzyme in the transition state), a with an enhanced resistance to unfavourable factors. considerable compacting of the protein globule occurs, More active mutant enzymes will supply the cell with and FDH is transformed into the «closed» state. The de- the energy required to more efficiently overcome un- velopment of a highly efficient system of expression of favourable effects of stress with the same expression AthFDH and SoyFDH in E. coli cells enabled the tran- level of FDH. Soybean FDH is also considered to be an sition to their crystallization and identification of their exceptionally promising FDH for the design of a highly structure., Nowadays AthFDH structures both in open efficient biocatalyst for NAD(P)H regeneration in the and closed conformations (3NAQ and 3N7U, with reso- synthesis of optically active compounds using dehy- lution of 1.7 and 2.0 Å, respectively) have been identi- drogenases. The natural enzyme is notable for its high fied. Enzyme crystals were produced in space [75] in or- operational stability and has one of the lowest values der to obtain AthFDH structure in open conformation of the Michaelis constant with respect both to NAD+ with higher resolution (3JTM, 1.3 Å), Fig. 4 shows the and formate among all of the known FDHs. However, structures of apo- and holoFDHs (the open and closed in order to practically implement SoyFDH, its catalytic conformations, respectively). A more detailed analysis activity and thermal stability need to be enhanced. We of these structures will be provided in individual arti- are currently performing active research in this direc- cles. The crystals of the SoyFDH–NAD+–azide ternary tion. complex of FDH from soybean G. max have been ob- tained both on the Earth and in space. The structures This study was supported by the Ministry of Education of these complexes were identified with resolution of and Science of the Russian Federation (State Contract 1.9 Å; the deposition of these structures into the PDB № 16.512.11.2148) and the Russian Foundation for data bank is in progress. Basic Research (grant № 11-04-00920).

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