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Highlights • Chloroplastic transgenic C. reinhardtii was developed that stably expressed malate synthase. • This transgenic strain showed a more 19% increase in dry cell weight than wild-type strain. • Transcripts of MDH4 , ACS3 , ICL1 , and MAS1 were increased, and F v /F m was decreased. • Upregulation of heterotrophic metabolism might be involved in biomass increase. • This could serve as a valuable strain for treating wastewater containing acetate and glyoxylate. Abstract High uptake of malate and efficient distribution of intracellular malate to organelles contributed to biomass increase, reducing maintenance energy. In this study, transgenic Chlamydomonas reinhardtii was developed that stably expresses malate synthase in the chloroplast. The strains under glyoxylate treatment showed 19% more increase in microalgal biomass than wild-type. By RNA analysis, transcript levels of malate dehydrogenase (MDH4) and acetyl-CoA synthetase (ACS3), isocitrate lyase (ICL1) and malate synthase (MAS1), were significantly more expressed (17%, 42%, 24%, and 18% respectively), which was consistent with reported heterotrophic metabolism flux analysis with the objective function maximizing biomass. Photosynthetic F v /F m was slightly reduced. A more meticulous analysis is necessary, but, in the transgenic microalgae with malate synthase overexpression, the metabolism is likely to more rely on heterotrophic energy production via TCA cycle and glyoxylate shunt than on photosynthesis, resulting in the increase in microalgal biomass. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Polymalic acid (PMA) is a water-soluble biopolymer produced by the yeast-like fungus Aureobasidium pullulans. In this study, the physiological response of A. pullulans against exogenous alcohols stress was investigated. Interestingly, ethanol stress was an effective inducer of enhanced PMA yield, although cell growth was slightly inhibited. The stress-responsive gene malate synthase ( mls ), which is involved in the glyoxylate shunt, was identified and was found to be regulated by exogenous ethanol stress. Therefore, an engineered strain, YJ-MLS, was constructed by overexpressing the endogenous mls gene, which increased the PMA titer by 16.2% compared with the wild-type strain. Following addition of 1% (v/v) of ethanol, a high PMA titer of 40.0 ± 0.38 g/L was obtained using batch fermentation with the mutant YJ-MLS in a 5-L fermentor, with a strongest PMA productivity of 0.56 g/L h. This study was the interesting report to show strengthening of the carbon metabolic flow from the glyoxylate shunt for PMA synthesis, and also provided a new sight for re-recognizing the regulatory behavior of alcohol stress in eukaryotic microbes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 H 2 O 2 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 H 2 O 2 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Expression of rice malate synthase (OsMS), one of the two key genes in the glyoxylate cycle, is highly upregulated under salt stress. In this study, we aimed to investigate the role of OsMS in salt stress responses using the Arabidopsis T-DNA insertional mutant line of malate synthase (AtMS), an OsMS orthologous gene, for ectopic expression. Germination of the Atms mutant under salt stress was lower than that of Arabidopsis Col-0 wildtype (WT); furthermore, the two Atms mutant lines ectopically expressing OsMS reversed the salt-sensitive phenotype. Atms mutants salt-treated for 3 days exhibited higher electrolyte leakage, higher Na+/K+ ratio, lower expression of stress-responsive genes, and lower soluble sugar content than WT and the two OsMS -expressing Atms mutant lines. Consistently, Atms mutants salt-treated for 3 days followed by a 5-day recovery period displayed greater fresh-weight reduction. Notably, leaf greenness and chlorophyll and total carotenoid contents were higher in the Atms mutant lines than in the WT under stress. OsMS -expressing Atms mutants exhibited a change in pigment content closer to that of WT. During dark-induced senescence, an Atms mutant, Aticl , mutant (the other key gene in the glyoxylate cycle), and three double mutant lines of Atms and Aticl exhibited lower decreases in leaf greenness than the WT and OsMS -expressing Atms mutant lines. Furthermore, SAG12 expression levels in the Atms mutant, Aticl mutant, and three double mutant lines were lower than those in OsMS -expressing Atms mutant lines. Altogether, our data indicate that OsMS likely plays a key role in salt stress responses, possibly through the induction of leaf senescence. [ABSTRACT FROM AUTHOR]

Fragment screening and high throughput screening are complementary approaches that combine with structural biology to explore the binding capabilities of an active site. We have used a fragment-based approach on malate synthase (GlcB) from Mycobacterium tuberculosis and discovered several novel binding chemotypes. In addition, the crystal structures of GlcB in complex with these fragments indicated conformational changes in the active site that represent the enzyme conformations during catalysis.Additional structures of the complexwith malate and of the apo form of GlcB supported that hypothesis. Comparative analysis of GlcB structures in complex with 18 fragments allowed us to characterize the preferred chemotypes and their binding modes. The fragment structures showed a hydrogen bond to the backbone carbonyl of Met-631. We successfully incorporated an indole group from a fragment into an existing phenyl-diketo acid series. The resulting indole-containing inhibitor was 100-foldmore potent than the parent phenyl-diketo acid with an IC50 value of 20 nM. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary Elicitation of CD4 IFN-gamma T cell responses to Mycobacterium tuberculosis (MTB) is a rational vaccine strategy to prevent clinical tuberculosis. Diagnosis of MTB infection is based on T-cell immune memory to MTB antigens. The MTB proteome contains over four thousand open reading frames (ORFs). We conducted a pilot antigen identification study using 164 MTB proteins and MTB-specific T-cells expanded in vitro from 12 persons with latent MTB infection. Enrichment of MTB-reactive T-cells from PBMC used cell sorting or an alternate system compatible with limited resources. MTB proteins were used as single antigens or combinatorial matrices in proliferation and cytokine secretion readouts. Overall, our study found that 44 MTB proteins were antigenic, including 27 not previously characterized as CD4 T-cell antigens. Antigen truncation, peptide, NTM homology, and HLA class II tetramer studies confirmed malate synthase G (encoded by gene Rv1837) as a CD4 T-cell antigen. This simple, scalable system has potential utility for the identification of candidate MTB vaccine and biomarker antigens. [ABSTRACT FROM AUTHOR]

Members of the DRE-TIM metallolyase superfamily rely on an active-site divalent cation to catalyze various reactions involving the making and breaking of carbon–carbon bonds. While the identity of the metal varies, the binding site is well-conserved at the superfamily level with an aspartic acid and two histidine residues acting as ligands to the metal. Previous structural and bioinformatics results indicate that the metal can adopt an alternate architecture through the addition of an asparagine residue as a fourth ligand. This asparagine residue is strictly conserved in all members of the DRE-TIM metallolyase superfamily except fungal homocitrate synthase (HCS-lys) where it is replaced with isoleucine. The role of this additional metal ligand in α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been investigated using site-directed mutagenesis. Substitution of the asparagine ligand with alanine or isoleucine results in inactive enzymes with respect to α-isopropylmalate formation. Control experiments suggest that the substitutions have not drastically affected the enzyme's structure indicating that the asparagine residue is essential for catalysis. Interestingly, all enzyme variants retained acetyl CoA hydrolysis activity in the absence of α-ketoisovalerate, similar to the wild-type enzyme. In contrast to the requirement of magnesium for α-isopropylmalate formation, hydrolytic activity could be inhibited by the addition of magnesium chloride in wild-type, D81E, and N321A MtIPMS, but not in the other variants studied. Attempts to rescue loss of activity in N321I MtIPMS by mimicking the fungal HCS active site through the D81E/N321I double variant were unsuccessful. This suggests epistatic constraints in evolution of function in IPMS and HCS-lys enzymes. [ABSTRACT FROM AUTHOR]

Abstract: Peroxisomes play a crucial role in regulating cellular metabolism, providing compartments where metabolic pathways can be contained and controlled. Their importance is underlined by the developmental brain disorders caused by peroxisome malfunction, while disturbances in peroxisome function also contribute to ageing. As peroxisomes do not contain DNA, they rely on an active transport system to obtain the full quota of proteins required for function. Organelle protein transport however, is rarely a one-way process and exciting recent data have demonstrated that peroxisomes can selectively export membrane and matrix proteins to fulfil specific functions. This review will summarise the current knowledge on peroxisomal membrane and matrix protein export, discussing the mechanisms underlying export as well as the role of peroxisomal protein export in peroxisomal and cellular function. [Copyright &y& Elsevier]

Highlights: [•] 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. [Copyright &y& Elsevier]

Abstract: In this study, we synthesized (±)-tylophorine malate (NK-007), an analog of tylophorine (DCB3503), and analyzed its anti-inflammatory effect in vivo using a dextran sulfate sodium (DSS)-induced colitis model and an acetic acid-induced colitis model. As indicated by disease activity index (DAI) and degree of macroscopic colonic damage, NK-007 can significantly suppress colitis. To delineate the underlying mechanism, we have explored the influence of NK-007 on the production of TNF-α by murine primary bone marrow-derived dendritic cells (BMDCs) as well as monocyte/macrophage cell line Raw 264.7 triggered by lipopolysaccharide (LPS). For both types of innate immune cells, NK-007 showed a potent TNF-α inhibitory effect, and has in addition reduced the expression of IL-12 in BMDCs. Moreover, Raw cells treated with NK-007 also showed decreased phosphorylation of NF-κB, which may explain the protective immune-regulatory effect of NK-007 for experimental colitis. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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

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

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

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

• T. circumcincta isocitrate lyase (Tc ICL) gene encoded 444 amino acid proteins. • T. circumcincta malate synthase (Tc MS) gene encoded 525 amino acid proteins. • Combined bifunctional Tc ICL-MS showed 70–90 % similarity to other nematode sequences. • Antibodies in both serum and saliva from field-immune sheep recognised recombinant TcICL. A 1332 bp full length cDNA encoding Teladorsagia circumcincta isocitrate lyase (Tci ICL) and a 1575 bp full length cDNA encoding T. circumcincta malate synthase (Tci MS) were cloned, expressed in Escherichia coli and the recombinant proteins purified. The predicted Tci ICL protein of 444 amino acids was present as a single band of about 52 kDa on SDS-PAGE and the recombinant Tci MS of 525 amino acids formed a single band about 62 kDa. Multiple alignments of the combined bifunctional Tci ICL-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 Tci ICL and Tci MS and shown to be highly conserved. Substrate and metal ion binding sites were identified and were completely conserved in other homologues. Tci ICL was confirmed as a functional enzyme. At 30 °C, the optimum pH was pH 7.5, the V max was 275 ± 23 nmoles.min−1. mg-1 protein and the apparent K m 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 Tci ICL activity by 60–90 %. Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant Tci ICL in ELISA, supporting similar antigenicity to that of the native enzyme. [ABSTRACT FROM AUTHOR]