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Trichophyton mentagrophytes secretes Metallocarboxypeptidase A and B of the M14 family as endoproteases and exoprotease. T. mentagrophytes produce Metalloprotease 3 and 4 which degrades the protein into the short peptides and amino acids. To understand the host fungal relationship and identification of such genes expressed during infection is utmost important. T. mentagrophytes encodes some proteins which are associated with the glyoxylate cycle. The glyoxylate cycle enzymes have been involving in virulence of dermatophytes and their up-regulation during dermatophytes growth on keratin. On comparing the expression level of virulence protease and non-protease genes, we observed, among exoprotease protease genes, Metallocarboxypeptidase B was strongly up regulated (134.6 fold high) followed by Metallocarboxypeptidase A (115.6 fold high) and Di-peptidyl-peptidases V (10.1 fold high), in dermatophytic patients as compared to ATCC strain. Furthermore, among endoprotease, Metalloprotease 4 was strongly up regulated (131.6 fold high) followed by Metalloprotease 3 (16.7 fold high), in clinical strains as compared to T. mentagrophytes ATCC strain. While among non-protease genes, Citrate Synthase was highly expressed (118 fold high), followed by Isocitrate Lyase (101.6 fold high) and Malate Synthase (52.9 fold high). All the studied virulence genes were considered the best suitable ones by geNorm, Best keeper, Norm Finder and Ref finder.

Background Methylocella silvestris is a facultative aerobic methanotrophic bacterium which uses not only methane, but also other alkanes such as ethane and propane, as carbon and energy sources. Its high metabolic versatility, together with the availability of tools for its genetic engineering, make it a very promising platform for metabolic engineering and industrial biotechnology using natural gas as substrate. Results The first Genome Scale Metabolic Model for M. silvestris is presented. The model has been used to predict the ability of M. silvestris to grow on 12 different substrates, the growth phenotype of two deletion mutants (ΔICL and ΔMS), and biomass yield on methane and ethanol. The model, together with phenotypic characterization of the deletion mutants, revealed that M. silvestris uses the glyoxylate shuttle for the assimilation of C1 and C2 substrates, which is unique in contrast to published reports of other methanotrophs. Two alternative pathways for propane metabolism have been identified and validated experimentally using enzyme activity tests and constructing a deletion mutant (Δ1641), which enabled the identification of acetol as one of the intermediates of propane assimilation via 2-propanol. The model was also used to integrate proteomic data and to identify key enzymes responsible for the adaptation of M. silvestris to different substrates. Conclusions The model has been used to elucidate key metabolic features of M. silvestris, such as its use of the glyoxylate shuttle for the assimilation of one and two carbon compounds and the existence of two parallel metabolic pathways for propane assimilation. This model, together with the fact that tools for its genetic engineering already exist, paves the way for the use of M. silvestris as a platform for metabolic engineering and industrial exploitation of methanotrophs.

Simple Summary In this study, three longan cultivars, including non-aroma types ‘Shixia’ (SX), ‘Lidongben’ (LDB), and strong aroma type ‘Xiangcui’ (XC), were selected to analyze free amino acids (FAAs) variations at six distinct growth stages. The genome-wide identification and expression analysis of genes related to the branched-chain amino acids (BCAA) synthesis pathway were carried out. Results showed that thirty-six FAAs were identified, which increased drastically with fruit development until ripening. During the period of rapid fruit expansion, the aroma of XC changed from light to strong, and the contents of L-alanine and L-leucine were significantly higher than those of SX and LDB. The content of Leu was negatively correlated with the expression of DilBCAT1, -6, and -9 in three varieties, but positively correlated with DilBCAT16, indicating that these four genes may be responsible for the different synthesis and degradation of Leu among cultivars. Abstract Amino acids are important component of fruit nutrition and quality. In this study, three longan cultivars, including non-aroma types ‘Shixia’ (SX), ‘Lidongben’ (LDB), and strong aroma type ‘Xiangcui’ (XC), were selected to analyze free amino acids (FAAs) variations at six distinct growth stages (S1–S6). The genome-wide identification and expression analysis of genes related to the branched-chain amino acids (BCAA) synthesis pathway were carried out. Results showed that 36 FAAs were identified, and the total FAAs content ranged from 2601.0 to 9073.5 mg/kg, which increased drastically with fruit development until ripening. L-glutamic acid (Glu), L-alanine (Ala), L-arginine (Arg), γ-Aminobutyric acid (GABA), L-aspartic acid (Asp), L-leucine (Leu), hydroxyl-proline (Hypro), and L-serine (Ser) were the predominant FAAs (1619.9–7213.9 mg/kg) in pulp, accounting for 62.28–92.05% of the total amino acids. During the period of rapid fruit expansion (S2–S4), the aroma of XC changed from light to strong, and the contents of L-alanine (Ala) and L-leucine (Leu) were significantly higher than those of SX and LDB. Furthermore, a total of two 2-isopropyl malate synthase (IPMS), two 3-isopropyl malate dehydrogenase (IPMD), and 16 BCAA transferase (BCAT) genes were identified. The expression levels of DilBCAT1, -6, and -9 genes in XC were significantly higher than those in SX and LDB, while DilBCAT16 in XC was lower. The content of Leu was negatively correlated with the expression of DilBCAT1, -6, and -9 in three varieties, but positively correlated with DilBCAT16, indicating that these four genes may be responsible for the different synthesis and degradation of Leu among cultivars.

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%.

Geobacillus thermoglucosidasius NY05 catalyzes calcite single crystal formation at 60 °C by using acetate and calcium. Endospores are embedded at the central part of the calcite single crystal and carbon atoms in the calcite lattice are derived from acetate carbon. Here, we synthesized 21-mer antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs and evaluated the effect of these oligonucleotides on calcite formation in G. thermoglucosidasius NY05. G. thermoglucosidasius NY05 cells containing antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs had reduced calcite single crystal formation by 18.7, 50.6, 55.7, and 82.3%, respectively, compared with cells without antisense DNA oligonucleotides. These results support that calcite formation needs endospores as the nucleus to grow, and carbon dioxide generated from acetate, which is metabolized via the glyoxylate pathway and glucogenesis, is supplied to the crystal lattice.

The number and type of isoforms of superoxide dismutase (SOD) and their activities were compared in mitochondria and peroxisomes isolated from cotyledons of three different oilseed seedlings. Mitochondrial and peroxisomal isoforms of SOD could be distinguished in nondenaturing polyacrylamide gels by their differential sensitivities to KCN and/or H2O2. The type of SOD was not the same for each organelle in each of the three oilseed species. For example, a single Mn–SOD was found in cotton and cucumber mitochondria, whereas four CuZn–SODs were present in mitochondria from sunflower. At least one CuZn–SOD isoform was found in the peroxisomes of all three species. Cucumber peroxisomes contained both a CuZn–SOD and a Mn–SOD, cotton peroxisomes contained a single CuZn–SOD, whilst four separate CuZn–SODs, but no Mn–SOD were found in sunflower peroxisomes. Using antibodies against CuZn–SOD from watermelon peroxisomes or from chloroplasts of Equisetum, a single polypeptide of c. 16·5 kDa was detected on immunoblots of peroxisomal fractions from the three species. Post-embedment, electron-microscopic double immunogold-labelling showed that CuZn–SOD, with malate synthase used as marker enzyme of peroxisomes, was localized in the matrix of these organelles of all three species. These results suggest that CuZn–SOD is a characteristic matrix enzyme of peroxisomes in oilseed cotyledons.

A cDNA clone encoding malate synthase (MS; EC 4.1.3.2) was isolated from a 48-h postharvest asparagus (Asparagus officinalis L.) spear cDNA library using a MS clone from Brassica napus. The asparagus MS (AoMS1) cDNA hybridized to a 1.9-kb transcript that increased in abundance preferentially in spear-tip tissue during postharvest storage. The AoMS1 transcript also accumulated during natural foliar senescence of asparagus fern. The cDNA consists of 1960 nucleotides with an open reading frame of 1665 nucleotides or 555 amino acids, and encodes a deduced protein with a predicted Mr of 63 kDa and a pI of 8.1. The deduced amino acid sequence of AoMS1 showed high identity with the B. napus MS clone (77.2%) used to isolate it, and with MS from cucumber (77%). Genomic Southern analysis suggests that a single gene in asparagus encodes AoMS1. Controlled- atmosphere treatments aimed at reducing deterioration of harvested asparagus spears reduced the expression of AoMS1. The reduction was correlated with the reduced oxygen level, and reduced MS enzyme activity was also observed. Asparagus cell cultures were used to test the role of sugar status in regulating AoMS1 gene expression. In cultures without sucrose there was an accumulation of AoMS1 transcript that was absent in cultures containing sucrose.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.

Location of the proteases would affect on protease stability and photorespiratory bypass pathway, while it is unsolved. Potato rbcS signal peptide was analyzed and constructed into the protease for study of their localization site. The tartronate semialdehyde reductase (EcTSR) proteins could be accurately and efficiently located in chloroplast only when this signal peptide was extended to 80 amino acids. The signal peptide would help malate synthase (CmMS) locate to the surface of chloroplast, to form granules on the outer membrane of chloroplast. The whole spectrum scanning showed that these proteins could enter chloroplast. A signal peptide named PCS1 (Peptide of self-cleavage site 1) carrying a self-cleavage site was designed, and sixteen amino acids from the blue pigment precursor protein of chloroplast positioning signal of Silene pratensis were added to the C-terminal of PCS1. Transient expression, Western blot analysis and full-spectrum scanning showed that PCS1 could locate the EcTSR to the chloroplast, after the removal of the signal peptide.

International audience; Methyl groups are valuable probes for solution NMR, allowing the investigation of large protein complexes. Among the six methyl containing residues, threonine has one of the less hydrophobic side chain, and can reside both within the interior of a protein or on the protein surface. This article presents an efficient mixed chemical/enzymatic synthesis scheme, enabling the preparation of threonine with natural configuration on the two stereogenic centers together with an optimal introduction of 1H/2H and 12C/13C atoms at specific sites. Such specifically labelled amino acid can be efficiently incorporated in overexpressed proteins without scrambling or in combination with any other types of 13CH3 probes. Additionally we report application to the 82 kDa Malate Synthase G. Our findings demonstrate that structural meaningful long range nOes can be detected between threonine methyl probes and methionine and isoleucine methyl groups distant by 12 Å.

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.

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.

Background: Resveratrol naturally occurring antioxidant in peanut (Legume: Arachis hypogaea) has phytochemical human health dietary effects associated with reduced inflammatory cancer risks. Its levels in peanut are ultra-low and variable (0 to 26 μg·g-1), which has made it difficult to market as a consistent high resveratrol produce. Objective: Understanding the regulation of resveratrol accumulation in peanut might lead to development of new techniques for optimizing and stabilizing its yield. Method: Peanuts were cultivated in horticultural field plots and treated with solutions of mineral salts (sulfate, potassium, phosphate, ammonium ion) that were optimized in stoichiometric (reactive) ratios. Peanut seed’s RNAs were subjected to Northern blot analysis for profiling the RNAs synthesized by glutamate dehydrogenase (GDH), and mRNAs encoding resveratrol synthase. The seed’s extracts were analyzed by GC-MS for determination of the resveratrol and fatty acid compositions. Result: Stoichiometric mixes of mineral ions induced the peanut GDH to synthesize some RNA that silenced the mRNAs encoding resveratrol synthase, phosphoglucomutase, isocitrate lyase, malate synthase, enolase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and phosphoglycerate mutase in the control, KN-, and NPKS-treated but not in the NPPK-treated peanut. These resulted to decreased resveratrol content (6.0 μg·g-1) in the control peanut but maximized it (1.15 mg·g-1) in the NPPK-treated peanut. Therefore, resveratrol accumulation was optimized by coupling of glycolysis and citric-glyoxylic acid cycles to resveratrol biosynthesis. Fatty acid content of control (55.6 g·kg-1) was higher than the NPKS-treated (48.5 g·kg-1) and NPPK-treated peanut (44.9 g·kg-1) meaning that malonyl-CoA intermediate in both fatty acid and stilbenoid pathways was diverted to support maximum resveratrol biosynthesis in the NPPK-treated peanut. Conclusion: The functional coupling of citric-glyoxylic acid cycles and glycolysis to optimize resveratrol biosynthesis may encourage development of horticultural technology specific for production of ultra-high resveratrol peanuts.

Specialized metabolites are structurally diverse and cell- or tissue-specific molecules produced in restricted plant lineages. In contrast, primary metabolic pathways are highly conserved in plants and produce metabolites essential for all of life, such as amino acids and nucleotides. Substrate promiscuity - the capacity to accept non-native substrates - is a common characteristic of enzymes, and its impact is especially apparent in generating specialized metabolite variation. However, promiscuity only leads to metabolic diversity when alternative substrates are available; thus, enzyme cellular and subcellular localization directly influence chemical phenotypes. We review a variety of mechanisms that modulate substrate availability for promiscuous plant enzymes. We focus on examples where evolution led to modification of the 'cellular context' through changes in cell-type expression, subcellular relocalization, pathway sequestration, and cellular mixing via tissue damage. These varied mechanisms contributed to the emergence of structurally diverse plant specialized metabolites and inform future metabolic engineering approaches.

This research aimed to investigate the effects of progesterone, a mammalian steroid sex hormone, on the mitochondrial respiration in germinating maize seeds. For this purpose, maize seeds were divided into four different groups (control, 10−6, 10−8, and 10−10 mol·L−1 progesterone) and were grown in a germination cabinet in the dark at 24.5 ± 0.5 °C for 4 d. The changes in gene expression levels of citrate synthase (CS), cytochrome oxidase (COX19), pyruvate dehydrogenase (Pdh1), and ATP synthase (ATP6), which is involved in mitochondrial respiration, were studied in root and cotyledon tissues. Significant increases were recorded in the gene expression levels of all studied enzymes. In addition, progesterone applications stimulated activities of malate synthase (MS), isocitrate lyase (ICL), and alpha-amylase, which are important enzymes of the germination step. The changes in gene expression levels of mas1 and icl1 were found parallel to the rise in these enzymes’ activities. It was determined similar increases in root and coleoptile lengths and total soluble protein and total carbohydrate contents. The most remarkable changes were detected in 10−8 mol·L−1 progesterone-treated seedlings. These results clearly indicate that progesterone stimulates mitochondrial respiration by inducing biochemical and molecular parameters and thus accelerates seed germination thanks to the activation of other pathways related to mitochondrial respiration.

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.

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.

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.

Organic acids are important components of overall fruit quality through flavor, taste, nutritional and medicinal values. Pollinated fig (Ficus carica L.) fruit quality is enhanced by increased acidity. We quantified the major organic acids and characterized the expression pattern of organic acid metabolic pathway-related genes in the reproductive part - inflorescence and non-reproductive part - receptacle of parthenocarpic and pollinated fig fruit during ripening. Essentially, pollinated fruit contains seeds in the inflorescence, as opposed to no seeds in the parthenocarpic inflorescence. The major organic acids - citrate and malate - were found in relatively high quantities in the inflorescence compared to the receptacle of both parthenocarpic and pollinated fig fruit. Notably, pollination increased citric acid content significantly in both inflorescence and receptacle. Genes related to the phosphoenolpyruvate carboxylase (PEPC) cycle, tricarboxylic acid cycle, citrate catabolism and glyoxylate cycle were identified in fig fruit. Expression levels of most of these genes were higher in inflorescences than in receptacles. In particular, FcPEPC and FcFUM (encoding fumarase) had significantly higher expression in the inflorescence of pollinated fruit. Most importantly, expression of the glyoxylate cycle genes FcMLS and FcICL (encoding malate synthase and isocitrate lyase, respectively) was induced to strikingly high levels in the inflorescence by pollination, and their expression level was highly positively correlated with the contents of all organic acids. Therefore, the glyoxylate cycle may be responsible for altering the accumulation of organic acids to upgrade the fruit taste during ripening, especially in the pollinated, seeded inflorescence.

The Gram-positive bacterium Corynebacterium glutamicum represents a promising platform for the production of amino acids, organic acids, and other bio-products. However, the availability of only few expression vectors limits its use for production purposes, using metabolic engineering approaches when co-expression of several target genes is desired. To widen the scope for co-expression, the pCG1/p15A and pBL1/colE1 replicons were employed to construct the two differentially-inducible and compatible expression vectors pRG_Duet1 and pRG_Duet2. To functionally validate these newly constructed expression vectors, target genes for easily measurable enzymes were cloned and over-expression of these genes was investigated using respective enzyme assays. Furthermore, functionality and co-existence of the pCG1-based C. glutamicum - E. coli shuttle vector pRG_Duet1 were confirmed with pBL1-based expression vectors pRG_Duet2 and pEKEx2, using co-transformation and enzyme assays. The novel shuttle expression vectors pRG_Duet1 and pRG_Duet2 are attractive additions to the existing set of vectors for co-expression studies and metabolic engineering of C. glutamicum.

International audience; Chaperonins are ubiquitous protein assemblies present in bacteria, eukaryota, and archaea, facilitating the folding of proteins, preventing protein aggregation, and thus participating in maintaining protein homeostasis in the cell. During their functional cycle, they bind unfolded client proteins inside their double ring structure and promote protein folding by closing the ring chamber in an adenosine 5'-triphosphate (ATP)-dependent manner. Although the static structures of fully open and closed forms of chaperonins were solved by x-ray crystallography or electron microscopy, elucidating the mechanisms of such ATP-driven molecular events requires studying the proteins at the structural level under working conditions. We introduce an approach that combines site-specific nuclear magnetic resonance observation of very large proteins, enabled by advanced isotope labeling methods, with an in situ ATP regeneration system. Using this method, we provide functional insight into the 1-MDa large hsp60 chaperonin while processing client proteins and reveal how nucleotide binding, hydrolysis, and release control switching between closed and open states. While the open conformation stabilizes the unfolded state of client proteins, the internalization of the client protein inside the chaperonin cavity speeds up its functional cycle. This approach opens new perspectives to study structures and mechanisms of various ATP-driven biological machineries in the heat of action.

The glyoxylate cycle is an anaplerotic pathway of the tricarboxylic acid cycle that allows bacteria to grow using acetate, fatty acids, or poly-β-hydroxybutyrate (PHB). In Sinorhizobium meliloti, activities of the glyoxylate cycle enzymes isocitrate lyase (AceA) and malate synthase (GlcB) are present during growth on these kinds of carbon sources, but a study of the importance of these enzymes in utilizing these carbon compounds under starvation conditions has not been done. We therefore evaluated the role of AceA and GlcB in the utilization of PHB by determining the PHB degradative and growth capacities of the S. meliloti wild type and aceA and glcB mutants under carbon starvation conditions in culture. We found that only the aceA gene product was essential for bacterial growth and PHB degradation under these conditions, presumably by generating succinate from the acetyl-CoA derived from PHB catabolism, thus allowing the cells to grow in the absence of an external carbon source.

The ovate mutation has frequently been used to study changes in fruit shape but not fruit quality. A deterioration in fruit quality associated with the ovate mutation was discovered in this study. To elucidate how ovate influences the quality of fruit, we performed a proteomics analysis of the fruits of the ovate mutant (LA3543) and wild-type (“Ailsa Craig”, LA2838A) using tandem mass tag analysis. The results indicated that the ovate mutation significantly influences fruit quality in a number of ways, including by reducing the expression of 1-aminocyclopropane-1-carboxylic acid oxidase 3 (ACO3) in ethylene biosynthesis, improving firmness by reducing the amount of pectinesterase and polygalacturonase, reducing sugar accumulation by downregulating the abundance of mannan endo-1,4-β-mannosidase 4, β-galactosidase, and β-amylase, and reducing the malic acid content by downregulating the accumulation of malic enzymes and malate synthase. These findings could inform future improvements in fruit quality.

Malic acid (MA), one of the major organic acid exudates from roots, plays a significant role in the chemotaxis of beneficial bacteria to the plant’s rhizosphere. In this study, the effect of a plant-growth-promoting rhizobacterium, Bacillus subtilis RR4, on the synthesis and exudation of MA from roots is demonstrated in rice. To test the chemotactic ability of strain RR4 towards MA, a capillary chemotaxis assay was performed, which revealed a positive response (relative chemotactic ratio of 6.15 with 10 μmol/L MA); with increasing concentrations of MA, an elevated chemotactic response was observed. Quantitative polymerase chain reaction, performed to analyze the influence of RR4 on the MA biosynthetic gene, malate synthase (OsMS), and the transporter gene, aluminium-activated malate transporter (OsALMT), demonstrated significant differential expression, with 1.8- and –0.58-fold changes, respectively, in RR4-treated roots. The gene expression pattern of OsMS corroborated the data obtained by high-performance liquid chromatography, which showed elevated MA levels in roots (1.52-fold), whereas the levels of MA in root exudates were not altered significantly although expression of OsALMT was reduced. Our results demonstrate that B. subtilis RR4 is chemotactic to MA and can induce biosynthesis of MA in rice roots.

Defending phagocyte generated oxidants is the key for survival of Salmonella Typhimurium (S. Typhimurium) inside the host. Met residues are highly prone to oxidation and convert into methionine sulfoxide (Met-SO). Methionine sulfoxide reductase (Msr) can repair Met-SO to Met thus restoring the function(s) of Met-SO containing proteins. Using pull down method we have identified several MsrA interacting proteins in the S. Typhimurium, one of them was malate synthase (MS). MS is an enzyme of glyoxylate cycle. This cycle is essential for survival of S. Typhimurium inside the host under nutrient limiting conditions. By employing in vitro cross-linking and blot overlay techniques we showed that purified MsrA interacted with pure MS. Treatment of pure malate synthase with H2O2 resulted in reduction of MS activity. However, MsrA along with thioredoxin-thioredoxin reductase system partially restored the activity of oxidized MS. Our mass spectrometry data demonstrated H2O2 mediated oxidation and MsrA mediated repair of Met residues in MS. Further in comparison to S. Typhimurium, the msrA gene deletion (∆msrA) strain showed reduced (60%) malate synthase specific activity. Oral inoculation with wild type, ∆msrA and ∆ms strains of S. Typhimurium resulted in colonization of 100, 0 and 40% of the poultry respectively.

Early reports have demonstrated the occurrence of glyoxylate cycle enzymes in several Leishmania species. However, these results have been underestimated because genes for the two key enzymes of the cycle, isocitrate lyase (ICL) and malate synthase (MS), are not annotated in Leishmania genomes. We have re-examined this issue in promastigotes of Leishmania amazonensis. Enzyme activities were assayed spectrophotometrically in cellular extracts and characterized partially. A 40 kDa band displaying ICL activity was visualized on zymograms of the extracts. By immunoblotting with mouse antibodies against ICL from Bacillus stearothermophilus, a band of approximately 40 kDa was identified, coincident with the relative molecular mass of the activity band revealed on zymograms. Indirect immunofluorescence of intact promastigotes showed that the recognized antigen is distributed as a punctuated pattern, mainly distributed beneath the subpellicular microtubules, over a diffused cytoplasmic stain. These results clearly demonstrate the existence of an apparent ICL activity in L. amazonensis promastigotes, which is associated to a 40 kDa polypeptide and distributed both diffused and as punctuate aggregates in the cytoplasm. The relevance of this activity is discussed.

International audience; As part of our continuing chemical and biological analyses of Rubiaceae species from Cerrado, we isolated novel alkaloids 1 and 2, along with known compounds epicatechin, ursolic acid, and oleanolic acid, from Galianthe ramosa. Alkaloid 2 inhibited malate synthase from the pathogenic fungus Paracoccidioides spp. This enzyme is considered an important molecular target because it is not found in humans. Molecular docking simulations were used to describe the interactions between the alkaloids and malate synthase.

In a previous report, the pepper receptor-like kinase 1 (CaRLK1) gene was shown to be responsible for negatively regulating plant cell death caused by pathogens via accumulation of superoxide anions. Here, we examined whether this gene also plays a role in regulating cell death under abiotic stress. The total concentrations of free amino acids in CaRLK1-overexpressed cells (RLKox) increased by twofold compared with those of the wild-type Nicotiana tabacum BY-2 cells. Additionally, alanine and pyruvate concentrations increased by approximately threefold. These accumulations were associated with both the expression levels of the isocitrate lyase (ICL) and malate synthase genes and their specific activities, which were preferentially up-regulated in the RLKox cells. The expression levels of ethylene biosynthetic genes (ACC synthase and ACC oxidase) were suppressed, but those of both the metallothionein and lesion simulating disease 1 genes increased in the RLKox cells during submergence-induced hypoxia. The specific activity of catalase, which is involved in protecting ICL from reactive oxygen species, was also induced threefold in the RLKox cells. The primary roots of the transgenic plants that were exposed to hypoxic conditions grew at similar rates to those in normal conditions. We propose that CaRLK1 maintains a persistent hypoxia-resistant phenotype.

Despite their prevalence in biological systems, information about the folding pathways of large and multidomain proteins is meager, as they often unfold irreversibly under in vitro conditions which make their folding studies difficult or even impossible. The folding mechanism of a large (82 kDa) and multidomain protein Malate synthase G (MSG) has been demonstrated in the present study using intrinsic tryptophan fluorescence, enzymatic activity, and extrinsic fluorophore ANS as probes for monitoring the refolding process. Refolding of MSG is found to occur in three kinetic phases. Denatured MSG forms a collapsed state in the burst phase of refolding, which then gives rise to an active intermediate having the same tryptophan fluorescence and enzymatic activity as native MSG in the slow phase. Native topology of MSG is formed from the active intermediate in the very slow phase of refolding which is silent to tryptophan fluorescence change and is susceptible to aggregation at higher protein concentrations. Dependence of rates of very slow phase on GdnHCl concentration suggests that it is not solely a cis/trans proline isomerization limited process but might involve an additional folding event of the domains, not forming the active site of the protein. In light of the above findings, the appearance of a functional intermediate during refolding of MSG was predicted to be an instance of weak interdomain cooperativity. This work has significant implications in the characterization of the refolding intermediates of multidomain proteins in general and MSG in particular, where weak interdomain cooperativity might contribute toward generation of a functional intermediate during its refolding.

Chloris virgata, an alkali-tolerant halophyte, was chosen as the test material for our research. The seedlings of C. virgata were treated with varying salt and alkali stress. First, the composition and content of organic acids in shoots were analyzed and the results indicated that there was not only a significant increase in total organic acids, but there were also obvious changes in different components of organic acids under alkali stress. The increments in citrate were the largest, followed by malate. However, none of the organic acids showed significant alterations in the content and components under salt stress. Also, activity of some enzymes (citrate synthase, malate synthase, NADP-isocitrate dehydrogenase, and isocitrate lyase) associated with such organic acids did not change significantly under alkali stress, but malate dehydrogenase activity markedly decreased under a stronger alkali stress (80 mM). Under salt stress as well as increased malate synthase (MS) activity, however, there was no significant change for other enzymes. These results strongly demonstrated that the enzymatic regulation of organic acid metabolism may be the biochemical basis of alkali tolerance for C. virgata. Citrate synthase (CS), MS and isocitrate lyase (ICL) might be the key enzymes that determine the alkali tolerance of C. virgata. Key words: Salinity, ion balance, enzyme activity, Chloris virgata.

Haloarchaea are extremely halophilic heterotrophic microorganisms belonging to the class Halobacteria ( Euryarchaeota ). Almost half of the haloarchaea possesses the genes coding for enzymes of the methylaspartate cycle, a recently discovered anaplerotic acetate assimilation pathway. In this cycle, the enzymes of the tricarboxylic acid cycle together with the dedicated enzymes of the methylaspartate cycle convert two acetyl coenzyme A (acetyl-CoA) molecules to malate. The methylaspartate cycle involves two reactions catalyzed by homologous enzymes belonging to the CitE-like enzyme superfamily, malyl-CoA lyase/thioesterase (haloarchaeal malate synthase [hMS]; Hah_2476 in Haloarcula hispanica ) and β-methylmalyl-CoA lyase (haloarchaeal β-methylmalyl-CoA lyase [hMCL]; Hah_1341). Although both enzymes catalyze the same reactions, hMS was previously proposed to preferentially catalyze the formation of malate from acetyl-CoA and glyoxylate (malate synthase activity) and hMCL was proposed to primarily cleave β-methylmalyl-CoA to propionyl-CoA and glyoxylate. Here we studied the physiological functions of these enzymes during acetate assimilation in H. hispanica by using biochemical assays of the wild type and deletion mutants. Our results reveal that the main physiological function of hMS is malyl-CoA (not malate) formation and that hMCL catalyzes a β-methylmalyl-CoA lyase reaction in vivo . The malyl-CoA thioesterase activities of both enzymes appear to be not essential for growth on acetate. Interestingly, despite the different physiological functions of hMS and hMCL, structural comparisons predict that these two proteins have virtually identical active sites, thus highlighting the need for experimental validation of their catalytic functions. Our results provide further proof of the operation of the methylaspartate cycle and indicate the existence of a distinct, yet-to-be-discovered malyl-CoA thioesterase in haloarchaea. IMPORTANCE Acetate is one of the most important substances in natural environments. The activated form of acetate, acetyl coenzyme A (acetyl-CoA), is the high-energy intermediate at the crossroads of central metabolism: its oxidation generates energy for the cell, and about a third of all biosynthetic fluxes start directly from acetyl-CoA. Many organic compounds enter the central carbon metabolism via this key molecule. To sustain growth on acetyl-CoA-generating compounds, a dedicated assimilation (anaplerotic) pathway is required. The presence of an anaplerotic pathway is a prerequisite for growth in many environments, being important for environmentally, industrially, and clinically important microorganisms. Here we studied specific reactions of a recently discovered acetate assimilation pathway, the methylaspartate cycle, functioning in extremely halophilic archaea.

© 2016 Ghosh, Ando, Gin, Runguphan, Denby, Wang, Baidoo, Shymansky, Keasling and García Martín. Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined13C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. These genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by ~70%.

Isocitrate lyase plays a key role for survival of Mycobacterium tuberculosis in the latent form during a chronic stage of infection. This enzyme is important for M. tuberculosis during steady stage growth when it converts isocitrate to succinate and glyoxylate. Then, the glyoxylate is condensed with acetyl-CoA to form malate by malate synthase. The carbon conserving glyoxylate pathway has not been observed in mammals; therefore, it has been determined as a potential drug target for discovery of a new antituberculosis agent. Novel active molecules should shorten the duration of therapy, prevent resistance development and eliminate latent disease. The review summarizes recent progresses in isocitrate lyase inhibitors, overviews structural analogues of several metabolic intermediates (3-nitropropionate, 3-bromopyruvate, itaconate, itaconic anhydride), peptide inhibitors, and recently developed inhibitors with various chemical structures. The largest inhibitory activity against isocitrate lyase (IC(50) of 0.10 ± 0.01 μM) and concomitantly a significant antimycobacterial activity has been presented by fluoroquinolone derivative 1-cyclopropyl-7-[3,5-dimethyl-4-(3-nitropropanoyl)piperazin-1-yl]-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, which has incorporated 3-nitropropionyl group as one of the structural analogue of succinate, a metabolic intermediate.

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.

The growth of wild type strain Yarrowia lipolytica VKM Y-2373 and its mutant Y. lipolytica NG40/UV7 as well as the biosynthesis of citric and isocitric acid on rapeseed oil were studied. It was indicated that the initial step of assimilation of rapeseed oil in the yeast Y. lipolytica is its hydrolysis by extracellular lipases with the formation of glycerol and fatty acids, which appear in the medium in the phase of active growth. The concentrations of these metabolites were changed insignificantly upon further cultivation. Lipase and the key enzymes of glycerol metabolism (glycerol kinase) and the glyoxylate cycle responsible for the metabolism of fatty acids (isocitrate lyase and malate synthase) are induced just at the beginning of the growth phase and remain active in the course of further cultivation. These results, taken together, suggest that glycerol and fatty acids available in the medium do not suppress the metabolism of each other. Citric acid production and a ratio between citric and isocitric acids depended on the strain used. It was revealed that the wild strain produced almost equal amounts of citric and isocitric acids while the mutant produced only citric acid (175 g/L) with a yield of 1.5 g of CA per g of oil.

The glyoxylate cycle is a modified form of the tricarboxylic acid cycle, which enables organisms to synthesize carbohydrates from C2 compounds. In the protozoan Euglena gracilis, the key enzyme activities of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), are conferred by a single bifunctional protein named glyoxylate cycle enzyme (Euglena gracilis glyoxylate cycle enzyme [EgGCE]). We analyzed the enzymatic properties of recombinant EgGCE to determine the functions of its different domains. The 62-kDa N-terminal domain of EgGCE was sufficient to provide the MS activity as expected from an analysis of the deduced amino acid sequence. In contrast, expression of the 67-kDa C-terminal domain of EgGCE failed to yield ICL activity even though this domain was structurally similar to ICL family enzymes. Analyses of truncation mutants suggested that the N-terminal residues of EgGCE are critical for both the ICL and MS activities. The ICL activity of EgGCE increased in the presence of micro-molar concentrations of acetyl-coenzyme A (CoA). Acetyl-CoA also increased the activity in a mutant type EgGCE with a mutation at the acetyl-CoA binding site in the MS domain of EgGCE. This suggests that acetyl-CoA regulates the ICL reaction by binding to a site other than the catalytic center of the MS reaction.

Pseudomonas putida JM37 metabolized glyoxylate at a specific rate of 55 g/g dry biomass/day. In order to investigate their role, three genes encoding enzymes that are potentially involved in the conversion of glyoxylate were disrupted; namely, tartronate semialdehyde synthase (gcl), malate synthase (glcB), and isocitrate lyase (aceA). Strains with transposon insertion in either of these genes were isolated from a 50,000 clone library employing a PCR-guided enrichment strategy. In addition, all three double mutants were constructed via targeted insertion of a knock-out plasmid. Neither mutation of gcl, glcB, and aceA nor any of the respective double mutations influenced glyoxylic acid conversion, indicating that P. putida JM37 may possess other enzymes and pathways for glyoxylate metabolism.

Methyl (13)CHD(2) isotopomers of all methyl-containing amino-acids can be observed in residually protonated samples of large proteins obtained from [U-(13)C,(1)H]-glucose/D(2)O-based bacterial media, with sensitivity sufficient for a number of NMR applications. Selective detection of some subsets of methyl groups (Ala(beta), Thr(gamma 2)) is possible using simple 'out-and-back' NMR methodology. Such selective methyl-detected 'out-and-back' NMR experiments allow complete assignments of threonine gamma 2 methyls in residually protonated, [U-(13)C,(1)H]-glucose/D(2)O-derived samples of an 82-kDa enzyme Malate Synthase G. [U-(13)C,(1)H]-glucose/D(2)O-derived protein samples are relatively inexpensive and are usually available at very early stages of any NMR study of high-molecular-weight systems.

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption.

A set of sensitive methyl-detected 'out-and-back' NMR experiments for simultaneous assignments of Alabeta and Ilegamma2 methyl positions in large proteins is described. The developed methodology is applied to an 82-kDa enzyme Malate Synthase G. Complete alanine beta and isoleucine gamma2 1H-13C methyl chemical shift assignments could be obtained from the set of new methyl-detected 'out-and-back' 3D experiments. The described methodology for methyl assignments should be applicable to protein molecules within approximately 100-kDa molecular weight range irrespective of the labeling strategy chosen to produce selectively protonated Alabeta and Ilegamma2 13CH3 sites on a deuterated background.

The genome sequence of the marine bacterium 'Candidatus Pelagibacter ubique' and subsequent analyses have shown that while it has a genome as small as many obligate parasites, it nonetheless possesses a metabolic repertoire that allows it to grow as one of the most successful free-living cells in the ocean. An early report based on metabolic reconstruction indicated that SAR11 cells are prototrophs for all amino acids. However, here we report experimental evidence that 'Cand. P. ubique' is effectively auxotrophic for glycine and serine. With glucose and acetate added to seawater to supply organic carbon, the addition of 125 nM to 1.5 microM glycine to growth medium containing all other nutrients in excess resulted in a linear increase in maximum cell density from 1.14 x 10(6) cells ml(-1) to 8.16 x 10(6) cells ml(-1) (R(2) = 0.992). Serine was capable of substituting for glycine at 1.5 microM. 'Cand. P. ubique' contains a glycine-activated riboswitch preceding malate synthase, an unusual genomic context that is conserved in the SAR11 group. Malate synthase plays a critical role in central metabolism by enabling TCA intermediates to be regenerated through the glyoxylate cycle. In vitro analysis of this riboswitch indicated that it responds solely to glycine but not close structural analogues, such as glycine betaine, malate, glyoxylate, glycolate, alanine, serine or threonine. We conclude that 'Cand. P. ubique' is therefore a glycine-serine auxotroph that appears to use intracellular glycine level to regulate its use of carbon for biosynthesis and energy. Comparative genomics and metagenomics indicate that these conclusions may hold throughout much of the SAR11 clade.

Enzymes of the glyoxylate shunt are important for the virulence of pathogenic organisms such as Mycobacterium tuberculosis and Candida albicans. Two isoforms have been identified for malate synthase, the second enzyme in the pathway. Isoform A, found in fungi and plants, comprises approximately 530 residues, whereas isoform G, found only in bacteria, is larger by approximately 200 residues. Crystal structures of malate synthase isoform G from Escherichia coli and Mycobacterium tuberculosis were previously determined at moderate resolution. Here we describe crystal structures of E. coli malate synthase A (MSA) in the apo form (1.04 A resolution) and in complex with acetyl-coenzyme A and a competitive inhibitor, possibly pyruvate or oxalate (1.40 A resolution). In addition, a crystal structure for Bacillus anthracis MSA at 1.70 A resolution is reported. The increase in size between isoforms A and G can be attributed primarily to an inserted alpha/beta domain that may have regulatory function. Upon binding of inhibitor or substrate, several active site loops in MSA undergo large conformational changes. However, in the substrate bound form, the active sites of isoforms A and G from E. coli are nearly identical. Considering that inhibitors bind with very similar affinities to both isoforms, MSA is as an excellent platform for high-resolution structural studies and drug discovery efforts.

In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid β-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae ; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C 2 carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid β-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid β-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.

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.

Comperative studies on the activities of isocitrate lyase (ICL) and malate synthase (MS) were carried out with Saccharomycopsis lipolytica incubating the yeast on media with different carbon sources. When cells were incubated in minimal medium with glucose, the activities of both enzymes were very low. In contrast, in minimal medium with acetate enhanced enzyme activities could be demonstrated. It is probably that the synthesis of ICL is repressed in presence of glucose. Furthermore the activity of ICL was inhibited by tricarboxylic acid cycle intermediates like succinic acid and oxalacetic acid. It was concluded that the syntheses of enzymes are derepressed. When cells of Sm. lipolytica were incubated in minimal medium with acetate, a high enzyme activity is evident. Synthesis of ICL on acetate was inhibited by cycloheximid and actinomycin D. The results were discussed comparing them with data obtained from other organisms.

Paracoccidioides is the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase of Paracoccidioides species ( Pb MLS). The three-dimensional (3D) structure of Pb MLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds with Pb MLS, it was possible to identify β-carboline moiety as a standard key structure. The compounds with β-carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket of Pb MLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of β-carboline moiety, was crucial for their stability in the binding pocket of Pb MLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four β-carboline alkaloid compounds showed the ability to inhibit recombinant Pb MLS ( Pb MLSr) activity, Paracoccidioides species growth, and adhesion of the fungus and Pb MLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated.

Mycobacterium tuberculosis, the causative agent of Tuberculosis, is intrinsically resistant to many first line antibiotics used to treat other common bacterial infections and resistance to effective drugs is increasing. The characterization of new targets and the development of new drugs are urgently needed. Mycobacteria, like many other bacteria, synthesize the branched-chain amino acids L-valine and L –leucine, and pantothenic acid, from α-ketoisovalerate1 (α–KIV). Studies using transposon mutagenesis in mycobacteria have demonstrated that the L-leucine biosynthetic pathway is essential for Mycobacterium bovis both in vitro and in vivo (1-3). These results suggest that mycobacteria, engulfed within the macrophage phagolysosome-like compartment, cannot obtain sufficient L-leucine from the surrounding environment. These findings also validate the L-leucine biosynthetic pathway as a target for inhibitor design and drug development to treat Tuberculosis. In spite of its importance, the L-leucine biosynthetic pathway is among the least studied amino acid biosynthetic pathways. The L-leucine branch of the branched-chain amino acid pathway (Scheme 1) starts with the AcCoA-dependent carboxymethylation of α–KIV catalyzed by α–isopropylmalate synthase (EC 2.3.3.13). This enzyme is subject to feedback inhibition by L-leucine in many organisms and the M. tuberculosis IPMS has recently been shown to exhibit slow-onset, feedback inhibition by L-leucine (4). α–Isopropylmalate is subsequently converted to β-isopropylmalate by α–isopropylmalate isomerase (EC 4.2.1.33) and then oxidatively decarboxylated to α–ketoisocaproate by β-isopropylmalate dehydrogenase (EC 1.1.1.85). α–Ketoisocaproate is converted directly to L-leucine by the action of a branched-chain amino acid transaminase (5). All of the genes involved in L-leucine biosynthesis have defined orthologues in the M. tuberculosis genome (6, 7). Scheme 1 α–Isopropylmalate synthase (IPMS) catalyzes a Claisen-type condensation between α–KIV and AcCoA. The most well-characterized members of this family are malate synthase (MS), a component of the glyoxylate pathway, citrate synthase (CS), a component of the Krebs cycle, and most recently homocitrate synthase (HCS), a component of the yeast α–aminoadipate pathway (8). Despite the similarities in their respective substrates and the chemical reactions catalyzed, MS and CS perform the condensation reaction using quite different catalytic mechanisms. MS from both yeast and M. tuberculosis use a Mg2+ ion (9, 10) to bind and polarize the carbonyl group of glyoxylate, while CS does not use a divalent metal, but rather a pair of histidine residues to polarize the carbonyl group of oxaloacetate. In addition, CS from different organisms can display quite different pH dependences, with the Thermoplasma acidophilum CS reaction being independent of pH and the pig heart CS reaction being dependent on two ionizable enzyme groups (11). IPMS from Salmonella enterica serovar typhimurium (12-15), Alcaligenes eutrophus H16 (16-18), Neurospora (19), and Saccharomyces (20) have been partially characterized. The common features of most α–IPM synthases are a requirement for monovalent cations for maximal activity, feedback inhibition by L-leucine, and narrow substrate specificity for analogs of α–KIV. In contrast, there are many differences revealed by these studies including the effects of divalent metals, inhibition by some α-keto acids, feedback regulation, and allosteric properties. Chanchaem and Palittapongarnpim have cloned, expressed and purified active MtIPMS, but no rigorous kinetic studies were performed (21). Recently, Koon et al. solved the three-dimensional crystal structure of IPMS from M. tuberculosis with Zn2+ and α–KIV bound at the active site (22). The insertion of two residues of one monomer into the active site of the other in the dimeric enzyme suggests how regulation by L-leucine may be achieved (22). Our own studies have revealed an absolute dependence for a monovalent cation for activity and potassium appears to be the physiologically relevant monovalent ion. Additionally, the monovalent cation is required for the binding of, and activation of catalysis by, a divalent metal that is used to coordinate the carbonyl and carboxyl oxygens of α-KIV (25). This paper describes the detailed biochemical characterization of the recombinant α-isopropylmalate synthase from M. tuberculosis H37Rv. Using a combination of steady-state kinetics, primary deuterium and solvent kinetic isotope effects, isotopic labeling, and 1H-NMR spectroscopy, the following questions were addressed. (i) What is the substrate specificity for α–keto acids and acyl-CoA analogs? (ii) What is the kinetic mechanism of MtIPMS? (iii) Does acid/base chemistry play an important role in catalysis? (iv) Does the enzyme hydrolyze acyl-CoA analogs? (v) What is the rate-limiting step in the reaction? (vi) What is the chemical mechanism used for intermediate cleavage?

Recently it has been found that Candida albicans harbours enzymes involved in the glyoxylate cycle (GC), which have a role in its virulence, especially the two key enzymes, isocitrate lyase (ICL) and malate synthase (MS). There are however, few studies on the GC enzyme activities isolated in the clinical isolates. Samples were collected from three groups of patients namely, HIV/AIDS, diabetic and burn patients suffering from candidiasis at different body locations. Isolation, identification and the antifungal susceptibility test of all the isolates of C. albicans were followed by the standard techniques. Measurements of all the GC enzyme activities were also carried out by the standard methods. Levels of the principal GC enzymes showed significant changes when calculated and compared taking control strains of C. albicans. The activity of the two key enzymes of the GC, ICL and MS were significantly higher in the isolates from diabetic patients. No significant relationship between the drug susceptibility and the level of enzymes of the GC was observed. As GC activity is absent in mammalian cells, a specific inhibitor for the GC could be developed and these enzymes therefore can be used as a new antifungal target.

During phototrophic growth on acetate and CO2Rhodospirillum rubrum 2R contained malate synthase but lacked isocitrate lyase. Acetate assimilation by R. rubrum cells was stimulated by pyruvate, propionate glyoxylate, CO2 and H2. Acetate photoassimilation by R. rubrum cells in the presence of bicarbonate was accompanied by glyoxylate secretion, which increased after addition of fluoroacetate and decreased after addition of malonate. When acetyl-CoA was incubated with pyruvate in cell-free extracts, citramalate was formed. Citramalate was also formed from propionyl-CoA and glyoxylate. The existence in R. rubrum of a CO2-dependent cyclic pathway of acetate oxidation to glyoxylate with citramalate as an intermediate is proposed. Inhibitor analysis of acetate and bicarbonate assimilation indicated that pyruvate synthase is not involved in acetate assimilation in R. rubrum. The possible anaplerotic sequences employed by R. rubrum during phototrophic growth on acetate are discussed.

Isocitrate lyase and malate synthase are the key enzymes of glyoxylate cycle that represents the most important stage on the pathway of conversion of fatty acids to carbohydrates. Until now, induction of enzymes of this metabolic pathway was considered to take place only in cells of prokaryotes, plants, fungi, and nematodes in response to arising demands in carbohydrates. However, the isocitrate lyase activities have been detected in the liver of food-starved rats in our previous work and in pupas of the butterfly Papilio machaon in the present study. The enzymes from both studied objects were purified to homogeneous condition. The main kinetic and physicochemical properties of isocitrate lyase were studied. Organisms of evolutionary distant taxa—mammal, insect, and plant—were chosen for comparative analysis of properties of the studied enzyme. A substantial similarity of kinetic and physicochemical properties of plant and animal isocitrate leases has been found. At the same time, the absence of specific for prokaryotic, plant, and nematode isocitrate lyase nucleotide sequences has been established in mRNA from liver of starved rats and swallowtail pupa. These results are completely confirmed by analysis of the complete genome sequences of the mouse, Drosophila, and human. The obtained data raise the question about the pathway of evolution of genes of the glyoxylate cycle key enzymes.