D-lactate Dehydrogenase Activity Research Articles

Overproduction of a 37-kilodalton cytoplasmic protein homologous to NAD+-linked D-lactate dehydrogenase associated with vancomycin resistance in Staphylococcus aureus.

We previously reported the isolation of a laboratory-derived Staphylococcus aureus mutant, 523k, that has constitutive low-level resistance to vancomycin (MIC = 5 micrograms/ml) and teicoplanin (MIC = 5 micrograms/ml) and elaborates a ca. 39-kDa cytoplasmic protein that was not detected in the parent strain 523 (MIC = 1 micrograms/ml). We have now detected the protein in strain 523 by immunoblotting with antiserum raised against the protein. Consistent with our initial observations, densitometric analysis of the immunoblots revealed an increased production of the protein in 523k compared with that of the susceptible parent 523. The 5' region of the gene encoding the protein of interest was identified by nucleotide sequencing a PCR product amplified from the genome of 523k with degenerate primers designed to encode the amino acid sequence of proteolytic peptides obtained from the protein. The remainder of the gene was identified by library screening, PCR, and nucleotide sequencing. The gene encodes a 36.7-kDa protein with homology to a family of bacterial NAD+-dependent, D-specific 2-hydroxyacid dehydrogenases which includes both D-lactate dehydrogenase and the enterococcal vancomycin resistance protein VanH and is therefore designated ddh. Increased production of the product of ddh, Ddh, was associated with increased D-lactate dehydrogenase activity in 523k, a finding which suggested that Ddh is likely to be the D-lactate dehydrogenase previously identified in S. aureus. The increased D-lactate dehydrogenase activity in strain 523k and the structural similarities among Ddh, D-lactate dehydrogenase, and VanH suggest that overproduction of Ddh might play a role in vancomycin resistance in this strain.

Pediococcus acidilactici ldhD gene: cloning, nucleotide sequence, and transcriptional analysis

The gene encoding D-lactate dehydrogenase was isolated on a 2.9-kb insert from a library of Pediococcus acidilactici DNA by complementation for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase and pyruvate-formate lyase double mutant. The nucleotide sequence of ldhD encodes a protein of 331 amino acids (predicted molecular mass of 37,210 Da) which shows similarity to the family of D-2-hydroxyacid dehydrogenases. The enzyme encoded by the cloned fragment is equally active on pyruvate and hydroxypyruvate, indicating that the enzyme has both D-lactate and D-glycerate dehydrogenase activities. Three other open reading frames were found in the 2.9-kb insert, one of which (rpsB) is highly similar to bacterial genes coding for ribosomal protein S2. Northern (RNA) blotting analyses indicated the presence of a 2-kb dicistronic transcript of ldhD (a metabolic gene) and rpsB (a putative ribosomal protein gene) together with a 1-kb monocistronic rpsB mRNA. These transcripts are abundant in the early phase of exponential growth but steadily fade away to disappear in the stationary phase. Primer extension analysis identified two distinct promoters driving either cotranscription of ldhD and rpsB or transcription of rpsB alone.

Cloning of the D-lactate dehydrogenase gene from Leuconostoc mesenteroides subsp. cremoris

A genomic library from Leuconostoc mesenteroides subsp. cremoris was screened for D-lactate dehydrogenase activity using a stereospecific lactate detection test on agar plate. Among 3500 clones tested, six positive colonies were found on D-lactate detection plate, displaying significantly higher D-LDH activity than Escherichia coli host strain.

D-lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family. Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum

The gene encoding D-lactate dehydrogenase (D-lactate: NAD+ oxidoreductase, EC 1.1.1.28) of Lactobacillus plantarum has been sequenced, and expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5'-noncoding region of the gene was replaced with the tac promoter. Comparison of the sequence of D-lactate dehydrogenase with L-lactate dehydrogenases, including the L. plantarum L-lactate dehydrogenase, showed no significant homology. In contrast, the D-lactate dehydrogenase is homologous to E. coli D-3-phosphoglycerate dehydrogenase and Lactobacillus casei D-2-hydroxyisocaproate dehydrogenase. This indicates that D-lactate dehydrogenase is a member of a new family of 2-hydroxyacid dehydrogenases recently proposed, being distinct from L-lactate dehydrogenase and L-malate dehydrogenase, and strongly suggests that the new family consists of D-isomer-stereospecific enzymes. In the reductive reaction, the enzyme showed a broad substrate specificity, although pyruvate was the most favorable of all 2-ketocarboxylic acids tested. In particular, hydroxypyruvate is effectively reduced by the enzyme, the reaction rate, and Km value being comparable to those in the case of pyruvate, indicating that the enzyme has not only D-lactate dehydrogenase activity but also D-glycerate dehydrogenase activity. The conserved residues in this family appear to be the residues involved in the substrate binding and the catalytic reaction, and thus to be targets for site-directed mutagenesis.

Studies on the anaerobic energy metabolism in the foot muscle of marine gastropod Patella caerulea (L.)

1. 1. The foot muscle of P. caerulea has a complete sequence of glycolytic enzymes. The low activitiy of hexokinase, in comparison with the activities of glycogen phosphorylase and phosphofructokinase, indicate that glycogen is the main fuel oxidized. 2. 2. The reduction of aspartate content in combination with the accumulation of alanine and the presence of considerable activities of glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase indicates a coupled metabolism of glycogen and aspartate during exposure to air. 3. 3. From the changes in the concentration of the metabolites during exposure to air it appears that up to the second hour of anaerobiosis alanine, lactate and glutamate are the end-products which accumulate in the foot muscle of P. caerulea, whereas from the second to the fourth hour only succinte and alanine accumulate. 4. 4. The low activities of the Krebs cycle enzymes as well as the absence of α-ketoglutarate dehydrogenase activity suggest that the Krebs cycle is not in operation. 5. 5. The absence of opine dehydrogenases shows that the end products octopine, alanopine and strombine are not accumulated in the foot muscle under anaerobiosis. 6. 6. The high activity of malate dehydrogenase in the direction of malate formation in combination with the low activity of d-lactate dehydrogenase and the absence of opine dehydrogenases suggests that the former dehydrogenase is coupled 1:1 to glyceraldehyde-3-phosphate dehydrogenase.

Inhibition of glycolate and D-lactate metabolism in a Chlamydomonas reinhardtii mutant deficient in mitochondrial respiration

The possibility that glycolate oxidation in unicellular green algae is linked to mitochondrial electron transport, rather than to peroxisomal metabolism as in higher plants and animals, was studied in a mutant of Chlamydomonas reinhardtii (dk97) deficient in cytochrome oxidase. This mutant had normal rates of dark respiration (40 +/- 15 mumol of O(2) uptake per hr per mg of chlorophyll) but had only 11% of wild-type levels of cytochrome oxidase activity. Salicylhydroxamic acid (SHAM) reduced the dark respiration rate of dk97 cells by 71%, but cyanide did not significantly inhibit this rate. During photosynthesis in the presence of SHAM, glycolate oxidation was blocked, resulting in glycolate accumulation and excretion by mutant cells but not by wild-type Chlamydomonas. D-Lactate, which accumulated after brief periods of anaerobiosis in Chlamydomonas, was reoxidized by air-grown cells only aerobically in the light, and reoxidation of D-lactate was blocked by SHAM in the dk97 cells. Thus, glycolate and D-lactate dehydrogenase activities are both linked to mitochondrial electron transport in Chlamydomonas. During photosynthetic (14)CO(2) fixation by dk97 cells in the presence of SHAM, (14)C-labeled tricarboxylic acid cycle intermediates accumulated, indicating that, in Chlamydomonas, mitochondrial respiration functions during photosynthesis.

Study of a time lag in the assay of Escherichia coli membrane-bound dehydrogenases based on tetrazolium salt reduction

The Escherichia coli membrane-bound d-lactate dehydrogenase and succinate dehydrogenase were assayed on the basis of the phenazine methosulfate- (PMS-) mediated reduction of the tetrazolium salt, MTT. An initial slower phase (lag) in the time-course of the reaction was observed and analyzed. The results were as follows. (1) The time lag in the assay of the d-lactate dehydrogenase was eliminated by preincubating the membranes with PMS plus d-lactate, with PMS plus succinate, or with PMS plus NADH (conditions which implicated PMS reduction). (2) When the d-lactate dehydrogenase was assayed by another method based on the measurement of the pyruvate formed, neither was a time lag observed nor was the enzyme activity affected by membrane preincubation with PMS plus d-lactate, (3) Although the superoxide radical was involved in MTT reduction, this radical seemed not to participate in the generation of the time lag. (4) Membranes whose d-lactate dehydrogenase activity had previously been destroyed by heating at 80°C for 1 min, were able to prolong the time lag in MTT reduction when added to the assay medium for the d-lactate dehydrogenase from untreated membranes, whereas membranes previously heated at 100°C instead of 80°C did not have this effect. It was concluded that the E. coli membranes interfered in the dehydrogenase assay based on the PMS-mediated reduction of MTT. The time lag was interpreted as a period during which the interfering substance reacted with reduced PMS inhibiting the reduction of MTT.

Effect of hyperthermia and gamma-radiation on Escherichia coli K1060 D-lactate dehydrogenase.

The response of E. coli K1060 D-lactate dehydrogenase (D-LDH), an enzyme located in the cytoplasmic membrane, was studied following 42.5 degrees C hyperthermia and/or gamma-irradiation. The inactivation of D-LDH following the above treatment was used as a tool to probe the role of membrane proteins in the radiation and/or heat sensitivity of cells. No correlation between loss of enzyme activity and cell killing was found, suggesting that D-LDH does not play an important role in hyperthermic cell survival. The results obtained in combined hyperthermia and gamma-irradiation treatments on loss of D-LDH activity and E. coli cell killing suggest that an interaction between heat and radiation occurs at the membrane structure level. Moreover, when cells were heated at 42.5 degrees C in the presence of 10 mM procaine-HCl, both cell killing and loss of D-LDH activity were enhanced. The involvement of membrane structure in the heat sensitivity of cells is strongly indicated by the latter observations. The opposite effect was observed when procaine was present during irradiation in oxic conditions, suggesting that procaine itself can also act as a scavenger towards OH-induced radicals.

Large scale production of d-lactate dehydrogenase for the stereospecific reduction of pyruvate and phenylpyruvate

To initiate studies of the stereospecific reduction of pyruvate and phenylpyruvate to the corresponding d-2-hydroxyacids a limited screening was carried out for microorganisms possessing a high NADH-dependet d-lactate dehydrogenase activity. Lactobacillus confusus was found to produce the desired dehydrogenase, which showed also relatively high activity towards phenylpyruvate, so this strain was selected for large scale production of the enzyme. A procedure for large scale purification of the enzyme starting with 24 kg wet cells is described including liquid-liquid extraction, ultrafiltration and chromatography on DEAE-cellulose, yielding a catalyst with specific activities of 216 U×mg−1 for pyruvate reduction and 15 U×mg−1 for phenyl-pyruvate reduction. A further tenfold purification can be achieved by affinity chromatography on Blue-Sepharose C-6B. Parameters which are important for industrial application of the enzyme were determined: substrate specifity, pH and temperature optimum, temperature stability, stability at different pH-values, and the storage stability of the enzyme in crude extracts.

Modulation of an 'equilibrium enzyme': ecological evidence.

The activity of D-lactate dehydrogenase in the foot of the snail,Helix pomatia, is closely correlated with time of year and mean daily temperature, and increases strikingly after the animals have been exposed to a nitrogen atmosphere for 24 h.

Effect of lipids on the reconstitution of D-lactate oxidase in Escherichia coli membrane vesicles.

An unsaturated fatty acid auxotroph lacking D-lactate dehydrogenase activity has been isolated from Escherichia coli ML 308-225 dld-3. While NADH oxidase activity in membrane vesicles prepared from the mutant cells grown in a variety of unsaturated fatty acids is comparable to that of previously isolated fatty acid auxotrophs, D-lactate oxidase activity is absent. However, D-lactate oxidase ativity can be restored when vesicles are incubated with a purified preparation of D-lactate dehydrogenase obtained from wild type cells. The effect of altering the fatty acid composition of the membrane on the reconstitution of D-lactate oxidase activity was examined. Binding of purified D-lactate dehydrogenase was not affected by either the lipid composition of the membrane vesicles or the temperature during reconstitution. However, the reconstitution of D-lactate oxidase activity was strongly influenced by the fatty acid composition of the membrane lipids. The temperature dependence of the reconstituted activity was analyzed. Temperature transitions were not observed with membrane vesicles supplemented with oleic or linolenic acid but palmitelaidic acid-enriched vesicles exhibited a transition temperature at 30 degrees. Attempts to reconstitute the elaidic-supplemented vesicles at temperatures below 30 degrees failed to yield active D-lactate oxidase and the vesicles aggregated. At 42 degrees, aggregation did not occur and D-lactate oxidase activity was obtained to a level of 10% of that found with membranes from the parent strain (ML 308-225). These results suggest that although binding of D-lactate dehydrogenase is independent of the physical state of the membrane, reconstitution of the D-lactate oxidase activity in membrane vesicles is dependent upon the fatty acid composition of the phospholipid, hence the physical state of the membrane.

Localization of D-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles

In the preceding paper the preparation and characterization of antiserum to purified D-lactate are described. In this paper the effects of the antibody on D-lactate dehydrogenase activity and D-lactate-dependent active transport in native Escherichia coli ML 308-225 membrane vesicles and ML 308-225dld-3 vesicles reconstituted with D-lactate dehydrogenase are described. The results demonstrate that D-lactate dehydrogenase is inaccessible to antibody in native ML 308-225 vesicles, but readily accessible to antibody in reconstituted dld-3 vesicles. The findings indicate that D-lactate dehydrogenase is located on the inner surface of native ML 308-225 vesicles and on the outer surface of reconstituted dld-3 vesicles. The results with the native vesicle preparations also provide further evidence that virtually none of the vesicles is inverted or sufficiently damaged to allow access of antibody to D-lactate dehydrogenase. In addition, experiments are presented which demonstrate that an impermeable electron carrier, reduced 5-N-methylphenazonium-3-sulfonate, drives active transport in native ML 308-225 vesicles as well as its permeable analogue reduced phenazine methosulfate. Thus, reduction of the respiratory chain from either side of the vesicle membrane is able to drive active transport. Ca2+, Mg2+-stimulated ATPase is also inaccessible to antibody in ML 308-225 vesicles unless the preparation is subjected to ultrasonic sound, incubated in Tris buffer at pH 9.0, or homogenized vigorously. Moreover, as opposed to D-lactate dehydrogenase and cytochrome b1, ATPase is readily lost from the membrane during the preparation of vesicles.

Immunochemical properties of the membrane-bound D-lactate dehydrogenase from Escherichia coli

The preparation, characterization, and purification of antibody against the membrane-bound D-lactate dehydrogenase solubilized and purified from Escherichia coli ML 308-225 are described. The antibody is highly specific for the flavin-linked D-lactate dehydrogenase, and incubation of the enzyme with antiserum results in marked inhibition of enzymatic activity. By means of a radioimmune assay, it is demonstrated that membrane vesicles prepared from E. coli ML 308-225dld-3 contain catalytically inactive material which cross-reacts with native D-lactate dehydrogenase. In the following paper, the effects of this antiserum on D-lactate dehydrogenase activity and D-lactate-dependent active transport in native and reconstituted membrane vesicles are examined.

The localization of glycollate-pathway enzymes in Euglena.

Isolation of organelles from broken-cell suspensions of phototrophically grown Euglena gracilis Klebs was achieved by isopycnic centrifugation on sucrose gradients. 2. Equilibrium densities of 1.23g/cm3 for peroxisome-like particles, 1.22g/cm3 for mitochondria and 1.17g/cm3 for chloroplasts were recorded. 3. The enzymes glycollate dehydrogenase, glutamate-glyoxylate aminotransferase, serineglyoxylate aminotransferase, aspartate-alpha-oxoglutarate aminotransferase, hydroxy pyruvate reductase and malate dehydrogenase were present in peroxisome-like particles. 4. Unlike higher plants glycollate dehydrogenase and glutamate-glyoxylate aminotransferase were present in the mitochondria of Euglena. 5. Rates of glycollate and D-lactate oxidation were additive in the mitochondria, and, although glycollate dehydrogenase was inhibited by cyanide, D-lactate dehydrogenase activity was unaffected. 6. Glycollate oxidation was linked to O2 uptake in mitochondria but not in peroxisome-like particles. This glycollate-dependent O2 uptake was inhibited by antimycin A or cyanide. 7. The physiological significance of glycollate metabolism in Euglena mitochondria is discussed, with special reference to its role in photorespiration in algae.

The outer membrane of Proteus mirabilis I. Isolation and characterization of the outer and cytoplasmic membrane fractions

1. 1. The crude envelope preperation obtained by sonication of Proteus mirabilis cells in the presence of lysozyme was seperated into outer and cytoplasmic membrane fractions by sucrose density gradient centrifugation. The outer membrane fraction accounted for about two thirds of the dry weight of the envelope preparation. 2. 2. In thin sections, the outer and cytoplasmic membrane fractions were shown to consist of vesicles bounded by a singel trilaminar membrane, but those of the outer membrane were considerably smaller and were frequently open, forming C-shaped strutures. The cytoplasmic membrane vesicles were cleaved by freeze fracturing to expose fracture faces studded with particles, while the outer membrane fragments resisted cleavage. 3. 3. The outer membrane fraction consisted of protien (∼40%), lipopolysaccharide (∼36%) and lipid (∼18%) and had a density of about 1.22 g/cm 3 . The cytoplasmic membranse fraction consisted mostly of protein (∼56%) and lipid (∼38%), had a density of about 1.16 g/cm 3, and contained almost all the NADH oxidase. succinate and d-lactate dehydrogenase activities of the crude envelope preparation. 4. 4. Electrophoresis in polyacrylamide gels containing sodium dodecylsulfate revealed over 20 polypeptide bands in the cytoplasmic membrane fraction and only 6–7 in the outer membrane fraction. The outer membrane electrophorogram was dominated by a major band (mol. wt 40 000) which was resolved into two bands when electrophoresed in an acidic gel system. Amino acid analysis revealed a higher content of polar amino acids in the protein moiety of the outer membrane.

Comparative inhibition studies of the phosphotransferase and glycerophosphate acylation systems in membrane vesicles of Escherichia coli

Purified membrane vesicles were treated with various reagents specific for different amino acid side-chains. Titration of sulfhydryl groups with specific reagents shows that the sulfhydryl content of membrane vesicles as estimated directly is similar to that found by treating spheroplasts or cells and then isolating the membrane vesicles. The blocking of sulfhydryl groups specifically inhibits the α-methylglucoside transport system (phosphotransferase system), whereas the glycerophosphate acylation system is not affected. The kinetics of inhibition of the first system show that a high reactivity of the sulfhydryl groups is involved. Inhibition of the acyltransferase activity by sulfhydryl reagents occurs only on partial denaturation of the membranes induced by mild sonication, heat or toluene treatment. The Inhibition is at the level of the glycerol 3-phosphate:acyl thioester acyltransferase. The effects of sonication and/or sulfhydryl reagents were measured by sulfhydryl titration, by assays of NADH oxidase and d-lactate dehydrogenase activities, as well as by 1-anilino-8-naphthalene sulfonate binding. The results support the hypothesis that the acyltransferase system is embedded within the membrane and that the readily accessible permease system is closer to (or at) the surface of the membrane.

Mechanisms of Active Transport in Isolated Bacterial Membrane Vesicles: X. INACTIVATION OF d-LACTATE DEHYDROGENASE AND d-LACTATE DEHYDROGENASE-COUPLED TRANSPORT IN ESCHERICHIA COLI MEMBRANE VESICLES BY AN ACETYLENIC SUBSTRATE

Abstract The acetylenic hydroxy acid, 2-hydroxy-3-butynoate, is an irreversible inactivator of d-lactate dehydrogenase and d-lactate-dependent lactose, proline, and valinomycin-induced rubidium transport in isolated membrane vesicles from Escherichia coli. The compound serves as a substrate for d-lactate dehydrogenase which undergoes some 15 to 30 turnovers prior to inactivation. Similarly, a low activity, membrane-bound l-lactate dehydrogenase is also inactivated presumably by the l isomer of the acetylenic substrate. These effects are highly specific as evidenced by the following observations. 1. Inactivation of d-lactate dehydrogenase in membrane vesicles or in a solubilized, partially purified preparation of this enzyme exhibits similar properties. 2. Although d- and l-lactate oxidation by membrane vesicles and transport in the presence of these electron donors is inactivated by hydroxybutynoate, the oxidation of succinate and NADH, and succinate- and NADH-stimulated transport are not affected. Moreover, ascorbate-phenazine methosulfate-dependent transport is not inactivated by hydroxybutynoate. 3. 3-Butynoate inhibits neither d-lactate dehydrogenase activity nor d-lactate-dependent transport; and 2-hydroxy-3-butenoate (vinylglycolate) serves as a substrate for d-lactate dehydrogenase, and is thus an effective electron donor for transport. 4. Kinetics of inactivation of d-lactate dehydrogenase activity and d-lactate-dependent lactose, proline, and valinomycin-induced rubidium transport are virtually identical with respect to both inhibitor concentration and time. 5. Inactivation of d-lactate-dependent transport by 2-hydroxy-3-butynoate is blocked by d-lactate, but not by succinate and NADH. 6. α-Glycerol-P-dependent threonine transport in Staphylococcus aureus vesicles is not inactivated by hydroxy-butynoic acid. A possible mechanism of inhibition is discussed.

Solubilization and partial purification of amino acid-specific components of the D-lactate dehydrogenase-coupled amino acid-transport systems (E. coli-cell membranes-sephadex-detergent-solubilized-vesicles).

A protein-containing fraction has been solubilized from E. coli ML 308-225 membrane vesicles that has many of the properties of the amino acid "carrier proteins" of the D-lactate dehydrogenase-coupled amino acid-transport systems. Membrane vesicles were partially solubilized with the nonionic detergent Brij 36-T, and the solubilized material was fractionated by Sephadex G-100 chromatography in the presence of the same detergent. Three fractions possess binding activity for proline: one of relatively low molecular weight with a high specific activity, and two of higher molecular weight with low specific activities. The higher molecular weight fractions exhibit D-lactate dehydrogenase activity; however, there is no corresponding activity associated with the low molecular weight fraction. Moreover, proline-binding activity is highly specific as it is not inhibited by structurally-unrelated amino acids. In addition to proline, the low molecular weight fraction exhibits binding activities for serine, glycine, lysine, and tyrosine. The proline-binding activity of the low molecular weight fraction is not inhibited by electron transfer or by general metabolic inhibitors that inhibit the accumulation of proline by intact membrane vesicles. In contrast, binding activity is inhibited by p-chloromercuribenzoate and N-ethylmaleimide, and the inhibition observed with p-chloromercuribenzoate is reversed by the addition of dithiothreitol. The effect of these sulfhydryl reagents on binding corresponds to the effect of these compounds on proline transport by intact membrane vesicles.