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The successful management of tuberculosis (TB) requires efficient diagnosis and treatment. Further, the increasing prevalence of drug-resistant TB highlights the urgent need to develop novel inhibitors against both drug-susceptible and drug-resistant forms of disease. Malate synthase (MS), an enzyme of the glyoxylate pathway, plays a vital role in mycobacterial persistence, and therefore it is considered as an attractive target for novel anti-TB drug development. Recent studies have also ascribed an adhesin function to MS and established it as a potent diagnostic biomarker. In this study, a panel of Mycobacterium tuberculosis (Mtb) MS-specific single-stranded DNA aptamers was identified by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The best-performing G-quadruplex-forming 44-mer aptamer, MS10, was optimized post-SELEX to generate an 11-mer aptamer, MS10-Trunc. This aptamer was characterized by various biochemical, biophysical, and in silico techniques. Its theranostic activity toward Mtb was established using enzyme inhibition, host cell binding, and invasion assays. MS10-Trunc aptamer exhibited high affinity for MS (equilibrium dissociation constant [KD] ∼19 pM) and displayed robust inhibition of MS enzyme activity with IC50 of 251.1 nM and inhibitor constant (Ki) of 230 nM. This aptamer blocked mycobacterial entry into host cells by binding to surface-associated MS. In addition, we have also demonstrated its application in the detection of tuberculous meningitis (TBM) in patients with sensitivity and specificity each of >97%.

This work inserts in the research field regarding the effects of altered gravity conditions on biological plant processes. Pinus pinea seeds germination was studied in simulated microgravity (2x10−3g) and hypergravity (20g) conditions. The effects of simulated gravity were evaluated monitoring the levels of the key enzymes, involved in the main metabolic pathway during germination process of lipid–rich seeds (oilseeds): isocitrate lyase and malate synthase for glyoxylate cycle, 3-hydroxyacyl-CoA dehydrogenase for beta-oxidation, isocitrate dehydrogenase for Krebs cycle, pyruvate kinase for glycolysis and glucose 6 phosphate dehydrogenase for pentose phosphate shunt. The simulated micro and hypergravity conditions were obtained by a Random Position Machine and a Hyperfuge, respectively. Results show that the levels of some tested enzymes, at different lag times of the germination process, have the same trend of controls (g = 1), but with significant differences from quantitative point of view. They are higher in microgravity conditions and lower in hypergravity ones, suggesting that, from a biochemical point of view, the germination process results accelerated in microgravity conditions and delayed in hypergravity ones. These biochemical results show a good correlation with morphological ones, obtained with the measurement of the length of the seeds sprouting radicle. These results give promising indications regarding the possibility to grow plant with lipid-rich seeds in spatial environment, to obtain food sources for astronauts during long term space missions and to reconstitute new atmosphere.

Most protein folding studies until now focus on single domain or truncated proteins. Although great insights in the folding of such systems has been accumulated, very little is known regarding the proteins containing multiple domains. It has been shown that the high stability of domains, in conjunction with inter-domain interactions, manifests as a frustrated energy landscape, causing complexity in the global folding pathway. However, multidomain proteins despite containing independently foldable, loosely cooperative sections can fold into native states with amazing speed and accuracy. To understand the complexity in mechanism, studies were conducted previously on the multidomain protein malate synthase G (MSG), an enzyme of the glyoxylate pathway with four distinct and adjacent domains. It was shown that the protein refolds to a functionally active intermediate state at a fast rate, which slowly produces the native state. Although experiments decoded the nature of the intermediate, a full description of the folding pathway was not elucidated. In this study, we use a battery of biophysical techniques to examine the protein's folding pathway. By using multiprobe kinetics studies and comparison with the equilibrium behavior of protein against urea, we demonstrate that the unfolded polypeptide undergoes conformational compaction to a misfolded intermediate within milliseconds of refolding. The misfolded product appears to be stabilized under moderate denaturant concentrations. Further folding of the protein produces a stable intermediate, which undergoes partial unfolding-assisted large segmental rearrangements to achieve the native state. This study reveals an evolved folding pathway of the multidomain protein MSG, which involves surpassing the multiple misfolding traps during refolding.

Malate is regarded as one of the key building block chemicals which can potentially be produced from biomass at a large scale. Although glucose has been extensively studied as the substrate for malate production, its high price and potential competition with food production are serious limiting factors. In this study, Escherichia coli was metabolically engineered to effectively produce malate from xylose, the second most abundant sugar component of lignocellulosic biomass. First, the biosynthetic route of malate was constructed by overexpressing D -tagatose 3-epimerase, L-fuculokinase, L-fuculose-phosphate aldolase, and aldehyde dehydrogenase A. Second, genes encoding malic enzyme, malate dehydrogenase, and fumarate hydratase were knocked out to eliminate malate consumption, resulting in a titer of 1.99 g/l malate and a yield of 0.47 g malate/g xylose. Third, glycolate oxidase and malate synthase were overexpressed to strengthen the conversion of glycolate to malate, which led to a titer of 4.33 g/l malate and a yield of 0.83 g malate/g xylose, reaching 93% of the theoretical yield. Finally, catalase HPII was overexpressed to decompose H2O2 and alleviate its toxicity, which improved cell growth and further boosted malate titer to 5.90 g/l with a yield of 0.80 g malate/g xylose. To the best of our knowledge, this is the first study to report efficient malate production from xylose as the carbon source.

The NAD+-dependent glycerol 3-phosphate dehydrogenase (KlGpd1) is an important enzyme for maintenance of the cytosolic redox balance in the milk yeast Kluyveromyces lactis. The enzyme is localized in peroxisomes and in the cytosol, indicating its requirement for the oxidation of NADH in both compartments. Klgpd1 mutants grow more slowly on glucose than wild-type cells and do not grow on ethanol as a sole carbon source. We studied the molecular basis of the latter phenotype and found that Gpd1 is required for high expression of KlICL1 and KlMLS1 which encode the key enzymes of the glyoxylate pathway isocitrate lyase and malate synthase, respectively. This regulation is mediated by CSRE elements in the promoters of these genes and the Snf1-regulated transcription factors KlCat8 and KlSip4. To study the transactivation function of these factors we developed a modified yeast one-hybrid system for K. lactis, using the endogenous s-galactosidase gene LAC4 as a reporter in a lac9 deletion background. In combination with ChIP analyses we discovered that Gpd1 controls both the specific binding of Cat8 and Sip4 to the target promoters and the capacity of these factors to activate the reporter gene expression. We propose a model in which KlGpd1 activity is required for maintenance of the redox balance. In its absence, genes which function in generating redox balance instabilities are not expressed. A comparison of mutant phenotypes further indicates, that this system not only operates in K. lactis, but also in Saccharomyces cerevisiae.

The metabolic pathway such as glyoxylate cycle (GC) enables Candida albicans, to survive under glucose deficient conditions prevalent in the hostile niche. Thus its key enzymes (Isocitrate lyase; ICL and malate synthase; MLS) represent attractive targets against C. albicans. We have previously reported the antifungal potential of a natural monoterpenoid perillyl alcohol (PA). The present study uncovers additional role of PA as a potent GC inhibitor. We explored that PA phenocopied ICL1 deletion mutant and were hypersensitive under low carbon utilizing conditions. The effect of PA on GC was substantiated by molecular docking analyses, which reveals the in-silico binding affinity of PA with ICL and MLS and explored that PA binds to the active sites of both proteins with better binding energy in comparison to their known inhibitors 3-nitropropionate and bromopyruvate respectively. Enzyme kinetics by Lineweaver-Burk plot unravels that PA inhibits ICL and MLS enzymes in competitive and non-competitive manner respectively. Moreover, semi-quantitative RT-PCR indicated that PA inhibits ICL1 and MLS1 mRNA expressions. Lastly, we demonstrated the antifungal efficacy of PA by enhanced survival of Caenorhabditis elegans model and less hemolytic activity (10.6%) on human blood cells. Further studies are warranted for PA to be considered as viable drug candidate.

Glyoxylate is an important chemical building block which is currently produced by chemical or electrochemical oxidation methods We herein described the achievement of microbial glyoxylate production from renewable biomass by metabolic engineering of Escherichia coli. The synthetic ribulose-1-phosphate pathway was expressed to produce glycolate from xylose. Additional genomic modifications including the inactivation of malate synthase genes aceB/glcB and glyoxylate carboligase gene gcl were performed to prevent the consumption of glyoxylate. The constructed strain was found to accumulate 0.13 g/L glyoxylate in shake flask cultivations. Further overexpression of glycolate oxidase and catalase significantly improved glyoxylate production to 0.74 g/L, which is the highest titer reported to date. These results demonstrate that microbial fermentation has a promising potential to manufacture glyoxylate from renewable feedstocks.

Malate synthase is a condensing enzyme responsible for conversion of glyoxylate to malate in the presence of acetyl-CoA. This reaction helps in bypassing the TCA cycle reactions involving carbon loss and leads to diverting some of the carbon skeletons to gluconeogenic events while rest can continue to provide TCA cycle intermediates. Malate synthase (GlcB) is encoded by MRA_1848 of Mycobacterium tuberculosis H37Ra (Mtb-Ra). We developed a knockdown (KD) Mtb-Ra strain by down-regulating GlcB. The survival studies suggested increased susceptibility to oxidative and nitrosative stress as well as to rifampicin. The susceptibility profile was reversed in the presence of free radical scavengers. Also, KD showed reduced biofilm maturation, failed to enter persistent state, and showed reduced growth inside macrophages. The study of post-endocytosis events showed differences in late stage endosomal maturation behavior in macrophages infected with KD compared to WT. Increased iNOS, LAMP1 and cathepsin D expression was observed in macrophages infected with KD compared to WT.

There are enormous evidences and previous reports standpoint that the enzyme of glyoxylate pathway malate synthase G (MSG) is a potential virulence factor in several pathogenic organisms, including Brucella melitensis 16M. Where the lack of crystal structures for best candidate proteins like MSG of B. melitensis 16M creates big lacuna to understand the molecular pathogenesis of brucellosis. In the present study, we have constructed a 3-D structure of MSG of Brucella melitensis 16M in MODELLER with the help of crystal structure of Mycobacterium tuberculosis malate synthase (PDB ID: 2GQ3) as template. The stereo chemical quality of the restrained model was evaluated by SAVES server; remarkably we identified the catalytic functional core domain located at 4th cleft with conserved catalytic amino acids, start at ILE 59 to VAL 586 manifest the function of the protein. Furthermore, virtual screening and docking results reveals that best leadmolecules binds at the core domain pocket of MSG catalytic residues and these ligand leads could be the best prospective inhibitors to treat brucellosis.

The aim of this study was to analyze the metabolism of storage materials in germinating tomato (Solanum lycopersicum) seeds and to determine whether it is regulated by light via phytochromes. Wild type, single and multiple phytochrome A, B1 and B2 mutants were investigated. Imbibed seeds were briefly irradiated with far-red or far-red followed by red light, and germinated in darkness. Triacylglycerols and starch were quantified using biochemical assays in germinating seeds and seedlings during the first 5 days of growth. To investigate the process of fat-carbohydrate transformation, the activity of the glyoxylate cycle was assessed. Our results confirm the role of phytochrome in the control of tomato seed germination. Phytochromes A and B2 were shown to play specific roles, acting antagonistically in far-red light. While the breakdown of triacylglycerols proceeded independently of light, phytochrome control was visible in the next stages of the lipid-carbohydrate transformation. The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were regulated by phytochrome(s). This was reflected in a greater increase of starch content during seedling growth in response to additional red light treatment. This study is the first attempt to build a comprehensive image of storage material metabolism regulation by light in germinating dicotyledonous seeds.

The glyoxylate cycle is essential for growth on C2 compounds such as acetate. In this investigation, for the first time, we have elucidated the subcellular localization of the enzymes of the glyoxylate cycle in the green microalga Chlamydomonas reinhardtii. Acetyl-CoA synthase and malate dehydrogenase exist as multiple isoforms in this microalga, therefore, we first identified those implicated in the glyoxylate cycle based on the observation that lack of isocitrate lyase (ICL) in a previously identified icl deficient mutant was correlated with specific loss of the other enzymes of the glyoxylate cycle. In this work, we determined that five of the six enzymes associated with the glyoxylate cycle were found to be within peroxisomal microbodies. Citrate synthase, aconitase, malate synthase, malate dehydrogenase, and acetyl-CoA synthase are located in peroxisomal microbodies while isocitrate lyase is cytosolic. Our findings implicate a key role for these cellular compartments in acetate assimilation for Chlamydomonas. Microbodies have only recently been discovered in C. reinhardtii and their existence had been previously debated. The isoform specific subcellular localization determined here suggests that peroxisomal microbodies should be considered in the design of metabolic models for carbon assimilation in C. reinhardtii.

The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the tricarboxylic acid cycle. The GS bypasses the carbon dioxide-producing steps of the tricarboxylic acid cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be up-regulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli. In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including up-regulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N2O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer; that is, a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress.

S-Adenosyl- l -methionine (SAM), with diverse pharmaceutical applications, is biosynthesized from l -methionine and ATP. To enhance SAM accumulation in Saccharomyces cerevisiae CGMCC 2842 (2842), a new strategy based on yeast acetyl-CoA metabolism combined with introducing a methionine adenosyltransferase (metK1) from Leishmania infantum, was presented here. It was found that over-expressing acs2 (encoding acetyl-CoA synthase) and deleting mls1 (encoding malate synthase) increased SAM by 0.86- and 1.30-fold, respectively. To eliminate feedback inhibition of SAM synthase, a codon-optimized metK1 was introduced into 2842, and an increase of 1.45-fold of SAM was observed. Subsequently, metK1 and acs2 were co-expressed in the mls1 deleted strain, obtained the highly SAM-productive strain Ymls1 △GAPmK, and 2.22 g/L of SAM accumulated, which was 3.36-fold that in 2842. Moreover, the Ymls1 △GAPmK strain yielded 6.06 g/L SAM, which was 9.18-fold that in 2842, by fed-batch fermentation in a 10-L fermenter. Finally, the isolation and purification of SAM from yeast cell and preparation of SAM sulfate were preliminarily investigated. This study demonstrated that up-regulating acs2 and deleting mls1, which elevated intracellular acetyl-CoA levels, effectively enhanced the intracellular methionine biosynthesis. The elevated intracellular acetyl-CoA levels ultimately enhanced SAM accumulation, whereas the introduction of metK1 enhanced the redirection of acetyl-CoA to SAM biosynthesis in Ymls1 △GAPmK strain.

A 1332 bp full length cDNA encoding Teladorsagia circumcincta isocitrate lyase (TciICL) and a 1575 bp full length cDNA encoding T. circumcincta malate synthase (TciMS) were cloned, expressed in Escherichia coli and the recombinant proteins purified. The predicted TciICL protein of 444 amino acids was present as a single band of about 52 kDa on SDS-PAGE and the recombinant TciMS of 525 amino acids formed a single band about 62 kDa. Multiple alignments of the combined bifunctional TciICL MS protein sequence with homologues from other nematodes showed that the greatest similarity (89-92%) to the homologues of Ancylostoma ceylanicum, Haemonchus contortus and Haemonchus placei and 71-87% similarity to the other nematode sequences. The 3-dimensional structures, binding and catalytic sites were determined for TciICL and TciMS and shown to be highly conserved. Substrate and metal ion binding sites were identified and were completely conserved in other homologues. TciICL was confirmed as a functional enzyme. At 30 °C, the optimum pH was pH 7.5, the Vmax was 275 ± 23 nmoles.min-1.mg-1 protein and the apparent Km for the substrate isocitrate was 0.7 ± 0.01μM (mean ± SEM, n = 3). Addition of 10 mM metal ions (except Mg2+) or 1 mM inhibitors reduced the recombinant TciICL activity by 60-90%. Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciICL in ELISA, supporting similar antigenicity to that of the native enzyme.

Ricinus communis L. seeds can germinate at high temperatures, but further development of the seedlings is negatively affected. This mainly caused by impairment of energy-generating pathways when seeds are germinated at 35 °C. Ricinus communis malate synthase (RcMLS) is a key responsive gene in lipid mobilization and gluconeogenesis and as such might have a role in sustaining successful seed germination and seedling growth. Herein, we raised the question whether RcMLS might be involved in the biochemical and molecular mechanisms required for R. communis seed germination under unfavourable environmental conditions. For that, we used a robust approach that encompassed bioinformatics analysis, transgenic Arabidopsis thaliana (L.) Heynh seeds overexpressing RcMLS, along with phenotypical characterization of seed germination under abiotic stress. The phylogenetic tree revealed important evolutionary relationship amongst MLS sequences from R. communis and from other crop species/model plants. Overexpression of RcMLS enhanced A. thaliana seed germination under high temperature and salt stress. For example, wild-type A. thaliana Columbia seeds (Col-0) showed 37 % of maximum germination at 35 °C, whereas A. thaliana seeds overexpressing RcMLS showed up to 71%. When salt stress was applied (75 mM NaCl), maximum germination of Col-0 seeds reached 37%, whereas for A. thaliana seeds overexpressing RcMLS it reached up to 93%. Nuclear Magnetic Resonance (NMR) and Gas Chromatography coupled to Time-Of-Flight Mass Spectrometry (GC-TOF-MS) metabolomics analysis showed a robust metabolic signature of A. thaliana seeds overexpressing RcMLS in response to abiotic stress. They accumulated high levels of Met, Ile, fructose, glucose, and sucrose. Therefore, we suggested that overexpression of RcMLS has modulated the glyoxylate cycle and gluconeogenesis pathway in order to maintain cellular homeostasis under unfavorable environmental conditions. Our results provide important leads into the contribution of RcMLS to the underlying mechanism of R. communis seed germination under adverse environmental conditions. This might be helpful for breeding programs to develop more resistant R. communis cultivars which are more likely to sustain growth and high yield under the severe conditions found in arid and semi-arid areas worldwide.

Background The structure-function relationships for large protein complexes at the atomic level would be comprehensively understood, if hitherto unexplored aromatic ring NMR signals became accessible in addition to the currently used backbone amide and side-chain methyl signals. Methods The 82 kDa malate synthase G (MSG) proteins, selectively labeled with Trp and Phe bearing relaxation optimized isotope-labeled rings, were prepared to investigate the optimal conditions for obtaining the aromatic TROSY spectra. Results The MSG proteins, selectively labeled with either [δ1,e1,e3,η2]-SAIL Trp or ζ-SAIL Phe, provided well-separated, narrow TROSY signals for the 12 Trp and 19 Phe residues in MSG. The signals were assigned sequence-specifically, using the set of single amino acid substitution mutants. The site-specific substitution of each Phe with Tyr or Leu induced substantial chemical shifts for the other aromatic ring signals, allowing us to identify the aromatic clusters in MSG, which were comparable to the structural domains proposed previously. Conclusions We demonstrated that the aromatic ring 13CH pairs without directly bonded 13C and adjacent 1H spins provide surprisingly narrow TROSY signals, if the rings are surrounded by fully deuterated amino acids. The observed signals can be readily assigned by either the single amino acid substitution or the NOEs between the aromatic and methyl protons, if the methyl assignments are available. General significance The method described here should be generally applicable for difficult targets, such as proteins in lipid bilayers or possibly in living cells, thus providing unprecedented opportunities to use these new probes in structural biology.

Glycolic acid is the smallest member of the α-hydroxy acid family. In order to produce glycolate from glucose via the glyoxylate shunt stably, one malate synthase gene aceB in Escherichia coli BW25113 was deleted by homologous recombination; another malate synthase gene glcB was then replaced by a DNA cassette WAK harboring isocitrate lyase gene (aceA), glyoxylate reductase gene (ycdW) and isocitrate dehydrogenase kinase/phosphatase gene (aceK). The above three genes were over-expressed in the chromosome of E. coli EYX-1WAK. This strain was then transferred 20 times on M9 medium to have a mutant strain: EYX-2 with a significantly improved growth rate. The glycolate yields of EYX-2 in the shaken flasks and the 5-L bioreactor using batch fermentation strategy under 2 vvm aeration and 800 rpm stirring speed were 0.33 g/g-glucose and 0.48 g/g-glucose, respectively. The fed-batch fermentation of EYX-2 on 120 g/L glucose had the highest titer of 56.44 g/L with 0.52 g/g-glucose yield in 120 h, and this is the highest reported glycolate yield ever.

Ricinus communis seeds germinate to a high percentage and faster at 35 °C than at lower temperatures, but with compromised seedling establishment. However, seedlings are able to cope with high temperatures at later stages of seedling establishment if germination occurred at lower temperatures. Our objective was to assess the biochemical and molecular requirements of R. communis germination for successful seedling establishment at varying temperatures. For that, we performed metabolite profiling (GC-TOF-MS) and measured transcript levels of key genes involved in several energy-generating pathways, such as storage oil mobilization, β-oxidation and gluconeogenesis of seeds germinated at three different temperatures. We identified a thermo-sensitive window during seed germination in which high temperatures compromise seedling development, most likely by down-regulating some energy-generating pathways. Overexpression of malate synthase (MLS) and glycerol kinase (GK) genes resulted in higher starch levels in Nicotiana benthamiana leaves, which highlights the importance of these genes in energy-generating pathways for seedling establishment. Additionally, we showed that GABA, which is a stress-responsive metabolite, accumulated in response to the water content of the seeds during the initial phase of imbibition. Herewith, we provide new insights into the molecular requirements for vigorous seedling growth of R. communis under different environmental conditions.

In Aspergillus nidulans, the utilization of acetate as sole carbon source requires several genes (acu). Most of them are also required for the utilization of fatty acids. This is the case for acuD and acuE, which encode the two glyoxylate cycle-specific enzymes, isocitrate lyase and malate synthase, respectively, but also for acuL that we have identified as AN7287, and characterized in this study. Deletion of acuL resulted in the same phenotype as the original acuL217 mutant. acuL encodes a 322-amino acid protein which displays all the structural features of a mitochondrial membrane carrier, and shares 60% identity with the Saccharomyces cerevisiae succinate/fumarate mitochondrial antiporter Sfc1p (also named Acr1p). Consistently, the AcuL protein was shown to localize in mitochondria, and partial cross-complementation was observed between the S. cerevisiae and A. nidulans homologues. Extensive phenotypic characterization suggested that the acuL gene is involved in the utilization of carbon sources that are catabolized via the TCA cycle, and therefore require gluconeogenesis. In addition, acuL proves to be co-regulated with acuD and acuE. Overall, our data suggest that AcuL could link the glyoxylate cycle to gluconeogenesis by exchanging cytoplasmic succinate for mitochondrial fumarate.

Despite a vast amount information on the interplay of GroEL, GroES, and ATP in chaperone-assisted folding, the molecular details on the conformational dynamics of folding polypeptide during its GroEL/GroES-assisted folding cycle is quite limited. Practically no such studies have been reported to date on large proteins, which often have difficulty folding in vitro. The effect of the GroEL/GroES chaperonin system on the folding pathway of an 82-kDa slow folding protein, malate synthase G (MSG), was investigated. GroEL bound to the burst phase intermediate of MSG and accelerated the slowest kinetic phase associated with the formation of native topology in the spontaneous folding pathway. GroEL slowly induced conformational changes on the bound burst phase intermediate, which was then transformed into a more folding-compatible form. Subsequent addition of ATP or GroES/ATP to the GroEL-MSG complex led to the formation of the native state via a compact intermediate with the rate several times faster than that of spontaneous refolding. The presence of GroES doubled the ATP-dependent reactivation rate of bound MSG by preventing multiple cycles of its GroEL binding and release. Because GroES bound to the trans side of GroEL-MSG complex, it may be anticipated that confinement of the substrate underneath the co-chaperone is not required for accelerating the rate in the assisted folding pathway. The potential role of GroEL/GroES in assisted folding is most likely to modulate the conformation of MSG intermediates that can fold faster and thereby eliminate the possibility of partial aggregation caused by the slow folding intermediates during its spontaneous refolding pathway.

SCB related virtual screening and docking studies can provide mechanistic insight on inhibition.Unlike glyoxlate response, inhibitor impact on malate seems microenvironment dependent.For similar microenvironments, ICL versus MS inhibition is metabolically more consequential.Strong ICL inhibition causes metabolite levels in persistent Mtb toward levels in non-viable Mtb.SCB points to a complex interplay between network structure, kinetics, and therapeutic outcome. The ability of Mycobacterium tuberculosis (Mtb) to survive in low oxygen environments enables the bacterium to persist in a latent state within host tissues. In vitro studies of Mtb growth have identified changes in isocitrate lyase (ICL) and malate synthase (MS) that enable bacterial persistence under low oxygen and other environmentally limiting conditions. Systems chemical biology (SCB) enables us to evaluate the effects of small molecule inhibitors not only on the reaction catalyzed by malate synthase and isocitrate lyase, but the effect on the complete tricarboxylic acid cycle (TCA) by taking into account complex network relationships within that system.To study the kinetic consequences of inhibition on persistent bacilli, we implement a systems-chemical biology (SCB) platform and perform a chemistry-centric analysis of key metabolic pathways believed to impact Mtb latency. We explore consequences of disrupting the function of malate synthase (MS) and isocitrate lyase (ICL) during aerobic and hypoxic non-replicating persistence (NRP) growth by using the SCB method to identify small molecules that inhibit the function of MS and ICL, and simulating the metabolic consequence of the disruption.Results indicate variations in target and non-target reaction steps, clear differences in the normal and low oxygen models, as well as dosage dependent response. Simulation results from singular and combined enzyme inhibition strategies suggest ICL may be the more effective target for chemotherapeutic treatment against Mtb growing in a microenvironment where oxygen is slowly depleted, which may favor persistence.

The dinoflagellates are an important group of eukaryotic, single celled algae. They are the sister group of the Apicomplexa, a group of intracellular parasites and photosynthetic algae including the malaria parasite Plasmodium. Many apicomplexan mitochondria have a number of unusual features, including the lack of a pyruvate dehydrogenase and the existence of a branched TCA cycle. Here, we analyse dinoflagellate EST (expressed sequence tag) data to determine whether these features are apicomplexan-specific, or if they are more widespread. We show that dinoflagellates have replaced a key subunit (E1) of pyruvate dehydrogenase with a subunit of bacterial origin and that transcripts encoding many of the proteins that are essential in a conventional ATP synthase/Complex V are absent, as is the case in Apicomplexa. There is a pathway for synthesis of starch or glycogen as a storage carbohydrate. Transcripts encoding isocitrate lyase and malate synthase are present, consistent with ultrastructural reports of a glyoxysome. Finally, evidence for a conventional haem biosynthesis pathway is found, in contrast to the Apicomplexa, Chromera and early branching dinoflagellates (Perkinsus, Oxyrrhis). Refereed/Peer-reviewed

Wild-type Acetobacter aceti NBRC 14818 possesses genes encoding isocitrate lyase (aceA) and malate synthase (glcB), which constitute the glyoxylate pathway. In contrast, several acetic acid bacteria that are utilized for vinegar production lack these genes. Here, an aceA-glcB knockout mutant of NBRC 14818 was constructed and used for investigating the role of the glyoxylate pathway in acetate productivity. In medium containing ethanol as a carbon source, the mutant grew normally during ethanol oxidation to acetate, but exhibited slower growth than that of the wild-type strain as the accumulated acetate was oxidized. The mutant grew similarly to that of the wild-type strain in medium containing glucose as a carbon source, indicating that the glyoxylate pathway was not necessary for glucose utilization. However, in medium containing both ethanol and glucose, the mutant exhibited significantly poorer growth and lower glucose consumption compared to the wild-type strain. Notably, the mutant oxidized ethanol nearly stoichiometrically to acetate, which was retained in the medium for a longer period of time than the acetate produced by wild-type strain. The features of the aceA-glcB knockout mutant revealed here indicate that the lack of the glyoxylate pathway is advantageous for industrial vinegar production by A. aceti.

Peroxisomes are functionally diverse organelles that are wholly dependent on import of nuclear-encoded proteins. The signals that direct proteins into these organelles are either found at the C-terminus (type 1 peroxisomal targeting signal; PTS1) or N-terminus (type 2 peroxisomal targeting signal; PTS2) of the protein. Based on a limited number of tests in heterologous systems, PTS1 signals appear to be conserved across species. To further test the generality of this conclusion and to establish the extent to which the PTS1 signals can be relied on for biotechnological purposes across species, we tested two PTS1 signals for their ability to target fluorescent proteins in diverse plant species. Transient assays following microprojectile bombardment showed that the six amino acid PTS1 sequence (RAVARL) from spinach glycolate oxidase effectively targets green fluorescent fusion protein to the leaf peroxisomes in all 20 crops tested, including four monocots (sugarcane, wheat, corn and onion) and 16 dicots (carrot, cucumber, broccoli, tomato, lettuce, turnip, radish, cauliflower, cabbage, capsicum, celery, tobacco, petunia, beetroot, eggplant and coriander). Similarly, results indicated that the 10 amino acid PTS1 sequence (IHHPRELSRL) from pumpkin malate synthase effectively targets red fluorescent fusion protein to the leaf peroxisomes in all four crops tested including monocot (sugarcane) and dicot (cabbage, celery and pumpkin) species. These signal sequences should be useful metabolic engineering tools to direct recombinant proteins to the leaf peroxisomes in diverse plant species of biotechnological interest.

For the 12 named taxa in the Gagea reticulata species complex, 609 cloned sequences of the low-copy nuclear gene malate synthase (MS) were used to investigate species relationships, using standard phylogenetic tools and network analyses. Three (homologous) copies of MS locus were present in each individual analyzed, and multiple alleles were present at most of these loci. Duplication of MS occurred after divergence of the G. reticulata complex. After comparisons, 591 sequence types (i.e. haplotypes) were identified, requiring implementation of novel statistical analyses to group haplotypes in a smaller number of groups/lineages to enable further study. Haplotype groups/lineages are not fully congruent with species limits with some widely present among species. MS genotypes at the root of the network are those of G. setifolia from central Iran, with more derived sequences in this species found in the west and northwest. Presence of ancestral genotypes in several other taxa may indicate either the retention of "ancestral" polymorphisms, more recent introgressive hybridization, or both. The relative DNA content of specimens was estimated with flow cytometry (FCM). The FCM analyses revealed two levels of DNA content (putatively "diploid" and "tetraploid"), but no correlation between number of MS gene copies and ploidy was found.

A forward cDNA-SSH library was constructed from a drought-tolerant cotton (Gossypium hirsutum L.) inbred line, Handan 177, to understand the expressions of drought-induced genes. Using suppression subtractive hybridization method, a total of 2300 positive clones were obtained, of which 300 clones were selected for PCR validation and sequencing. Among these clones, 284 were available sequences. According to clustering analysis of the expressed sequence tag (EST), 202 uniESTs were found, including 28 contigs and 174 singlets. The result of BlastN showed that 156 uniESTs had homologous sequences in GenBank database. The result of BlastX indicated that 116 uniESTs had high homology with proteins with known functions, and 40 uniESTs showed high similarities with unknown proteins or putative proteins. Thirty-three uniESTs were located into 55 KEGG pathways using KOBAS software, including 23 pathways at a significant level (P < 0.05). These significant pathways were mainly related to pyruvate metabolism (15%) and glyoxylate and dicarboxylate metabolism (12%). A large group of drought-induced genes were detected in the cDNA library, which were involved in signal transduction, energy metabolism, protein metabolism, nucleic acid metabolism, photosynthesis, and transmembrane transport. Some of them were associated with drought tolerance, such as malate synthase genes (MS1, 0001_C12 and MS2, 0002_F01), malate dehydrogenase genes (Md1, 001_C12 and Md2, 002_F01), NAC type transcription factor (001_C08), BZR1/BES1 (003_G04), zinc finger protein gene (zfp, 003_C06), and translationally controlled tumor protein gene (tctp, 002_C04).

SummaryGiven the success in engineering synthetic phenotypes in microbes and mammalian cells, constructing non-native pathways in mammals has become increasingly attractive for understanding and identifying potential targets for treating metabolic disorders. Here, we introduced the glyoxylate shunt into mouse liver to investigate mammalian fatty acid metabolism. Mice expressing the shunt showed resistance to diet-induced obesity on a high-fat diet despite similar food consumption. This was accompanied by a decrease in total fat mass, circulating leptin levels, plasma triglyceride concentration, and a signaling metabolite in liver, malonyl-CoA, that inhibits fatty acid degradation. Contrary to plants and bacteria, in which the glyoxylate shunt prevents the complete oxidation of fatty acids, this pathway when introduced in mice increases fatty acid oxidation such that resistance to diet-induced obesity develops. This work suggests that using non-native pathways in higher organisms to explore and modulate metabolism may be a useful approach.

Incidence and mortality rates of invasive aspergillosis clearly indicate the need of novel antifungals to treat patients suffering from this disease. Fungal proteins playing a crucial role in pathogenesis and with no orthologue in human cells are considered as primary therapeutic targets for the development of new antifungals with a high therapeutic index, one of the major drawbacks of the standard antifungal therapy, so far. In this work, we have analyzed the role in pathogenesis of the key enzymes of the Aspergillus fumigatus glyxoxylate cycle, isocitrate lyase and malate synthase, two possible candidates to primary therapeutic targets in this fungus. Deletion strains lacking isocitrate lyase (DeltaacuD strains) or malate synthase (DeltaacuE mutants) were constructed in this work. The Neurospora crassa pyr-4 gene was used as the replacing marker in gene deletion experiments. The pathogenicities of DeltaacuD and DeltaacuE mutants were tested in neutropenic mice and compared with those of two reference wild-type isolates A. fumigatus 237 and A. fumigatus 293. Interestingly, virulence and cytological studies clearly indicated the dispensability of the A. fumigatus glyoxylate cycle for pathogenicity. In addition, these results suggested the suitability of the pyr-4 gene as a valuable replacing marker for virulence studies in this fungus, a fact that was further confirmed by gene expression analyses. Finally, growth tests were performed to investigate the germination and growth of the DeltaacuD and DeltaacuE strains in nutrient deprivation environments, resembling the conditions that A. fumigatus conidia face after phagocytosis. Results obtained in this work strongly suggest that the ability to grow on lipids (triglycerides) of A. fumigatus isocitrate lyase and malate synthase deletion strains accounts for their fully virulent phenotype.

The X-ray damage of the sulfhydryl enzyme malate synthase from yeast has been investigated by physicochemical studies including time-resolved small-angle X-ray scattering. Our previous studies already suggested the role of cysteine-sulfenic acid and disulfides in the damage process and established a simple model for the occurring aggregation. The preceding model is now confirmed by ab initio modeling approaches, illustrating the evolution to a 2D aggregation process. Information on the involvement of the sulfur-containing groups was obtained from recourse to crystallographic and sequence data of the E. coli enzyme, using surface and hydration algorithms; the applied approaches prove the existence and localization of sulfhydryl and sulfenic acid sites in the enzyme.

This study evaluated the effects of priming on emergence responses of purple coneflower ( Echinacea purpurea L. Moench) seeds. The seeds that differ in seed size were either primed with moistened vermiculite (solid matrix priming) or primed in non-aerated −0.5 MPa polyethylene glycol 6000 solution at 25 °C for 6 days (osmopriming), followed by air-drying to their initial moisture level. The tetrazolium staining tests indicated that both large and small seeds were biochemically viable. No notable difference in germination percentage was found between large and small seeds. However, extensive cavity was visible in portions of small seeds in comparison with large seeds. Large seeds accumulated more antioxidants and had greater activities of anti-oxidative enzymes than small seeds. They also had greater isocitrate lyase and malate synthase activities than small seeds. As a result, large seeds had higher emergence percentage and faster emergence speed as compared to that of small seeds. Both solid matrix priming and osmopriming increased emergence percentage and shortened mean emergence time of purple coneflower seeds by increasing the activities of anti-oxidative enzymes and decreasing the levels of malondialdehyde and total peroxide accumulation. Moreover, priming also enhanced the anti-oxidative activities of treated seeds. The activities of isocitrate lyase and malate synthase were also increased in primed seeds. The enhanced anti-oxidation and lipid-carbohydrate conversion activities might explain in part why primed purple coneflower seeds emerged better than non-primed seeds.

The metabolic effect of fadR gene knockout on Escherichia coli was investigated based on comparative analyses of protein expressions, enzyme activities and intracellular metabolite concentrations between the parent strain Escherichia coli BW25113 and its fadR knockout mutant JWK1176. The E. coli strains were grown in a minimal medium using glucose as the carbon source. Two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry were used to detect and identify the proteins expressed. Enzyme activities and intracellular metabolite concentrations were also measured. Results showed that the fadR knockout E. coli reduced acetate excretion by 27.3%, accompanied by 8.5% enhancement in biomass yield. Expressions of proteins involved in glucose transport, energy metabolism and some of amino acid biosynthesis pathways were upregulated, whereas those for fatty acids biosynthesis, such as AccB and FabD were downregulated. Besides, UspA and DnaK, the universal stress and heat shock proteins, were induced in response to fadR knockout. There was 3.7- and 1.9-fold increase in the enzyme activities of isocitrate lyase and malate synthase, respectively, in the fadR mutant compared to the parent. Intracellular concentrations of acetyl coenzyme A, pyruvate and phosphoenolpyruvate deceased, whereas the concentrations of isocitrate, α-ketoglutarate, malate, oxaloacetate and aspartate increased. These results suggest that the fadR knockout E. coli activates the glyoxylate shunt and enhances the capacity of energy metabolism and biosynthesis, which leads to the reduction of acetate excretion and improvement of the biomass yield.

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 (1.3 kb) and aceB (1.6 kb) were cloned and expressed as tagged fusion proteins in Escherichia coli BL21 (DE3). The molecular weights of the predicted enzymes (ICL, 47.7 kDa and MS, 59.1 kDa) 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 (589.27 nmol min −1 mg −1 ) and MS (485.54 nmol min −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.

Escherichia coli changes its metabolism in response to environmental circumstances, and metabolic adaptations are evident in hungry bacteria growing slowly in glucose-limited chemostats. The role of isocitrate lyase (AceA) was examined in E. coli growing under glucose limitation. AceA activity was elevated in a strain-dependent manner in the commonly used E. coli K-12 laboratory strains MG1655 and MC4100, but an aceA disruption surprisingly increased fitness under glucose limitation in both strains. However, in bacteria adapted to limiting glucose in long-term chemostats, mutations outside aceA changed its role from a negative to a positive influence. These results suggest that a recently proposed pathway of central metabolism involving the glyoxylate cycle enzymes is redundant in wild-type bacteria, but may take on a beneficial role after context adaptation. Interestingly, the aceA gene sequence did not alter during prolonged selection, so mutations in unidentified genes changed the metabolic context of unaltered AceA from a negative to a positive influence in bacteria highly adapted to limiting glucose.

Glucosinolates are a group of sulfur-rich thioglucoside natural products common in the Brassicaceae and related plant families. The first phase in the formation of many glucosinolates involves the chain extension of the amino acid methionine. Additional methylene groups are inserted into the side chain of methionine by a three-step elongation cycle involving 2-oxo acid intermediates. This investigation demonstrated the first step of this chain elongation cycle in a partially-purified preparation from arugula (Eruca sativa). The 2-oxo acid derived from methionine, 4-methylthio-2-oxobutanoic acid, was shown to condense with acetyl-CoA to form 2-(2'-methylthioethyl)malate. The catalyst, designated as a 2-(omega-methylthioalkyl)malate synthase, belongs to a family of enzymes that mediate the condensation of acyl-CoAs with 2-oxo acids, including citrate synthase of the citric acid cycle, and 2-isopropylmalate synthase of leucine biosynthesis. The 2-(omega-methylthioalkyl)malate synthase studied here shares properties with other enzymes of this class, but appears chromatographically distinct and is found only in extracts of plant species producing glucosinolates from chain-elongated methionine derivatives. Although the principal glucosinolates of arugula are formed from methionine that has undergone two rounds of chain elongation to form dihomomethionine, studies with substrates and substrate analogs of different chain lengths showed that the isolated enzyme is responsible only for the condensation step of the first round of elongation.

Acetate accumulation is a common problem observed in aerobic high cell density cultures of Escherichia coli. It has been hypothesized in previous reports that the glyoxylate shunt is active in E. coli BL21, the low acetate producer, and inactive in E. coli JM109, the high acetate producer. This hypothesis was further strengthened by incorporating 13C from uniformly labeled glucose into TCA cycle intermediates. Using northern blot analyses, the current report demonstrates that the reason for the inactivity of the glyoxylate pathway in E. coli JM109 is the no apparent transcription of isocitrate lyase (aceA) and malate synthase (aceB), and transcription of the isocitrate lyase repressor (iclR). The reverse is seen in E. coli BL21 where the glyoxylate pathway is active due to constitutive transcription of aceA and aceB and no transcription of the iclR. In addition, there is a difference between the two strains in the transcription of the acetyl-CoA synthetase (acs), phosphotransacetylase-acetate kinase (pta-ackA) pathway, and pyruvate oxidase (poxB), pathway. The transcript of acs is higher in E. coli BL21 and lower in the E. coli JM109, while the reverse is true for poxB transcription.

The genome size, complexity, and ploidy of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was determined using flow cytometry, reassociation kinetics, and genomic reconstruction. Nuclei of G. intraradices from in vitro culture, were analyzed by flow cytometry. The estimated average length of DNA per nucleus was 14.07+/-3.52 Mb. Reassociation kinetics on G. intraradices DNA indicated a haploid genome size of approximately 16.54 Mb, comprising 88.36% single copy DNA, 1.59% repetitive DNA, and 10.05% fold-back DNA. To determine ploidy, the DNA content per nucleus measured by flow cytometry was compared with the genome estimate of reassociation kinetics. G. intraradices was found to have a DNA index (DNA per nucleus per haploid genome size) of approximately 0.9, indicating that it is haploid. Genomic DNA of G. intraradices was also analyzed by genomic reconstruction using four genes (Malate synthase, RecA, Rad32, and Hsp88). Because we used flow cytometry and reassociation kinetics to reveal the genome size of G. intraradices and show that it is haploid, then a similar value for genome size should be found when using genomic reconstruction as long as the genes studied are single copy. The average genome size estimate was 15.74+/-1.69 Mb indicating that these four genes are single copy per haploid genome and per nucleus of G. intraradices. Our results show that the genome size of G. intraradices is much smaller than estimates of other AMF and that the unusually high within-spore genetic variation that is seen in this fungus cannot be due to high ploidy.

Tuberculosis remains a leading cause of mortality worldwide into the 21st century. Among the main obstacles to the global control of the disease are emerging multi-drug resistant strains and the recalcitrance of persistent infections to treatment with conventional anti-TB drugs. Here we review recent developments in our understanding of some of the pathways involved in a persistent infection and pathogenesis of Mycobacterium tuberculosis, which reveal new targets for drug development. We describe the high-resolution crystal structures of enzymes of the glyoxylate shunt, isocitrate lyase and malate synthase, and of the cyclopropane synthases of mycolic acid biosynthesis. Structure-based drug design is now underway with the potential to lead to the development of new anti-tuberculars effective against persistent and resistant Mycobacterium tuberculosis infections.

We investigated oxidative processes in mitochondria of Saccharomyces cerevisiae grown on ethanol in the course of chronological aging. We elaborated a model of chronological aging that avoids the influence of exhaustion of medium, as well as the accumulation of toxic metabolites during aging. A decrease in total respiration of cells and, even more, of the contribution of respiration coupled with ATP-synthesis was observed during aging. Aging is also related with the decrease of the contribution of malonate-insensitive respiration. Activities of citrate-synthase (CS), alpha-ketoglutarate dehydrogenase (KGDH) and malate dehydrogenase (MDH) were threefold decreased. The activity of NADP-dependent isocitrate dehydrogenase (NADP-ICDH) decreased more significantly, while the activity of NAD-dependent isocitrate dehydrogenase (NAD-ICDH) fell even greater, being completely inactivated on the third week of aging. In contrast, succinate dehydrogenase (SDH), enzymes of glyoxylate cycle (GCL) (isocitrate lyase (ICL) and malate synthase (MLS)), and enzymes of ethanol oxidation (alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ACDH)), were activated by 50% or more. The behavior of oxidative enzymes and metabolic pathways are apparently inherent to a more viable, long-lived cells in population, selected in the course of chronological aging. This selection allows cells to reveal the mechanism of their higher viability as caused by shunting of complete Krebs cycle by glyoxylate cycle, with a concomitant increased rate of the most efficient energy source, namely succinate formation and oxidation. Thiobarbituric-reactive species (TAR species) increased during aging. We supposed that to be the immediate cause of damage of a part of yeast population. These data show that a greater succinate contribution to respiration in more active cells is a general property of yeast and animal tissues.

Summary Soybean ( Glycine max . L.) nodular senescence results in the dismantling of the peribacteroid membrane (PBM) and in an increase of soybean isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2) mRNA and protein levels. This suggests that in senescing soybean nodular cells, the specific glyoxylate cycle enzyme activities might be induced to reallocate carbon obtained from the PBM degradation. In order to evaluate as well the carbon metabolism of the nitrogen-fixing Bradyrhizobium japonicum endosymbiotic bacteroids during nodular senescence, their glyoxylate cycle activities were also investigated. To this end, partial DNA sequences were isolated from their icl and ms genes, but the corresponding mRNAs were not detected in the microorganisms. It was also observed that the bacteroid ICL and MS activities were negligible during nodular senescence. This suggests that glyoxylate cycle activities are not reinitiated in the bacteroids under these physiological conditions. In case the microorganisms nevertheless feed on the PBM degradation products, this might occur via the citric acid cycle exclusively.

Herein, we present a new enzyme-linked spectrophotometric assay for glyoxylate that detects glyoxylate via the formation of an intensely colored formazan. This glyoxylate-specific assay is reliant upon the enzymatic conversion of glyoxylate to oxaloacetate coupled to the reduction of oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide (NADH). The NADH-dependent reduction of a tetrazolium to the formazan enables the measurement of nanomole quantities of glyoxylate in an assay that is amenable to high-throughput screening methods. Assay validation was accomplished using two methods for glyoxylate generation, the base-catalyzed N-dealkylation of alpha-hydroxyhippurate to benzamide and glyoxylate and the oxidative cleavage of the glycyl Calpha-N bond in N-benzoylglycine (hippurate) by peptidylglycine alpha-amidating monooxygenase to again yield benzamide and glyoxylate. For both reactions, analysis of benzamide produced by reverse-phase high-performance liquid chromatography compared with glyoxylate measured using our glyoxylate assay showed a 1:1 molar ratio of benzamide to glyoxylate. These results indicate that the enzyme-linked spectrophotometric assay can quantitatively measure submicromole quantities of glyoxylate.

Establishment or maintenance of a persistent infection by Mycobacterium tuberculosis requires the glyoxylate pathway. This is a bypass of the tricarboxylic acid cycle in which isocitrate lyase and malate synthase (GlcB) catalyze the net incorporation of carbon during growth of microorganisms on acetate or fatty acids as the primary carbon source. The glcB gene from M. tuberculosis, which encodes malate synthase, was cloned, and GlcB was expressed inEscherichia coli. The influence of media conditions on expression in M. tuberculosis indicated that this enzyme is regulated differentially to isocitrate lyase. Purified GlcB hadK m values of 57 and 30 μm for its substrates glyoxylate and acetyl coenzyme A, respectively, and was inhibited by bromopyruvate, oxalate, and phosphoenolpyruvate. The GlcB structure was solved to 2.1-A resolution in the presence of glyoxylate and magnesium. We also report the structure of GlcB in complex with the products of the reaction, coenzyme A and malate, solved to 2.7-A resolution. Coenzyme A binds in a bent conformation, and the details of its interactions are described, together with implications on the enzyme mechanism.

The glyoxylate cycle comprising isocitrate lyase (ICL) and malate synthase (MS) is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, which encodes malate synthase has been previously cloned from Streptomyces clavuligerus NRRL 3585 and characterized. In this study, the aceA gene, encoding ICL from S. clavuligerus NRRL 3585, was obtained via genome walking experiments and PCR. The fully sequenced open reading frame encodes 436 amino acids with a deduced M r of 47.5 kDa, consistent with the observed M r (49–67.5 kDa) of most ICL enzymes reported so far. The cloned aceA gene was expressed in Escherichia coli BL21(λDE3) cells, from which ICL was purified as a His-tagged product and its functionality demonstrated. Furthermore, the relationship between the carbon sources, growth and ICL activity in S. clavuligerus were investigated. Rapid growth was observed when the cells were cultured on 0.5% (w/v) glycerol, while delayed growth was observed when cells were grown on 0.5% (w/v) acetate. However, in both cases, high levels of ICL activity coincided with a cessation of growth, suggesting a late physiological role played by ICL in the natural host, S. clavuligerus .

There is currently a shortage of DNA regions known to be useful for phylogenetic research in palms (Arecaceae). We report the development and use of primers for amplifying and sequencing regions of the nuclear gene malate synthase. In palms the gene appears to be single-copy, with exon regions that are phylogenetically informative within the family. We constructed a phylogeny of 45 palms and five outgroup taxa using 428 bp of malate synthase exon regions. We found that some major clades within the family were recovered, but there was a lack of resolution among the genera in subfamilies Arecoideae, Ceroxyloideae, Coryphoideae, and Phytelephantoideae. In a second analysis, malate synthase exon regions totaling 1002 bp were sequenced for 16 palms and two outgroup taxa. There was increased bootstrap support for some groups and for the placement of the monotypic genus Nypa as sister to the rest of the family. A comparison with data sets from noncoding regions of the chloroplast genome indicates that malate synthase sequences are more variable and potentially contain more phylogenetic information. We found no evidence of multiple copies of the malate synthase gene in palm genomes.

Summary Isocitrate lyase and malate synthase are two key enzymes of the glyoxylate cycle, and are associated with lipid degradation and gluconeogenesis in microorganisms, plants, nematodes, and animals. Although genes of isocitrate lyase have been cloned from seedling cotyledons of several plants, none were isolated from the senescent leaves. In this study, sweet potato yellow leaves with disassembled chloroplast thylakoids and remarkable osmiophilic globule accumulation in stroma were used as material. Using both PCR-based subtractive hybridization and RACE PCR, a 2.1 kb full-length sp/CL cDNA was cloned. The open reading frame contained 1728 nucleotides (576 amino acids) and exhibited more than 75% sequence identity with plant cotyledon isocitrate lyases of tomato, upland cotton, cucumber, castor bean, rape, soybean, and loblolly pine. The sp/CL-encoded protein contained the Leu-169, Lys-170, Pro-171 (LKP) and Thr-210, Lys-211, Lys-212 (TKK) motifs, with a reported substrate binding domain function, as well as a putative peroxisomal targeting signal (PTS) Ala-Arg-Met (ARM) tripeptide at the C-terminus. Its mANA accumulated exclusively in the senescing and completely yellow leaves, but not in the green leaves, roots, or stems. Hence, the sweet potato sp/CL is the first isocitrate lyase isolated from senescent leaves. The data may provide molecular evidence to support the notion that glyoxylate cycle, a metabolic pathway utilized in cotyledons for postgerminative growth of oilseeds, is also involved in lipid degradation and gluconeogenesis of senescent leaves.

During our study of the glyoxylate cycle in soybean (Glycine max. L. var. Maple arrow), two mitochondrial and three cytosolic aconitase molecular species (EC 4.2.1.3) were detected, designated as M1, M2, C1, C2 and C3 isoforms, respectively, according to their intracellular locations and electrophoretic mobilities. Using the glyoxylate cycle marker enzymes isocitrate lyase (ICL, EC 4.1.3.1) and malate synthase (MS, EC 4.1.3.2), the activity of this pathway providing the essential link between β-oxidation and gluconeogenesis was confirmed during germination (cotyledons) and senescence (leaves). It was then established that, in both cases, the activity of the C1 aconitase isoform developed concomitantly with the transcription and translation levels of the icl and ms genes. This strongly suggests that C1 aconitase is constitutive of the glyoxylate cycle. In addition, the same isoform was found to be active during pathogenic attack as well (hypocotyls). It might be assumed that in such a case the glyoxylate cycle is reinitiated as a part of a carbon reallocation system feeding on the diseased tissue cellular components.

The metabolic pathways specified by the glc and ace operons in Escherichia coli yield glyoxylate as a common intermediate, which is acted on by two malate synthase isoenzymes: one encoded by glcB and the other by aceB. Null mutations in either gene exhibit no phenotype, because of cross-induction of the ace operon by glycolate and the glc operon by acetate. In this study, the regulation of the glc operon, comprising the structural genes glcDEFGB, was analyzed at the molecular level. This operon, activated by growth on glycolate, is transcribed as a single message and is under the positive control of GlcC encoded by a divergent gene. Expression of the glc operon is strongly dependent on the integration host factor (IHF) and is repressed by the global respiratory regulator ArcA-P. In vitro gel-shift experiments demonstrated direct binding of the promoter DNA to IHF and ArcA-P. Mutant analysis indicated that cross-induction of the glc operon by acetate is mediated by the GlcC protein that recognizes the compound as an alternative effector. The similar pattern of regulation of the Glc and Ace systems by IHF and ArcA-P ensures their effective cross-induction.

The presence of the two glyoxylate cycle marker enzymes, isocitrate lyase (ICL, EC 4.1.3.1) and malate synthase (MS, EC 4.1.3.2), was investigated in cotyledons during the post-germinative growth of tomato (Lycopersicon esculentum Mill., cv Chico III) seedlings. Both ICL and MS increased in the dark and declined after illumination. Cotyledons from seedlings grown in the dark for 7 days were employed for sucrose density gradient fractionation of cell organelles. It was shown that ICL and MS are localized in glyoxysomal fractions. The presence of the two key enzymes of the glyoxylate cycle in tomato cotyledons was confirmed by immunoblotting both in crude extracts and in glyoxysomal fractions. The occurrence of such enzymes was also investigated in different tissues of tomato plants (leaves, fruits, roots and flowers). Although it was difficult to detect ICL and MS activities by spectrophotometric assays, immunoblot analysis showed the presence of both enzymes in senescent leaves and, surprisingly, in young expanding leaves, but not in mature leaves. The role of glyoxylate cycle enzymes in leaves and the value of further studies with tomato are discussed.

Summary Glyoxylate cycle enzymes isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2) are synthesised during the terminal irreversible phase of cucumber cotyledon senescence when the endomembrane system (endoplasmic reticulum, Golgi and tonoplast) is disassembled. This follows the period of reversible senescence when most chloroplast thylakoid membrane disassembly occurs. During the terminal stage of senescence, nuclear integrity is maintained for gene transcription, and mitochondria and peroxisomes are thought to participate in reactions of the glyoxylate cycle and gluconeogenesis.

Lipid levels, glyoxylate-cycle enzymes and respiratory quotients were measured in 3 cm tips of asparagus spears (Asparagus officinalis L.) during 5 days of storage at 20°C, to determine whether lipids are mobilised as in some other senescing plant tissues. Within 12 h, the respiratory quotient dropped from near unity to 0.66 before stabilising around 0.75 after 2 days. Over 5 days of storage, almost half of the total lipid in spear tips was lost. Malate synthase (EC 4.1.3.2) activity, which was not detectable at harvest, appeared after 1–2 days and continued to increase throughout the remaining 3 days. Malate concentration increased 3- to 4-fold in 4–5 days. Isocitrate lyase (EC 4.1.3.1) activity was not detected at any time during storage, and it is unlikely that the glyoxylate cycle functions in tips of harvested asparagus spears. These results are discussed in relation to other postharvest metabolic events in asparagus.