Evidence for a relationship between malate metabolism and activity of 1-sinapoylglucose: L-malate sinapoyltransferase in radish (Raphanus sativus L.) cotyledons

The control of malate metabolism and stimulation of 1-sinapolyglucose: L-malate sinapoyltransferase (SMT) activity in radish (Raphanus sativus L. var. sativus) cotyledons has been studied. The light-induced and nitrate-dependent activity of SMT catalyzes the formation of O-sinapoly-L-malate via 1-O-sinapoyl--D-glucose. When dark-grown radish seedlings, cultivated in quartz sand with nutrient solution containing NO ₃ ^- as the sole N source, were treated with light, SMT activity increased concomitantly with free malate in the cotyledons. This light effect was suppressed in seedlings grown in a culture medium which contained in addition to NO ₃ ^- also NH ₄ ⁺ . However, treatment with methionine sulfoximine neutralized this ammonium effect, resulting again in both rapid accumulation of malate and rapid increase in SMT activity. When seedlings grown on NO ₃ ^- nitrogen were subsequently supplied with NH ₄ ⁺ nitrogen, the accumulated level of L-malate rapidly dropped and the SMT increase ceased. The enzyme activity decreased later on, reaching the low activity level of plants which were grown permanently on NO ₃ ^- /NH ₄ ⁺ -nitrogen. An external supply (vacuum infiltration) of malate to excised cotyledons and intact seedings, grown on NO ₃ ^- /NH ₄ ⁺ -nitrogen medium, specifically promoted a dose-dependent increase in the activity of SMT. In summary these results provide evidence indicating that the SMT activity in cotyledons of Raphanus sativus might be related to the metabolism of malic acid.Abbreviation MSO L-methionine sulfoximine - SinGlc 1-O-sinapoyl--D-glucose - SinMal O-sinapoyl-L-malate - SMT 1-O-sinapoyl--D-glucose:L-malate sinapolytransferase     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively     

Sequence homologies between glyoxysomal and mitochondrial malate dehydrogenase

The comparison of mitochondrial and glyoxysomal malate dehydrogenase (EC 1.1.1.37) from cotyledons of germinating watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) by means of serological methods and peptide patterns revealed a high degree of homology. The N-terminal sequence analysis yielded a distinct presequence of eight or nine amino-acid residues, respectively, which is followed by an almost identical stretch of at least 20 amino-acid residues. A very similar domain has been recognized for mitochondrial malate dehydrogenase from porcine heart and yeast, and for Escherichia coli malate dehydrogenase.Abbreviations gMDH glyoxysomal malate dehydrogenase - mMDH mitochondrial malate dehydrogenase - SDS sodium dodecyl sulfate     

Import of in-vitro-synthesized glyoxysomal malate dehydrogenase into isolated watermelon glyoxysomes

Glyoxysomal malate dehydrogenase (gMDH; EC 1.1.1.37) is synthesized by a reticulocyte system in the presence of watermelon mRNA (Citrullus vulgaris Schrad., var. Kleckey's Sweet No 6) as a cytosolic, higher-molecular-weight precursor (41 kdalton). We now show that this precursor is posttranslationally sequestered by a crude glyoxysomal fraction or by glyoxysomes purified on a Percoll^R gradient to a proteolytically protected form (60 min proteinase-K treatment at 4° C) with the size of the gMDH subunit (33 kdalton). In the presence of buffer instead of organelles a complete degradation of the precursor is obtained. The in-vitro organelle import, however, depends upon the presence of proteases such as proteinase K or trypsin. After short proteolytic treatments (e.g. 10 min proteinase K at 4° C), the correct processing of the MDH precursor is obtained even in the absence of organelles. This product, however, is not sequestered in vitro to a protease-resistant form by glyoxysomes. The possibility is discussed that under in-vivo conditions pre-gMDH is processed on the outside of the glyoxysomal membrane and transferred immediately after processing into the organelle presumably as a gMDH monomer followed by refolding and dimerization.Abbreviations gMDH glyoxysomal malate dehydrogenase - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - TPCK-trypsin trypsin treated with l-1-tosylamide-2-phenylethyl chloromethyl ketone Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Two malate dehydrogenases in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum (strain Marburg) was found to contain two malate dehydrogenases, which were partially purified and characterized. One was specific for NAD⁺ and catalyzed the dehydrogenation of malate at approximately one-third of the rate of oxalacetate reduction, and the other could equally well use NAD⁺ and NADP⁺ as coenzyme and catalyzed essentially only the reduction of oxalacetate. Via the N-terminal amino acid sequences, the encoding genes were identified in the genome of M. thermoautotrophicum (strain ΔH). Comparison of the deduced amino acid sequences revealed that the two malate dehydrogenases are phylogenetically only distantly related. The NAD⁺-specific malate dehydrogenase showed high sequence similarity to l-malate dehydrogenase from Methanothermus fervidus, and the NAD(P)⁺-using malate dehyrogenase showed high sequence similarity to l-lactate dehydrogenase from Thermotoga maritima and l-malate dehydrogenase from Bacillus subtilis. A function of the two malate dehydrogenases in NADPH:NAD⁺ transhydrogenation is discussed. Received: 29 December 1997 / Accepted: 4 March 1998     

Import of glyoxysomal malate dehydrogenase precursor into glyoxysomes: A heterologous in-vitro system

A heterologous in-vitro system is described for the import of the precursor to glyoxysomal malate dehydrogenase from watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) cotyledons into glyoxysomes from castor-bean (Ricinus communis L.) endosperm. The 41-kDa precursor is posttranslationally sequestered and correctly processed to the mature 33-kDa subunit by a crude glyoxysomal fraction or by glyoxysomes purified on a sucrose gradient. The import and the cleavage of the extrasequence is not inhibited by metal chelators such as 1,10-phenanthroline and ethylenediaminetetraacetic acid. Uncouplers (carbonylcyanide m-chlorophenylhydrazone), ionophores (valinomycin), or inhibitors of oxidative phosphorylation (oligomycin) and ATP-ADP translocation (carboxyatractyloside) do not interfere, thus indicating the independence of the process of import by the organelle from the energization of the glyoxysomal membrane.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - EDTA ethylenediaminetetraacctic acid - gMDH glyoxysomal malate dehydrogenase - PMSF phenylmethylsulfonyl fluoride     

Sinapoylglucose: malate sinapoyltransferase activity in seeds and seedlings of rape

Protein preparations from seeds and seedlings (cotyledons) of rape (Brassica napus subsp. napus [L.] DC.) catalyzed the transfer of sinapic acid from 1-Osinapoyl--glucose to malate in the formation of O-s-inapoylmalate. The enzyme involved, 1-O-sinapoyl--glucose: l-malate O-sinapoyltransferase (SMT; EC 2.3.1), catalyzes the key step in the overall conversion of the seed constituent sinapine (O-sinapoylcholine) to the accumulating O-sinapoylmalate by way of the intermediate 1-O-sinapoyl--glucose. The present paper describes this phenomenon focussing on SMT activity.Abbreviations Sin-Glc 1-O-sinapoyl--glucose - Sin-Mal O-sinapoylmalate - SMT 1-O-sinapoyl--glucose: l-malate sinapoyltransferase (EC 2.3.1) This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie and the Ontario Ministry of Agriculture and Food.     

The metabolism of malate by cultured rat brain astrocytes

Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of¹⁴CO₂ production froml-[U-¹⁴C]malate in primary cultures of rat brain astrocytes. The¹⁴CO₂ production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the¹⁴CO₂ production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased¹⁴CO₂ production from 0.01 mM and 0.5 mM malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, -ketoglutarate and succinate decreased¹⁴CO₂ production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, -cyano-4-hydroxycinnamate and ouabain. Both the biphasic kinetics and the differential action of many of the effectors on the¹⁴CO₂ production from 0.01 mM and 0.5 mM malate provide evidence for the presence of more than one pool of malate metabolism in cultured rat brain astrocytes.This data was presented in part at the meeting of the Federation of American Societies for Experimental Biology in Las Vegas, Nevada, May 1988.     

Heterotrophic nitrification by Arthrobacter sp. (strain 9006) as influenced by different cultural conditions,growth state and acetate metabolism

An Arthrobacter sp. (strain 9006), isolated from lake water, accumulated nitrite up to about 15 mg N/l, but no nitrate. In a mineral medium supplemented with tryptone, yeast extract, acetate and ammonium, the cells released nitrite into the medium parallel to growth or when growth had virtually ceased. The nitrite formed was proportional to the initial acetate concentration, indicating an involvement of acetate metabolism with nitrification. The organism grew with a wide variety of organic carbon sources, but washed cells formed nitrite from ammonium only in the presence of citrate, malate, acetate or ethanol. Magnesium ions were required for nitrification of ammonium and could not be replaced by other divalent metal ions. Analysis of the glyoxylate cycle key enzymes in washed suspensions incubated in a minimal medium revealed that isocitrate lyase and malate synthase were most active during the nitrification phase. Nitrite accumulation but not growth was inhibited by glucose, tryptone and yeast extract. A possible explanation for the different nitrification patterns during growth is based on the regulatory properties of glyoxylate cycle enzymes.Abbreviations IL Isocitrate lyase [threo-D_s-isocitrate glyoxylate-lase, E.C. 4.1.3.1.] - MS malate synthase [l-malate glyoxylate-lyase (CoA-acetylating), E.C. 4.1.3.2.]     

Malic enzyme of Chromatium vinosum

Malic enzyme of the phototrophic bacterium Chromatium vinosum strain D that lacks malate dehydrogenase was partially purified yielding a specific activity of 55 units/mg protein. The constitutive enzyme with a molecular weight of 110,000 and a pH optimum of 8.0 was absolutely dependent on the presence of a monovalent cation (NH ₄ ⁺ , K⁺, Cs⁺, or Rb⁺) as well as a divalent cation (Mn²⁺, or Mg²⁺). The enzyme was inhibited by oxaloacetate, glyoxylate, and NADPH. The K _0.5 value for L-malate and the inhibition constants for oxaloacetate and glyoxylate are dependent on the concentration of the monovalent cation, whereas the K _m value for NADP (18 M) and the K ₁ value for NADPH (42 M) are independent. Throughout all kinetic measurements hyperbolic saturation curves and linear double reciprocal plots were obtained.Abbreviations OAA oxaloacetate - OD optical density     

Functional analysis and regulation of the malate synthase from<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Malate synthase (EC 2.3.3.9, formerly EC 4.1.2.2) has been investigated in the unicellular green algae Chlamydomonas reinhardtii. The molecular characteristics and the regulation of gene expression have been investigated for the enzyme. A full-length malate synthase cDNA has been isolated, containing an open reading frame of 1,641 bp encoding a polypeptide of 546 amino acids. This protein shares the conserved signature of the malate synthase family, along with the catalytic residues essential for enzymatic activity and a C-terminal motif that matches the consensus for glyoxysome import. Functionality studies have been facilitated by heterologous expression of the malate synthase cDNA in Escherichia coli. The remarkable metabolic versatility of the alga has been used to analyse the metabolic control of malate synthase gene expression. The data strongly support the role of acetate and light as the main regulatory effectors, and the existence of cross-talk between the two signalling pathways.Abbreviations IPTG Isopropyl -d-thiogalactopyranoside - MS Malate synthase - PCR Polymerase chain reaction - PTS Peroxisomal targeting sequence - RACE Rapid amplification of cDNA ends - TAP Tris–acetate–phosphate medium - TCA Tricarboxylic acid cycle     

Expression of a single gene encoding microbody NAD-malate dehydrogenase during glyoxysome and peroxisome development in cucumber

A full-length cDNA clone encoding microbody NAD⁺-dependent malate dehydrogenase (MDH) of cucumber has been isolated. The deduced amino acid sequence is 97% identical to glyoxysomal MDH (gMDH) of watermelon, including the amino terminal putative transit peptide. The cucumber genome contains only a single copy of this gene. Expression of this mdh gene increases dramatically in cotyledons during the few days immediately following seed imbibition, in parallel with genes encoding isocitrate lyase (ICL) and malate synthase (MS), two glyoxylate cycle enzymes. The level of MDH, ICL and MS mRNAs then declines, but then MDH mRNA increases again together with that of peroxisomal NAD⁺-dependent hydroxypyruvate reductase (HPR). The mdh gene is also expressed during cotyledon senescence, together with hpr, icl and ms genes. These results indicate that a single gene encodes MDH which functions in both glyoxysomes and peroxisomes. In contrast to icl and ms genes, expression of the mdh gene is not activated by incubating detached green cotyledons in the dark, nor is it affected by exogenous sucrose in the incubation medium. The function of this microbody MDH and the regulation of its synthesis are discussed.     

The relationship of biomass,polysaccharide and H2 formation in the wild-type and nifA/nifB mutants of Rhodobacter capsulatus

The production of biomass, polysaccharide storage material and H₂ from malate was studied in the wild-type and mutants RdcI, RdcII and RdcI/cII of Rhodobacter capsulatus. The mutants are defective in either copy I, copy II or both copies of the nitrogenase genes nifA and nifB. Stationary phase levels of biomass, polysaccharide and H₂ were determined in phototrophic batch cultures grown with 30 mM of d,l-malate and either 2, 5, or 8 mM of ammonium or 7 mM of glutamate. Calculation of the amounts of malate converted into the three products revealed that, at 8 mM of ammonium and 7 mM of glutamate, malate consumption and product formation were balanced. But with decreasing ammonium concentrations malate not converted into biomass was utilized with decreasing efficiency in polysaccharide and H₂ formation. This suggests formation of unknown products at the lower ammonium concentrations. Under conditions of optimal N supply, 80% of the malate not used for biomass production was converted by the wild-type and strain RdcII to H₂ and CO₂. Mutant RdcI exhibited slightly decreased H₂ production. The double mutant did not evolve H₂ but accumulated increased amounts of polysaccharide. However, the amounts of polysaccharide were lower than should be expected if all of the spare malate, not utilized by the double mutant for H₂ production, was converted into storage material. This and incomplete conversion of malate into known products at low ammonium supplies suggests that polysaccharide accumulation does not compete with the process of H₂ formation for malate.     

Purification of an endopeptidase involved with storage-protein degradation in Phaseolus vulgaris L. cotyledons

Phaseolin, the major seed storage protein of Phaseolus vulgaris L., is degraded in the cotyledons in the first 7–10 d following seed germination. We assayed cotyledon extracts for protease activity by using [³H]phaseolin as a substrate and then fractionated the digestion mixtures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in order to identify the cleavage products. The cotyledons of 4-d-old seedlings contain an endopeptidase which cleaves the polypeptides of [³H]phaseolin (apparent molecular weights=51 000, 48 000, 46 000 and 43 000) into three discrete clusters of proteolytic fragments (M _rs=27 000, 25 000 and 23 000). Endopeptidase activity is not detected in the cotyledons until the protein content of these organs starts to decline, shortly after the first day of seedling growth. Endopeptidase activity increases to a maximum level in the cotyledons of 5-d-old seedlings and then declines to a minimum value by day 10. The enzyme was purified 335-fold by ammonium-sulfate precipitation, organomercurial-agarose chromatography, gel filtration and ion-exchange chromatography. The endopeptidase constitutes 0.3% of the protein content in the cotyledons of 4-d-old seedlings. It is a cysteine protease with a single polypeptide chain (M _r=30 000). Optimum hydrolysis of [³H]phaseolin occurs at pH 5. The enzyme is irreversibly inactivated at pH values above 7 and at temperatures above 45° C. The endopeptidase attacks only a limited number of peptide bonds in [³H]phaseolin, without causing any appreciable change in the native molecular weight of the storage protein. The endopeptidase is also able to hydrolyze the bean-seed lectin, phytohemagglutinin. Thus, this enzyme may play a general role in degrading cotyledon proteins of P. vulgaris following seed germination.Abbreviations Da dalton - DTT dithiothreitol - M _r apparent molecular weight - PAGE polyacrylamide gel electrophoresis - PHA phytohemagglutinin - SDS sodium dodecyl sulfate     

Tissue distribution of phenylpropanoid metabolism in cotyledons of Raphanus sativus L.

The tissue distributions of sinapic acid esters (1-sinapoylglucose, sinapolyl-l-malate, 6,3-disinapoylsucrose), kaempferol glycosides, free malic acid and of the enzyme involved in the synthesis of sinapoyl-l-malate, 1-sinapoylglucose: l-malate sinapoyltransferase (SMT), have been investigated in cotyledons of Raphanus sativus L. seedlings. The kaempferol glycosides were mainly localized in the upper epidermis. The sinapoyl esters were found in all tissues, but differed markedly in their concentrations. While disinapoylsucrose was localized predominantly in the mesophyll, most sinapoylmalate was found in the epidermal layers, as was most SMT activity. Ultraviolet microscopy and microfluorospectrophotometry of isolated epidermal peels indicated that the epidermal sinapoyl esters were restricted to guard cells, guard mother cells and adjacent epidermal cells. Upon excitation by UV light (365 nm) these exhibited strong blue fluorescence with an emission maximum at about 480 nm. Our results indicate a highly tissue-and cell-specific secondary metabolism in Raphanus cotyledons and indicate that the biosynthesis of sinapoylmalate is intimately related to the malic-acid metabolism of the guard cells.Abbreviations HPLC high-performance liquid chromatography - SMT 1-sinapoylglucose: l-malate sinapoyltransferase     

The role of malate in ammonia assimilation in cotyledons of radish (Raphanus sativus L.)

The relationships between the metabolism of malate, nitrogen assimilation and biosynthesis of amino acids in response to different nitrogen sources (nitrate and ammonium) have been examined in cotyledons of radish (Raphanus sativus L.). Measurements of the activities of some key enzymes and pulse-chase experiments with [¹⁴C]malate indicate the operation of an anaplerotic pathway for malate, which is involved in the synthesis of glutamine during increased ammonia assimilation. It is most likely that the tricarboxylicacid cycle is supplied with carbon through entry of malate, formed via the phosphoenolpyruvate (PEP)-carboxylation pathway, when 2-oxoglutarate leaves the cycle to serve as precursor for an increased synthesis of glutamine via glutamate. This might occur predominantly in the cytosol via the activity of the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle, the NADH-dependent GOGAT being the rate-limiting activity.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - GDH glutamate dehydrogenase - GOGAT glutamate synthase (glutamine: 2-oxoglutarate aminotransferase) - GOT aspartate aminotransferase (glutamate: oxaloacetate transaminase) - GS glutamine synthetase - HPLC high-performance liquid chromatography - MCF extraction medium of methanol: chloroform: 7M formic acid, 12:5:3, by vol. - MDH malate dehydrogenase - MSO L-methionine, sulfoximine - PEPCase phosphoenolpyruvate carboxylase - TLC thin-layer chromatography     

Vacuolar localization of 1-sinapolglucose: l-malate sinapoyltransferase in protoplasts from cotyledons of Raphanus sativus

The distribution of l-malate, sinapic acid esters and 1-sinapoylglucose: l-malate sinapoyltransferase (SMT) which catalyzes the synthesis of sinapoyl-l-malate were examined in preparations of protoplasts obtained from cotyledons of red radish (Raphanus sativus L. var. sativus). Vacuoles isolated from the protoplasts contained all of the SMT activity, all of the accumulated sinapic acid esters and about 50% of free l-malate present initially in the protoplasts. An esterase activity, acting on 1-sinapoyglucose, was found to be exclusively localized in the cytoplasm and a large proportion was found to be recoverable in a 100 000-g pellet obtained from protoplast lysates. The vacuoles were obtained after lysis of the protoplasts by osmotic shock and purification on a Ficoll gradient. The cytoplasmic contamination of vacuole preparations was found to be about 10%, as judged by enzymatic markers and microscopic inspection. No SMT activity was found in a 100 000-g pellet obtained from vacuole lysates. The results indicate that biosynthesis of sinapoyl-l-malate takes place within the central vacuoles of redradish cotyledons.Abbreviation SMT 1-sinapoylglucose: l-malate sinapol-transferase     

Properties and primary structure of a thermostable l-malate dehydrogenase from Archaeoglobus fulgidus

A thermostable l-malate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was isolated and characterized, and its gene was cloned and sequenced. The enzyme is a homodimer with a molecular mass of 70 kDa and catalyzes preferentially the reduction of oxaloacetic acid with NADH. A. fulgidus l-malate dehydrogenase was stable for 5 h at 90° C, and the half-life at 101° C was 80 min. Thus, A. fulgidus l-malate dehydrogenase is the most thermostable l-malate dehydrogenase characterized to date. Addition of K₂HPO₄ (1 M) increased the thermal stability by 40%. The primary structure shows a high similarity to l-lactate dehydrogenase from Thermotoga maritima and gram-positive bacteria, and to l-malate dehydrogenase from the archaeon Haloarcula marismortui and other l-lactate-dehydrogenase-like l-malate dehydrogenases. Received: 20 November 1997 / Accepted: 28 February 1997     

Formation of l(-)malate by Saccharomyces cerevisiae during fermentation

Summary When grown in a synthetic medium most of the 51 strains of the genera Saccharomyces, Saccharomycodes, Zygosaccharomyces and Schizosaccharomyces investigated formed l-malate during fermentation. The quantity varied between 0.1 and 2.6 g malate per liter. Two strains of Saccharomyces cerevisiae synthesized malate at a rate of about 1.5 g/l. Malate was liberated during the growth phase and not metabolized during the stationary phase. Optimum malate formation was observed at a sugar concentration of about 20% (w/v), at pH 5 and at suboptimal nitrogen concentrations of less than 300 mg N/liter. Of the amino acids aspartate and glutamate were most favourable. If ammonium salts were used as the nitrogen source, significant amounts of malate were formed when the pH was kept constant by buffering. Trace metals had no or only little influence on malate synthesis. Biotin and pantothenate were essential for growth. Added ¹⁴CO₂ led to the formation of approximately equal quantities of labelled malate and succinate by S. cerevisiae strain 52, whereas about ten times more malate than succinate was formed by Saccharomyces uvarum. Avidin strongly inhibited the formation of malate while the inhibiton of succinate synthesis and of growth was comparatively much less. Malate is obviously formed by reduction of oxalacetate, the synthesis of which is catalysed by a biotin-dependent pyruvate carboxylase.