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J. Biol. Chem., Vol. 278, Issue 38, 36169-36175, September 19, 2003
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From the
Instituto de Biología Molecular y Celular de Rosario-Consejo Nacional de Investigaciones Científicas y Técnicas, Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000-Rosario, Santa Fe, Argentina and the Research Unit for Tropical Diseases and ¶Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology, Université Catholique de Louvain, ICP-TROP/74.39, Avenue Hippocrate 74, B-1200 Brussels, Belgium
Received for publication, May 30, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Zinc-dependent medium-chain dehydrogenases constitute a large superfamily of enzymes widely found in vertebrates, plants, fungi, and bacteria, including polyol dehydrogenases, threonine dehydrogenase, quinone oxidoreductases, and other proteins (2). Currently, they can be divided into tetrameric (as NAD-dependent ADHs from fungi or some bacterial NADP-dependent enzymes) and dimeric NAD-linked ADH families. The last, in turn, are divided into the classes found in vertebrates (I–VII) or plants (III, C and P), according to substrate preference and amino acid identity (3, 4).
Although it is not uncommon for ADHs to exhibit a broad substrate spectrum, as a rule they are specific either for primary or for secondary alcohols. One interesting exception is the tetrameric multifunctional Ralstonia eutropha (formerly Alcaligenes eutrophus) ADH. This enzyme is able to use both primary and secondary alcohols, polyols such as 2,3-butanediol, acetaldehyde, acetone, diacetyl, or acetoin as substrates and to use NAD(H) or NADP(H) as cosubstrate (5).
Few trypanosomatid ADHs have been described. These are the cytosolic NADPH-linked aldehyde reductases from Leishmania donovani (6) and Trypanosoma cruzi (7), which use n-propyl alcohol as the preferred substrate or the NADH-dependent ADH from Crithidia fasciculata (8). Unfortunately, neither sequence information nor sufficient biochemical data are available to correctly classify these enzymes.
We have previously described the purification and characterization of the mitochondrial ADH from Phytomonas, a trypanosomatid isolated from the laticiferous plant Euphorbia characias. It is a dimeric NAD-linked enzyme able to use a broad range of both primary and secondary alcohols. We have named the enzyme isopropyl alcohol dehydrogenase (iPDH) because of its high affinity and specific activity with this substrate (9). To understand the physiological role of the enzyme and its peculiar properties, we decided to clone the genes and to express the corresponding proteins in a heterologous system. To our knowledge, this is the first trypanosomatid ADH so far sequenced. Both its sequence and kinetic parameters suggest that the Phytomonas iPDH is the result of an event of horizontal gene transfer from a strictly aerobic bacterium to a trypanosomatid. The possible physiological role of this multifunctional ADH is discussed.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Purification of Enzymes—Natural iPDH was purified from 1 liter of Phytomonas culture, as described (9). Briefly, a method was used involving hydrophobic interaction chromatography on phenyl-Sepharose followed by affinity chromatography on blue Sepharose, both at pH 7.8.
Enzymes expressed in Escherichia coli were purified from 1 liter of bacterial culture. Cells were washed twice and resuspended in buffer A (25 mM Tris-HCl, pH 7.8, 1 mM EDTA, 1 mM dithiothreitol). Extracts were prepared by using a French press. Cell debris was removed by centrifugation (10,000 x g). Ammonium sulfate and Triton X-100 were added to the supernatant to a final concentration of 1 M and 0.05% (w/v), respectively, and the supernatant was clarified by centrifugation at 40,000 x g, loaded onto a phenyl-Sepharose column (1.4 x 25 cm), and equilibrated in 1 M ammonium sulfate in buffer A. The column was washed with 50 ml of the same buffer and then with 50 ml of buffer B (50 mM ammonium sulfate in buffer A) and eluted with a gradient of ethylene glycol from 0 to 70% (v/v) in buffer B (300 ml). Fractions of 7 ml were collected, and those containing activity were pooled. The ethylene glycol concentration was adjusted to 45% (v/v) before loading the pooled active fractions onto a blue Sepharose column (1 x 7 cm) equilibrated with ethylene glycol 45% (v/v in buffer B). The column was washed with the same buffer followed by a 60-ml gradient of NAD+ (0–2 mM).
Protein Determination—Protein concentration was determined by the method of Bradford (11) using bovine serum albumin as a standard.
SDS-PAGE—Analytical SDS-PAGE was performed in 10% polyacrylamide gels (w/v) according to Chua (12). Protein was visualized by Coomassie Brilliant Blue R-250 staining.
Partial Amino Acid Sequence Determination—The mitochondrial iPDH was purified by SDS-PAGE. Bands were cut from the gel, concentrated in an agarose-based gel concentration system, and digested with trypsin (13). Peptides were analyzed by nanoelectrospray ionization-tandem mass spectrometry (MS). Briefly, 2 µl of the acidified and desalted trypsin digest was injected to an LCQ ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA) fitted with a nanoelectrospray probe (14). Spectra were taken in full MS and zoom scan mode to determine parent masses and their charge state. Selected peptides were fragmented by collision-induced dissociation to generate tandem mass spectra. Peptide sequences were derived de novo from tandem MS spectra by following the fragmentation paths of y-ion and b-ion series.
Cloning and Sequencing of Isopropyl Alcohol Dehydrogenase—Available zinc-dependent medium-chain ADH amino acid sequences were aligned, and two consensus sequences were established. Consensus GHE(G/A)(A/G)G was used to design primer UP2 (5'-ggicaygarggigcigg), and consensus E(V/Y)FIDFT found on fungi ADHs was used for primer DW1 (5'-gtraartcratiaagta). Primer UP1 (5'-gtrccsgtbccsaa) was designed on peptide P1 (DEKPVPVPK) obtained from amino acid sequencing of purified iPDH. A DNA fragment of 600 bp was obtained by PCR on genomic DNA from Phytomonas using primers UP1 and DW1. The fragment was cloned in the pGEM-T Easy vector (Promega), sequenced, and used as a hybridization probe to a Southern blot, which was carried out with Hybond-N membranes on 4 µg of total DNA from Phytomonas sp. digested with different restriction enzymes. A limited genomic library was made with Phytomonas total DNA digested with PstI. After electrophoresis in an agarose gel, a portion of the gel corresponding to the region of 3–4 kb was cut based on Southern blot information, and DNA was recovered by standard procedures (15). Recovered genomic fragments were ligated to vector pUC18 digested with PstI and introduced into E. coli XL1 blue by electroporation. 378 colonies were screened by colony hybridization (15) with the same probe. Two positive clones (215 and 345) were identified and isolated. Plasmid DNA was obtained from them, digested with different restriction enzymes, and subjected to Southern blot analysis using the same probe. Plasmids from clones 215 and 345 were totally or partially sequenced, respectively.
Determination of iPDH Isoenzyme Number—Based on bacterial and Phytomonas protein ADH sequence multiple alignment, two highly conserved peptide sequences were selected. The corresponding DNA sequences were used to design oligonucleotides UP3 (5'-atyacsaccaccac) and DW2 (5'-cgcatgcgytcctt). Amplification of genomic DNA from Phytomonas was performed with these primers using total DNA from Sinorhizobium meliloti as a positive control and Trypanosoma brucei and E. coli DNA as negative controls. Amplified fragments from Phytomonas of 
800 bp were cloned in pGEM-T Easy vector, and eight clones were totally sequenced. Sequences were compared with those from clones 215 and 345.
Phylogenetic Analyses—Available zinc-dependent medium-chain ADH protein sequences were aligned with the program ClustalX (16). Positions with gaps were removed, and pairwise distances were calculated by using a correction for multiple hits and back mutations according to Kimura (17) as implemented in the Protdist program (18). Phylogenetic analyses were carried out by the neighbor-joining method using the programs Neighbor and Consense (18) with 10,000 bootstrap samplings, or by maximum likelihood with 1000 quartet puzzle steps using the program TreePuzzle version 5.0 (19) and the JTT matrix as the evolutionary model. Both methods gave similar but not identical tree topologies.
Expression in E. coli—Isopropyl alcohol dehydrogenase genes were amplified by PCR from clones 215 and 345 DNA using the oligonucleotide 5'-ggaattccatatgcttcgccgttccacc, which contains a 5'-NdeI artificial restriction site (underlined) followed by 18 nucleotides that correspond to the 5' end of the genes, and oligonucleotide 5'-cgggatccttatggcttgatagctacctt, which has 21 nucleotides complementary to the 3' end, including the TAA stop codon and an artificial BamHI restriction site. The amplified DNA fragments were cloned in the pT7–7.1 expression vector (20) and digested with NdeI and BamHI. The sequences were verified before introduction into E. coli BL-21 (DE3) containing the plasmid pCRF1 (21). Transformed cells were grown at 30 °C in Luria Bertani medium (15) supplemented with ampicillin (100 µg/ml) and chloramphenicol (25 µg/ml). Expression was induced at an A600 nm of 0.6 by adding isopropyl, 
-D-thiogalactopyranoside to a final concentration of 0.4 mM, and growth was continued overnight.
Kinetic Characterization—Dehydrogenases were assayed spectrophotometrically by following the ketone-dependent oxidation of NADH or the alcohol-dependent reduction of NAD+ at 340 nm and 25 °C. Optimal pH and buffers for forward and reverse reactions were used as described previously (9). One unit of activity is the amount of enzyme catalyzing the conversion of 1 µmol of substrate/min at 25 °C. Relative specific activity with different substrates was referred to that with D-2,3-butanediol for the forward reaction and that with acetoin for the reverse reaction.
Gas Chromatography-Mass Analysis—NADH-dependent reduction of acetoacetate (initial concentration = 1 mM) was followed spectrophotometrically, as indicated before, up to the exhaustion of the substrate. The reaction mixture (1 ml) was acidified with 200 µl of 3 N HCl and extracted twice with one volume of ethyl acetate. Organic phases were collected and dried under a stream of nitrogen. Pyridine (100 µl) and BSTFA (100 µl) were added and incubated at 80 °C for 1 h. 1 µl was injected into an AutoSystem XL gas chromatograph (PerkinElmer Life Sciences) equipped with a PE1 column (30 m x 0.25 mm internal diameter, PerkinElmer Life Sciences). Gas chromatographic analysis was performed at 180 °C isothermically. Gas chromatography-MS was carried out with a TurboMass mass detector (PerkinElmer Life Sciences) operated at an ionization voltage of 70 eV with a scan range of 20–500 Da. Retention times and mass spectra of peaks were compared with those of standards (Sigma) and information available in the data base NBS75K (National Bureau of Standards).
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RESULTS |
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Isolation and Sequence of Allelic Variants of iPDH—Our previous work suggested that Phytomonas iPDH belongs to the zinc-dependent medium-chain ADH family. This was based on our previous estimation of the subunit relative molecular mass of the purified enzyme (37–40 kDa), its absolute dependence on NAD(H) for enzymatic activity, and the inhibitory effect of Hg2+ (9). Moreover, 2 mM pyrazole completely inhibited the enzyme with an IC50 of 67 µM. Culture media containing 50–100 mM ZnSO4 stimulated the expression of iPDH in Phytomonas (data not shown). These properties are characteristic of zinc-dependent ADHs (22). Based on this information, a multiple sequence alignment was created with available protein sequences of zinc-dependent ADHs from plants, bacteria, vertebrates, and yeasts. This allowed us to determine two consensus sequences useful for the design of degenerate oligonucleotides (UP2 and DW1). A partial alignment (Fig. 1) indicates the position of these consensus sequences and the region of similarity with peptide P1 on which primer UP1 was based. PCR amplifications on genomic Phytomonas DNA were carried out using these primers. Amplification of a 600-bp fragment was possible only when using UP1 and DW1 as primers. Sequencing the resulting fragment revealed a high similarity with ADHs from a small group of bacteria. This fragment was used as a hybridization probe in a Southern blot of Phytomonas genomic DNA. As shown in Fig. 2A, the putative ADH appears to be present in Phytomonas in one copy per haploid genome and is contained in a PstI restriction fragment of 3.5 kb. A limited size library was made by using PstI-restricted genomic DNA and screened by using the same probe. Two positive clones with identical restriction patterns (Fig. 2B) were isolated. The PstI insert of clone 215 was completely sequenced and was found to contain 3493 bp. Only one open reading frame of 1086 bp was detected, which encodes a putative protein of 361 amino acids (allele 1 or iPDH1). A hypothetical N-terminal mitochondrial transit peptide was also detected (Fig. 3). This peptide contains 13 amino acids and has a high content of basic amino acids and a high degree of similarity with other mitochondrial transit peptides reported for trypanosomatids (23). A central region of the 3.5-kb insert of clone 345 (from the first NdeI to the second EcoRI site, Fig. 2B) was sequenced. It contained an open reading frame that is 99% identical to that present in clone 215. The inferred protein (iPDH2) differs only in one amino acid; lysine instead of the glutamic acid at position 60 in iPDH1 (Fig. 3). When the trypanosomatid sequences were compared with the bacterial ADHs, a high degree of similarity was always observed, starting from the methionine at position 14 in the iPDHs. Assuming that Met14 is the N terminus of the mature proteins, the calculated isoelectric points are 6.7 and 7.5 for iPDH1 and iPDH2, respectively, with a molecular mass of 37 kDa. This result is in good agreement with the estimated Mr by SDS-PAGE and with pI values obtained for the three dimeric isoenzymes previously isolated from crude extracts of Phytomonas sp. (9).
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To confirm the copy number of the iPDH genes, two highly conserved regions of bacterial and Phytomonas ADHs were used to design oligonucleotides UP3 and DW2 (Fig. 3). After PCR amplification on genomic DNA from Phytomonas and cloning of an 800-bp fragment, eight clones were chosen at random and sequenced. Five clones contained a sequence identical to that of allele 1, and three others were identical to allele 2. This result supports the notion that only two allelic variants of iPDH are present in the genome of Phytomonas sp.
Phylogenetic Relationship between iPDH and Zinc-dependent Alcohol Dehydrogenases—The multiple alignment containing 19 sequences served to create a phylogenetic tree using both distance and maximum likelihood methods (Fig. 4). The Phytomonas iPDH appeared to be robustly monophyletic with the ADHs from Mycoplasma penetrans and several proteobacteria with which it shared between 60 and 66% identical residues. This clustering is supported by the fact that the Phytomonas sequence shared with the other members of this clade the presence of a unique Thr4 tract at positions 67–70 in the alignment, as well as a deletion at position 78. The presence of the Phytomonas sequence within this bacterial clade can only be explained by a horizontal gene transfer event. Interestingly, a similar event of horizontal gene transfer can be observed in the other robust clade (100% supported by bootstrap analysis) of (facultative) anaerobes. Here again, a prokaryote must have provided the ADH1 gene for the anaerobic protists Trichomonas vaginalis and Entamoeba histolytica. The two protist enzymes, which share 77% identical residues, also share between 60 and 66% identical residues with the bacterial ADH sequence of this clade.
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Expression in E. coli and Purification of iPDHs from Bacteria—The iPDHs derived from clones 215 (iPDH1) and 345 (iPDH2) were ligated into vector pT7–7.1 and expressed in E. coli. We used an E. coli strain harboring the plasmid pCRF1, which allows the co-expression of the GroESL chaperonin system from Chromatium vinosum (21). Although 80% of the expressed protein was trapped in inclusion bodies, a 2-fold increase in specific activity of iPDH was measured in crude extracts of transformed E. coli/pCRF1 as compared with commonly used E. coli expression strains.
Enzymatic Characterization of Bacterially Expressed and Natural iPDH—Enzymes purified from E. coli presented specific activities of 9.7 and 12.5 units/mg of protein for iPDH1 and iPDH2, respectively. Activity with ethanol for iPDH1 and iPDH2 was 20 ± 2- and 11 ± 2-fold less than with isopropyl alcohol, respectively. These respective ratios were highly reproducible and therefore could be useful in isoenzyme identification. For the enzyme purified from cultured Phytomonas, the ratio of isopropyl/ethyl alcohol activity was much more variable and ranged from 15 to 21, which is suggestive of a variable expression level of one or both alleles.
The relative specific activity with and affinity for different substrates for both natural and bacterially expressed iPDHs were compared with the data available from the multifunctional fermentative ADH of R. eutropha (5). As shown in Table I, a similar pattern of relative activities with the various substrates was found for the Phytomonas and R. eutropha enzymes. Significant differences were found for the reverse reaction only with acetone as substrate, with iPDH showing the higher relative activity. The Phytomonas enzymes also appear to oxidize acetaldehyde, indicating that iPDH is a multifunctional enzyme able to dismutate this substrate, similar to what has been described for the R. eutropha ADH (5).
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The apparent affinities for the various substrates, although more variable, are still comparable. The differences were larger for acetaldehyde reduction and ethanol oxidation with 6-fold higher Km values for iPDH purified from the parasites (Table II). Unfortunately no data are available for the affinities for acetone or isopropyl alcohol of the R. eutropha ADH to extend the comparison. The Phytomonas enzymes have the lowest Km values for these compounds, indicating that they are the preferred substrates for the reverse and the forward reactions, respectively.
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The enzymatic properties for the Phytomonas and bacterially expressed iPDHs compared very well. Relative activity and affinity of iPDH from the parasite for the different substrates were close to the averaged values for both isoforms produced by the bacteria. It is interesting to note that the heterologously expressed enzymes correspond to homodimers of the respective putative pre-iPDHs and not the mature enzymes. On the other hand, the enzyme purified from Phytomonas will, most likely, represent a mixture of homodimers and heterodimers of mature polypeptides.
Our finding that the apparent rate of acetoacetate reduction was similar to that observed for acetone (Table I) was further studied by analyzing the products of the reaction by gas chromatography-MS. The expected reaction product, 3-hydroxybutanoate, could not be detected after 1–6 h of incubation, whereas a progressive loss of acetoacetate did occur. On the other hand, 3-hydroxybutanoate was not a substrate for the oxidation reaction (Table I). This indicates that the apparent oxidation of NADH by iPDH with acetoacetate as substrate is most likely because of the spontaneous decarboxylation of acetoacetate, a reaction which is strongly stimulated by the presence of proteins (24), followed by a further reduction of the resulting acetone to isopropyl alcohol.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The phylogenetic analysis indicates that Phytomonas iPDH belongs to a small group of enzymes sharing amino acid similarities of 76–74% and identities of 62–58% with ADHs of strict aerobic bacteria from Pseudomonad, Rhizobial, and Burkholderial families. The substrate profile and kinetic parameters are very similar to those of the R. eutropha fermentative ADH, which is so far the only characterized enzyme from this group of bacterial ADHs (5). Zinc-dependent enzymes are specific for either primary or secondary alcohols. However, R. eutropha fermentative ADH and Phytomonas iPDH are multifunctional enzymes able to use both primary and secondary alcohols, polyols, aldehydes, and ketones. They dismutate acetaldehyde as well, but its rate of reduction is 1 order of magnitude higher than its rate of oxidation. Amino acids involved in substrate and NADH binding are conserved with respect to other zinc-dependent ADHs (22). Interestingly, residues related to the coordination of the catalytic and structural Zn2+ are highly conserved in iPDH but not in R. eutropha ADH, where only those residues involved in the coordination of the catalytic Zn2+ are present. Both enzymes possess an Ile-Thr4-Ile amino acid motif immediately in front of the conserved Cys50 in the substrate-binding domain (Fig. 3). This motif is only present in ADHs belonging to this phylogenetic group. In a preliminary structural study, this threonine tract seems to replace a region of hydrophobic amino acids at the bottom of the active site cleft present in other ADHs (22, 25). The binding of longer secondary alcohols or polyols could be stabilized by hydrogen bonds between threonines and functional groups on the substrates, justifying the broad pattern of substrate specificity, which is characteristic of these multifunctional ADHs.
The iPDH amino acid sequence and its kinetic properties suggest that this enzyme originated in the genus Phytomonas most likely as the result of a horizontal gene transfer between a strictly aerobic bacterium as donor organism and an ancestral trypanosomatid as recipient. The fact that this enzyme is now found in all phytomonads analyzed thus far, but not in any other representative of the trypanosomatid family (26), suggests that the acquisition of this gene may have resulted in a selective advantage for a trypanosomatid living in an environment such as that present in the phloemic or laticiferous tubes of colonized plants. In this respect, it is interesting to note that the two sister taxa of Phytomonas are S. meliloti, which fixes nitrogen for the legumes alfalfa, sweet clover, and fenugreek, and Pseudomonas fluorescens, a plant growth-promoting bacterium that colonizes both leaves and roots of plants. Thus, gene transfer may have been facilitated by a close contact within a common host organism between these plant-colonizing bacteria and the Phytomonas ancestor. A similar explanation can be put forward for the appearance of the M. penetrans ADH within this clade, whereas Mycoplasma pneumoniae clusters with the Clostridia, as expected. Mycoplasmas are well known obligate parasites of eukaryotes, including plants, and could have picked up the ADH gene from the same or another plantcolonizing bacterium, as did the Phytomonas ancestor.
iPDH activity was present in 20 isolates of Phytomonas, whereas the enzyme was absent from 17 non-phytomonad species of trypanosomatids which belonged to the genera Trypanosoma, Leishmania, Leptomonas, Crithidia, Blastocrithidia, and Herpetomonas (26). This finding renders iPDH an interesting biochemical marker for the Phytomonas genus. Because of the high DNA sequence identity (60%) between iPDH and R. eutropha ADH, we expect an even higher percentage of identical residues between iPDH genes from different Phytomonas isolates. This would allow a detailed phylogenetic analysis of the genus as a first approach to establish Phytomonas as a species. Work in this direction is in progress.
Phytomonads are highly dependent on glycolysis. We have shown previously that they excrete hydrolytic enzymes involved in the degradation of polysaccharide constituents of plants and consume the resulting monosaccharides at a high rate (27). This high glycolytic rate is forced upon the organism by the absence of oxidative phosphorylation as the result of a complete repression of mitochondrial activity. Cytochromes cannot be detected, and a functional Krebs cycle is missing (27, 28). This high glycolytic rate can only be maintained by an effective regeneration of oxidative power. A mitochondrial cyanide-insensitive glycerol-3-phosphate oxidase is involved in the reoxidation of glycolytic NADH; most likely, the iPDH fulfills a similar role in the mitochondrion. In the strictly aerobic R. eutropha, the fermentative ADH has the function of regenerating oxidative power in the form of NAD+. This is done by the reduction of acetoin produced from pyruvate under limited oxygen supply and by the reduction of acetaldehyde formed from the accumulated acetyl-CoA (5). Under these conditions, the expression of the enzyme is induced in R. eutropha. In Phytomonas, oxidative phosphorylation is permanently suppressed, and iPDH is constitutive. As shown previously, one-third of the pyruvate is converted to ethanol under aerobic conditions; the remainder is transformed into acetate (29). Apparently the majority of the pyruvate produced from glycolysis enters the mitochondrion where it is converted to acetyl-CoA with concomitant reduction of NAD+ (27, 29). Acetyl-CoA may provide an additional molecule of ATP by passing through the acetate:succinate-CoA transferase/succinyl-CoA synthetase cycle (30). A minor part of the pyruvate is converted to acetaldehyde by a putative pyruvate decarboxylase. The subsequent reduction of the acetaldehyde by iPDH, producing ethanol as an end product of glycolysis, would regenerate part of the NAD+ consumed by the pyruvate dehydrogenase. Under anaerobic conditions, essentially all pyruvate is converted to ethanol. When energy sources are non-limiting, an excess of acetyl-CoA could be converted to acetoacetate, which is known to decarboxylate spontaneously to acetone. The latter can then be reduced to isopropyl alcohol by iPDH. For Phytomonas iPDH, in contrast to R. eutropha ADH, acetone seems to be the best substrate for the reverse reaction, with a Vmax of 6.8 units/mg and a Km of 32 µM.
Another potential source of acetone could be the acetoacetate produced by amino acid catabolism. Although virtually nothing is known about amino acid metabolism in Phytomonas, amino acids are considered to be the principal energy source for the parasite inside the insect vector, because of the lack of carbohydrates in this host. In other trypanosomatids, proline serves as the major energy source for the insect stage; however, this amino acid is metabolized via the Krebs cycle (31). Because Phytomonas lacks a functional Krebs cycle, it is expected that, in this organism, amino acids other than proline fulfil the role of energy substrate. In preliminary experiments, we have found evidence that the ketogenic amino acids leucine, lysine, and phenylalanine are able to support growth of Phytomonas. If in Phytomonas these amino acids are catabolized in a way similar to other eukaryotes (32), iPDH could be essential for the elimination of the resulting acetone, with the concomitant regeneration of oxidative power.
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FOOTNOTES |
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* This work was supported in part by Fondo Nacional de Ciencia y Tecnología and Secretaría de Ciencia y Tecnología, Argentina, Grant PICT 99 No. 1-7160, by Ministerio de Educación de la Nación, Argentina, FOMEC 138, by the Fund for Medical Scientific Research (Belgium), and by the Belgian Directorate General for Higher Education and Scientific Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
|| Member of Carrera del Investigador Científico, CONICET, Argentina. To whom correspondence should be addressed. Tel.: 54-341-4350661; Fax: 54-341-4390465; E-mail: toniuttaro{at}yahoo.com.ar.
1 The abbreviations used are: ADH, alcohol dehydrogenases; iPDH, isopropyl alcohol dehydrogenase; BSTFA, N,O-bis-(trimethylsilyl)-trifluoroacetamide; MS, mass spectrometry.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, those related to cosubstrate binding.
