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Enzymes of the glyoxylate shunt have been implicated as virulence factors in several pathogenic organisms, notably Mycobacterium tuberculosis and Candida albicans. Malate synthase has thus emerged as a promising target for design of anti-microbial agents. For this effort, it is essential to have reliable models for enzyme:substrate complexes. A 2.7 Å resolution crystal structure for M. tuberculosis malate synthase in the ternary complex with magnesium, malate, and coenzyme A has been previously described. However, some unusual aspects of malate and Mg++ binding prompted an independent determination of the structure at 2.3 Å resolution, in the presence of saturating concentrations of malate. The electron density map of the complex reveals the position and conformation of coenzyme A to be unchanged from that found in the previous study. However, the coordination of Mg++ and orientation of bound malate within the active site are different. The revised position of bound malate is consistent with a reaction mechanism that does not require reorientation of the electrophilic substrate during the catalytic cycle, while the revised Mg++ coordination is octahedral, as expected. The results should be useful in the design of malate synthase inhibitors. [ABSTRACT FROM AUTHOR]

In most bacteria acetate assimilation is accomplished via the glyoxylate pathway. Isocitrate lyase (ICL) and malate synthase (MS) are two key enzymes of this pathway, which results in the net generation of one molecule of succinyl-CoA from two acetyl-CoA molecules. Genetic and biochemical data have shown that genes encoding these key enzymes are present in streptomycetes, yet there has been no clear demonstration of the importance of these genes to acetate assimilation. In fact, for Streptomyces collinus an alternative butyryl-CoA pathway has been shown to be critical for growth on acetate as a sole carbon source. Crotonyl-CoA reductase (CCR) is a key enzyme in this pathway and catalyzes the last step of the conversion of 2-acetyl-CoA molecules to butyryl-CoA. In Streptomyces cinnamonensis C730.1, it has been shown that CCR and this butyryl-CoA pathway provide the majority of methylmalonyl-CoA and ethylmalonyl-CoA for monensin A biosynthesis in an oil-based fermentation medium. We have cloned a MS homologue gene from this strain. Reverse transcription and direct enzyme assays demonstrated that neither this nor other MS genes were expressed during fermentation in an oil-based fermentation of either the C730.1 or L1 strain (a ccr mutant). Similarly, no ICL activity could be detected. The C730.1 but not the L1 strain was able to grow on acetate as a sole carbon source. The Streptomyces coelicolor aceA and aceB2 genes encoding ICL and MS were cloned into a Streptomyces expression plasmid (a derivative of pSET152) to create pExIM1. Enzyme assays and transcript analyses demonstrated expression of both of these proteins in C730.1/pExIM1 and L1/pExIM1 grown in an oil-based fermentation and tryptic soy broth media. Nonetheless, L1/pExIM1, like L1, was unable to grow on acetate as a sole carbon source, and was unable to efficiently generate precursors for monensin A biosynthesis in an oil-based fermentation, indicating that the additional presence of these two enzyme activities does not permit a functional glyoxylate cycle to occur. UV mutagenesis of S. cinnamonensis L1 and L1/pExIM1 led to mutants which were able to grow efficiently on acetate despite a block in the butyryl-CoA pathway. Analysis of enzyme activity and monensin production from these mutants in an oil-based fermentation demonstrated that neither the glyoxylate cycle nor the butyryl-CoA pathway function, suggesting the possibility of alternative pathways of acetate assimilation. [ABSTRACT FROM AUTHOR]

Two proposals recommend substitution of surface lysine residues as a means to improve the quality of protein crystals. In proposal I, substitution of lysine by alanine has been suggested to improve crystallization by reducing the entropic cost of ordering flexible side chains at crystal contacts. In proposal II, substitution of lysine by residues more commonly found in crystal contacts, such as glutamine, has been proposed to improve crystallization. 15 lysine residues on the surface of Escherichia coli malate synthase G, distributed over a variety of secondary structures, were individually mutated to both alanine and glutamine. For 28 variants, detailed studies of the effect on enzymatic activity and crystallization were conducted. This has permitted direct comparison of the relative effects of the two types of mutations. While none of the variants produced crystals suitable for X-ray structural determination, small crystals were obtained in a wide variety of conditions, in support of the general approach. Glutamine substitutions were found to be more effective than alanine in producing crystals, in support of proposal II. Secondary structure at the site of mutation does not appear to play a major role in determining the rate of success. [ABSTRACT FROM AUTHOR]

Abstract: We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems. [Copyright &y& Elsevier]

Abstract: Euglena gracilis induced glyoxylate cycle enzymes when ethanol was fed as a sole carbon source. We purified, cloned and characterized a bifunctional glyoxylate cycle enzyme from E. gracilis (EgGCE). This enzyme consists of an N-terminal malate synthase (MS) domain fused to a C-terminal isocitrate lyase (ICL) domain in a single polypeptide chain. This domain order is inverted compared to the bifunctional glyoxylate cycle enzyme in Caenorhabditis elegans, an N-terminal ICL domain fused to a C-terminal MS domain. Purified EgGCE catalyzed the sequential ICL and MS reactions. ICL activity of purified EgGCE increased in the existence of acetyl-CoA at a concentration of micro-molar order. We discussed the physiological roles of the bifunctional glyoxylate cycle enzyme in these organisms as well as its molecular evolution. [Copyright &y& Elsevier]

Abstract: Peroxisomes are involved in oxidative metabolic reactions and have the capacity to generate large amounts of reactive oxygen species that could damage biomolecules including their own resident proteins. The purpose of this study was to determine whether peroxisomal proteins are susceptible to oxidation and whether oxidative damage affects their enzymatic activity. Peroxisomal proteins were subjected to metal-catalyzed oxidation (MCO) with CuCl2/ascorbate and derivatized with 2,4-dinitrophenylhydrazine which allowed for spectrophotometric quantification of carbonylation. Immunochemical detection of carbonylated peroxisomal proteins, resolved by gel electrophoresis and detected with anti-DNP antibodies, revealed five oxidatively modified proteins with the following molecular weights: 80, 66, 62, 55, and 50kDa. The proteins at 66, 62, and 55kDa were identified as malate synthase (MS), isocitrate lyase, and catalase (CAT), respectively. MS and CAT were estimated to contain 2–3mol of carbonyl/mol of protein as a result of MCO. Enzymatic assays revealed varying degrees of activity loss. Isocitrate lyase and malate synthase showed significant loss of activity while catalase and malate dehydrogenase were less inhibited by carbonylation. Our findings show that peroxisomal proteins are vulnerable to MCO damage and thus may also be affected by in vivo exposure to reactive oxygen species. [Copyright &y& Elsevier]

The glyoxylate cycle is a modified form of the tricarboxylic acid cycle that converts C2 compounds into C4 dicarboxylic acids at plant developmental stages. By studying submerged rice seedlings, we revealed the activation of the glyoxylate cycle by identifying the increased transcripts of mRNAs of the genes of isocitrate lyase (ICL) and malate synthase (MS), two characteristic enzymes of the glyoxylate cycle. Northern blot analysis showed that ICL and MS were activated in the prolonged anaerobic environment. The activity assay of pyruvate decarboxylase and ICL in the submerged seedlings indicated an 8.8-fold and 3.5-fold increase over that in the unsubmerged seedlings, respectively. The activity assay of acetyl-coenzyme A synthetase in the submerged seedlings indicated a 3-fold increase over that in the unsubmerged seedlings, which is important for initiating acetate metabolism. Consequently, we concluded that the glyoxylate cycle was involved in acetate metabolism under anaerobic conditions. Edited by: Hong-Xuan LIN [ABSTRACT FROM AUTHOR]

Abstract: Isocitrate lyase (ICL) and malate synthase (MS) are key glyoxylate cycle enzymes shown to be required for the persistence and virulence of Candida albicans and of Mycobacterium tuberculosis in macrophages because the up-regulation of glyoxylate genes and the corresponding enzymes could replenish C4 carbohydrates from C2 compounds in a persistent pathogen. In this study, the ace (acetate) genes (aceA and aceB) of a persistent pathogen, Burkholderia pseudomallei (ATCC 23343), encoding an ICL and a MS, respectively, were isolated and fully sequenced. The genes, aceA (1.3kb) and aceB (1.6kb) were cloned and expressed as tagged fusion proteins in Escherichia coli BL21 (DE3). The molecular weights of the predicted enzymes (ICL, 47.7kDa and MS, 59.1kDa) were consistent with ICLs and MSs reported so far. Phylogenetic analysis of these genes revealed significant identity (80–90%) with most bacterial ICLs and MSs. Comparative structural modeling and the localization of major ICL and MS family domains in the deduced peptide sequences showed interestingly significant similarity with isozymes from known pathogens. Specific activities of expressed ICL (589.27nmolmin−1 mg−1) and MS (485.54nmolmin−1 mg−1) were also demonstrated. Taken together, these results provide evidence for the functionality of glyoxylate cycle genes in B. pseudomallei and may thus be useful for designing antimicrobials targeted at the glyoxylate cycle. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were identified in pupas of the butterfly Papilio machaon L. The activities of these enzymes in pupas were 0.056 and 0.108 unit per mg protein, respectively. Isocitrate lyase was purified by a combination of various chromatographic steps including ammonium sulfate fractionation, ion- exchange chromatography on DEAE-Toyopearl, and gel filtration. The specific activity of the purified enzyme was 5.5 units per mg protein, which corresponded to 98-fold purification and 6% yield. The enzyme followed Michaelis-Menten kinetics (Km for isocitrate, 1.4 mM) and was competitively inhibited by succinate (Ki = 1.8 mM) and malate (R = 1 mM). The study of physicochemical properties of the enzyme showed that it is a homodimer with a subunit molecular weight of 68 ± 2 kD and a pH optimum of 7.5 (in Tris-HCl buffer). [ABSTRACT FROM AUTHOR]

The glyoxylate bypass genes aceA1 (isocitrate lyase 1, ICL1), aceA2 (isocitrate lyase 2, ICL2) and aceB1 (malate synthase, MS1) of Ralstonia eutropha HF39 were cloned, sequenced and functionally expressed in Escherichia coli. Interposon-mutants of all three genes (ΔaceA1, ΔaceA2 and ΔaceB1) were constructed, and the phenotypes of the respective mutants were investigated. Whereas R. eutropha HF39ΔaceA1 retained only 19% of ICL activity and failed to grow on acetate, R. eutropha HF39ΔaceA2 retained 84% of acetate-inducible ICL activity, and growth on acetate was not retarded. These data suggested that ICL1 is in contrast to ICL2 induced by acetate and specific for the glyoxylate cycle. R. eutropha HF39ΔaceB1 retained on acetate as well as on gluconate about 41–42% of MS activity and exhibited retarded growth on acetate, indicating the presence of a second hitherto not identified MS in R. eutropha HF39. Whereas in R. eutropha HF39ΔaceA1 and R. eutropha HF39ΔaceA2 the yields of poly(3-hydroxybutyric acid), using gluconate as carbon source, were significantly reduced, R. eutropha HF39ΔaceB1 accumulated the same amount of this polyester from gluconate as well as from acetate as R. eutropha HF39. [Copyright &y& Elsevier]

The tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase (IDH) and the glyoxylate bypass enzyme isocitrate lyase are involved in catabolism of isocitrate and play a key role in controlling the metabolic flux between the TCA cycle and the glyoxylate shunt. Two IDH genes icd1 and icd2 of Ralstonia eutropha HF39, encoding isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2), were identified and characterized. Icd1 was functionally expressed in Escherichia coli, whereas icd2 was expressed in E. coli but no activity was obtained. Interposon-mutants of icd1 (HF39Δicd1) and icd2 (HF39Δicd2) of R. eutropha HF39 were constructed and their phenotypes were investigated. HF39Δicd1 retained 43% of IDH activity, which was not induced by acetate, and HF39Δicd2 expressed 74% of acetate-induced IDH activity. Both HF39Δicd1and HF39Δicd2 kept the same growth rate on acetate as the wild-type. These data suggested that IDH1 is induced by acetate. The interposon-mutants HF39Δicd1 and HF39Δicd2 accumulated the same amount of poly(3-hydroxybutyric acid) as the wild-type. [Copyright &y& Elsevier]

Malate synthase, an enzyme of the glyoxylate pathway, catalyzes the condensation and subsequent hydrolysis of acetyl-coenzyme A (acetyl-CoA) and glyoxylate to form malate and CoA. In the present study, we present the 1.95 Å-resolution crystal structure of Escherichia coli malate synthase isoform G in complex with magnesium, pyruvate, and acetyl-CoA, and we compare it with previously determined structures of substrate and product complexes. The results reveal how the enzyme recognizes and activates the substrate acetyl-CoA, as well as conformational changes associated with substrate binding, which may be important for catalysis. On the basis of these results and mutagenesis of active site residues, Asp 631 and Arg 338 are proposed to act in concert to form the enolate anion of acetyl-CoA in the rate-limiting step. The highly conserved Cys 617, which is immediately adjacent to the presumed catalytic base Asp 631, appears to be oxidized to cysteine-sulfenic acid. This can explain earlier observations of the susceptibility of the enzyme to inactivation and aggregation upon X-ray irradiation and indicates that cysteine oxidation may play a role in redox regulation of malate synthase activity in vivo. There is mounting evidence that enzymes of the glyoxylate pathway are virulence factors in several pathogenic organisms, notably Mycobacterium tuberculosis and Candida albicans. The results described in this study add insight into the mechanism of catalysis and may be useful for the design of inhibitory compounds as possible antimicrobial agents. [ABSTRACT FROM AUTHOR]

Malate synthase (MS) is a key enzyme responsible for malic acid synthesis in the glyoxylate cycle, which functions to convert stored lipids to carbohydrates, by catalysing the glyoxylate condensation reaction with acetyl‐CoA in the peroxisome. In this study, the cloning of an MS cDNA, designated MaMS‐1, from the banana fruit is reported. MaMS‐1 was 1801 bp in length encoding a single polypeptide of 556 amino acid residues. Sequence analysis revealed that MaMS‐1 possessed the conserved catalytic domain and a putative peroxisomal targeting signal SK(I/L) at the carboxyl terminal. MaMS‐1 also shared an extensive sequence homology (79–81.3%) with other plant MS homologues. Southern analysis indicated that MS might be present as multiple members in the banana genome. In Northern analysis, MaMS‐1 was expressed specifically in ripening fruit tissue and transcripts were not detected in other organs such as roots, pseudostem, leaves, ovary, male flower, and in fruit at different stages of development. However, the transcript abundance in fruit was affected by stage of ripening, during which transcript was barely detectable at the early stage of ripening (FG and TY), but the level increased markedly in MG and in other fruits at advanced ripening stages. Furthermore, MaMS‐1 expression in FG fruit could be stimulated by treatment with 1 µl l–1 exogenous ethylene, but the stimulatory effect was abolished by the application of an ethylene inhibitor, norbornadiene. Results of this study clearly show that MS expression in banana fruit is temporally regulated during ripening and is ethylene‐inducible. [ABSTRACT FROM PUBLISHER]

A phylogenetic study of the largest tribe of palms, the Areceae, was conducted using sequences of two low-copy nuclear genes. Previous morphological and plastid DNA studies have not supported the monophyly of the tribe, but have placed its members in a large clade that includes the monophyletic tribes Geonomeae, Cocoeae, Podococceae, and Hyophorbeae. We analyzed this large clade to test the monophyly of tribe Areceae with nuclear data, to explore relationships among its subtribes, and to identify other monophyletic groups. For 54 palm species, including members of all 17 subtribes of tribe Areceae, we sequenced regions of the malate synthase (MS) and phosphoribulokinase (PRK) genes. Simultaneous analysis of these regions revealed 52 shortest trees, all of which resolved tribe Areceae as polyphyletic. Subtribes Iguanurinae, Dypsidinae, Oncospermatinae, and Arecinae were also resolved as polyphyletic. A clade of Indo-Pacific taxa was resolved with strong support, and would be a suitable target for more focused study. [ABSTRACT FROM AUTHOR]

Summary • The ectomycorrhiza is a symbiotic organ formed between a filamentous fungus and a plant root, mainly tree roots. Root colonization involves significant shifts in gene expression resulting in metabolic and structural changes in the fungus, including growth toward the plant root, penetration and establishment of the symbiotic organ. • The preinfection stage of the association is crucial as changes that occur throughout mycorrhiza formation are set in motion. Using an in vitro system for identifying preinfection stage symbiosis-regulated genes from the Laccaria bicolor–Pinus resinosa interaction we have identified a malate synthase from L. bicolor (Lb-MS ). • The glyoxylate pathway, of which malate synthase is an enzyme, acts as a tricarboxylic acid pathway bypass generating four-carbon compounds for biosynthesis. While it is anticipated that malate synthase would be a part of the genetic and metabolic machinery of any fungus, Lb-MS is of interest because it is symbiosis regulated. • Lb-MS is regulated through interaction between the fungus and the host, by glucose and by the presence of other carbon sources in the medium. Its proposed role in the symbiosis is in the utilization of two carbon compounds formed from catabolic processes in early interaction facilitating hyphal net growth. [ABSTRACT FROM AUTHOR]

Malate synthase is an essential metabolic enzyme of the glyoxylate bypass that makes possible the replenishment of carbon intermediates to cells grown on acetate. A polymerase chain reaction (PCR)-based molecular screening investigation of full-length malate synthase genes from Streptomyces spp. was initiated by our group. To this end, consensus primers were designed based on known streptomycete malate synthase sequences and successful amplification was obtained for Streptomyces griseus, S. fimbriatus and S. lipmanii. The putative full-length malate synthase gene from S. griseus was subsequently cloned, sequenced and expressed. Sequence analysis of this gene showed very high identity with other streptomycete malate synthase genes. Furthermore, high malate synthase activity was detected after heterologous expression in Escherichia coli, thus demonstrating successfully the rapid cloning and functional verification of a streptomycete malate synthase gene. Growth studies of S. griseus revealed that malate synthase activity was induced by the presence of acetate, which is a two-carbon source. Interestingly, the activity peaked during late growth phase when the biomass was declining, suggesting that the enzyme may have a late role in metabolism. [ABSTRACT FROM AUTHOR]

Structural and cytochemical comparisons were made between three peroxisome types in soybean [Glycine max (L.) Merr. cv. Centennial]. Leaf peroxisomes were densely granular organelles with an amorphous nucleoid and were generally located in close proximity to the chloroplasts. Catalase (EC 1.11.1.6) and glycolate oxidase (EC 1.1.3.1) were localized in these peroxisomes although glycolate oxidase was absent from the nucleoid region. Glyoxysomes, present in the etiolated cotyledons, were coarsely granular organelles that were generally in close proximity to lipid bodies. Malate synthase (EC 4.1.3.2), catalase, and glycolate oxidase were present throughout the matrix. Although peroxisomes were found in both infected and uninfected nodule tissue, uninfected interstitial cell peroxisomes. were the most developed. These organelles contained a core surrounded by a less electron-opaque periphery that frequently was in close association with (but distinct from) a network of smooth endoplasmic reticulum. Of the enzymes studied, only catalase and urate oxidase (EC 1.7.3.3) were detected in the nodule peroxisomes. Neither enzyme was detected in the peripheral area of the peroxisome. These data indicate that peroxisomes in the three tissue types have organelle associations., internal structures, enzyme constitutions and packaging that reflect their metabolic differences. [ABSTRACT FROM AUTHOR]

Two maize glyoxysomal genes expressed during germination, malate synthase (MS) and isocitrate lyase (ICL), were used to characterize the regulatory roles of the Viviparous-l (Vpl) regulatory gene and abscisic aicd (ABA) in the induction of embryo quiescence during kernel development. In wild-type maize embryo, MS and ICL transcripts were first detected at 2 (MS) or 3 (ICL) days after germination (DAG), peaked at 5 DAG, and decreased thereafter. By reverse transcriptasepolymerase chain reaction (RT-PCR), the germinationspecific genes were amplified in both ABA-insensitive (vpl) and ABA-deficient (vp7 and vplO) mutant embryos at 26 and 33 days after pollination (DAP), but not in wild-type embryos. The repression of these germination-specific genes thus r equires the Vpl gene product and normal levels of ABA to induce embryo quiescence during kernel development. This suggests that a genetic regulatory system exists to prevent vivipary in developing maize embryos. The involvement of the Vpl gene product and ABA in repressing germination-specific genes complements their previously defined roles in the induction of seed-specific genes such as Cl (Hattori et al., 1992; Paek et aI. , 1997), Em (McCarty et aI., 1991), and Glnl (Kriz et aI., 1990; Rivin and Grudt, 1991). [ABSTRACT FROM AUTHOR]

Treatment of the 1 + strain of Phycomyces blakesleeanus Bgff. with various cytokinins resulted in a stimulation of growth. The magnitude of growth stimulation depended on both the structure of the hormone used and the carbon source in the culture medium. Most of the cytokinin derivatives were active effect in glucose and oleic acid cultures. Benzyladenine (BA) and benzyladenosine stimulated the fungal growth only when oleic add was the sole carbon source, while they had no effect in glucose cultures within the tested range of concentrations. [14C]-BA was accumulated by the mycelium of oleic acid cultures. Therefore, differences in BA uptake between glucose and oleic acid cultures could account mainly for the specific growth-promoting effect of BA. In oleic acid cultures isocitrate lyase (EC 4.1 3.1) and malate synthase (EC 4.1.3.2) activities were enhanced by 40 and 34%, respectively, in the presence of BA. A time course of the hormone effect suggests that BA is not involved in induction, but in the regulation of the mentioned enzymes in Phycocmyces. In contrast, acetate when presented as the sole carbon source or after addition to a glucose culture medium, induced isocitrate lyase activity. This enzyme induction was prevented by simultaneous addition of cycloheximide. [ABSTRACT FROM AUTHOR]

Glycolic acid oxidase activity was present in all three organs, but ca 20-fold lower (per mg protein) in both nodule and cotyledon extracts compared to leaf extracts. Electrophoresis followed by activity staining on native gels indicated one enzyme form with the same mobility in nodule, cotyledon and leaf preparations. Urate oxidase activity was found in nodule extracts only. Native gel electrophoresis showed a single band of activity. Novel electrophoretic systems had to be developed to resolve the urate oxidase and glycolate oxidase activities; both of these enzymes moved cathodally in the gel system employed while most other proteins moved anodally. This multifaceted study of enzymes located within three specialized types of peroxisomes in a single species has not been undertaken previously, and the results indicate that previous comparisons between the enzyme content of specialized peroxisomes from different organisms are mostly consistent with that for a single species, soybean.

The glyoxylate cycle, catalysed by two unique enzymes: isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2), is necessary for the net conversion of acetate into glucose. This metabolic pathway operates in microorganisms, higher plants and nematodes. Two bacterial genes, encoding ICL and MS, were modified in order to introduce them into the mouse germ line. The ovine metallothionein-Ia (MT-Ia) promoter- aceB gene-ovine growth hormone (GH) gene (3′ GH sequence) construct was fused to the ovine MT-Ia promoter- aceA gene-ovine GH gene (3′ GH sequence). Therefore, in this single DNA sequence, both aceA and aceB are under independent MT-Ia promoter control and can be induced by zinc. Transgenic mice were generated by pronuclear microinjection of the aceB-aceA gene construct. We now report the establishment of four mouse lines carying these two transgenes. Studies on the progeny of these lines indicate that one line (No. 91) is expressing both genes at the mRNA and enzyme levels in the liver and intestine, whereas another line (No. 66) has a much lower expression. Both enzyme activities were detected in the liver and intestine at levels up to 25% of those measured in fully derepressed Escherichia coli cells. [ABSTRACT FROM AUTHOR]

In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurrs in cells growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a broad spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolase and proteins with the C-terminal SKL tripeptide. [ABSTRACT FROM AUTHOR]

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min–1 (mg protein)–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent K m of isocitrate lyase for d, l-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 μM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the V max nor the d, l-isocitrate K m of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum. [ABSTRACT FROM AUTHOR]

In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes ( aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum. [ABSTRACT FROM AUTHOR]

The malate synthase gene, MLS1, of the yeast Saccharomyces cerevisiae is transcriptionally regulated by the carbon source in the growth medium. A MLS1-lacZ fusion gene, expressed at a basal level in the presence of 2% glucose, is derepressed more than 100-fold under conditions of sugar limitation. No evidence for MLS1 induction by oleic acid was found. By deletion analysis of the MLS1 control region, we identified two sites, UAS1 and UAS2, as important for efficient derepression of the gene. Both sites contain sequences that resemble the previously characterized carbon source-responsive element (CSRE) found in the promoter of the isocitrate lyase gene ICL1. Indeed, UAS1 and UAS2 in the MLS1 upstream region turn out to be functional CSRE sequence variants. This finding allowed us to define a modified version of the CSRE consensus sequence (CCRTYSRNCCG). Protein binding to UAS1MLS1 was observed with extracts from derepressed but not from repressed cells, and could be competed for by an excess of the unlabelled CSRE( ICL1) sequence. No competition was observed with a mutated CSRE variant. Site-directed mutagenesis of both CSREs in the MLS1 promoter reduced gene activation under derepressing conditions to 20% of the wild-type level. The same decrease was observed with the wild-type MLS1 promoter in a cat8 mutant, lacking an activator of CSRE-dependent transcription. The CSRE/Cat8p-independent activation of MLS1 is mediated by constitutive UAS elements. The pleiotropic transcription factor Abf1p, which binds to the MLS1 upstream region, may contribute to constitutive activation. Thus, in order to ensure the severe glucose repression of MLS1 observed, repressor elements that respond to the carbon source must counteract constitutive activation. In summary, ICL1 and MLS1 share common cis-acting elements, although a distinct mechanism of carbon source control also contributes to MLS1 regulation. [ABSTRACT FROM AUTHOR]

Copyright of Chemical Monthly / Monatshefte für Chemie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Chemical Monthly / Monatshefte für Chemie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Chemical Monthly / Monatshefte für Chemie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Analytical ultracentrifugation is a powerful tool for investigating the size of proteins in solution, especially by measuring sedimentation and diffusion coefficients and molar masses. Several further molecular parameters such as frictional ratios, axial ratios of hydrodynamic models, and Stokes radii allow a rough estimate of the protein overall structure. Sedimentation analysis may also be applied efficaciously for monitoring conformational changes of proteins occurring upon ligand binding or denaturation. For the determination of very small changes in shape, however, great care and a series of precautions are required. We investigated the enzymes citrate synthase and malate synthase in the absence and in the presence of ligands, in order to study the structural properties of the proteins and their ligand complexes. We also compared the results of the ultracentrifugal analysis with the results of other solution techniques such as UV absorption, fluorescence spectroscopy, circular dichroism, and small-angle x-ray scattering on the one hand, and the crystallographic 3D structure of citrate synthase on the other. The spectroscopic methods may be used as efficient and rapid tools for screening the occurrence of conformational changes caused by alterations of chromophores and fluorophores. The structural information provided by small-angle scattering (e.g., radii of gyration, maximum particle diameters, vclumes and surface areas) can be used to establish quantitative correlations between solution scattering and hydrodynamic data. In this context, however, knowledge or qualified assumptions of partial specific volumes and hydration are additionally required. Good agreement was reached between small-angle scattering and ultracentrifugal data, and also with crystallographic data if protein hydration was considered properly. The given approaches may be used to predict hydrodynamic properties if x-ray data are available, and for many verifications of other structural data, e.g., Stokes radii, diffusion coefficients, axial and frictional ratios determined by independent methods. [ABSTRACT FROM AUTHOR]

The cucumber malate synthase (MS) gene, including 1856 bp of 5′ non-trnascribed sequence, has been transferred into Petunia (Mitchell) and Nicotiana plumbaginifolia plants using an Agrobacterium binary vector. The transferred gene is found in variable copy number in different transformants, and is stably transmitted in each case as a single Mendelian character. Transgene mRNA accumulates in the seedling during the first three days of germination, then declines in amount as the cotyledons emerge from the seed. The decline is more pronounced in light-grown seedlings than in dark-grown seedlings. Expression of the MS transgene is also detected at a low level in petals of transformed Petunia plants. In these respects the pattern of MS gene expression is similar in cucumber and in trnasformed plants, showing that the transferred DNA fragment contains a functional MS gene. A 1076 bp fragment of 5′ sequence was linked to the β-glucuronidase reporter gene and transferred into Nicotiana, where it was shown to direct temporal and spatial patterns of expression similar to that of the complete MS gene. However, histochemical localisation of β-glucuronidase activity demonstrated that the chimaeric gene is expressed not only in cotyledons of transgenic plants, but also in endosperm and some hypocotyl cells during early germination. The relevance of these findings to the control of malate synthase gene expression is discussed. [ABSTRACT FROM AUTHOR]

The temporal, nonconcerted development of activities of malate synthase (MS), isocitrate lyase (ICL), and catalase (Cat) was explored in more detail in maturing and germinated cotton ( Gossypium hirsutum L.) seeds. RNA was extracted at six intervals beginning at 17 days post anthesis (DPA) through 72 hours post imbibition (HPI). In vitro translations revealed that mRNAs for each enzyme were translatable at all intervals. Enzyme activities and immunoselected proteins also were found at all intervals. Similar specific activities throughout maturation indicated that embryo cells were not accumulating inactive protein. The steady-state level of mRNAs encoding each enzyme exhibited different patterns of change during seed maturation, and each peaked at least 24 h before peak enzyme activities in germinated seeds. All three enzymes occur together as early as 17 DPA in a coordinate manner; however, the subsequent, nonconcerted increases in protein, activity, and mRNA for each enzyme indicate that developmental expression in cotton seed embryos is regulated in a noncoordinate fashion by as yet unidentified specific control mechanism(s). [ABSTRACT FROM AUTHOR]