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Biodegradation of 1,4-dioxane (dioxane) contamination has gained much attention for decades. In our previous work, we isolated a highly efficient dioxane degrader, Xanthobacter sp. YN2, but the underlying mechanisms of its extraordinary degradation performance remained unresolved. In this study, we performed a comparative transcriptome analysis of YN2 grown on dioxane and citrate to elucidate its genetic degradation mechanism and investigated the transcriptomes of different dioxane degradation stages (T0, T24, T48). We also analyzed the transcriptional response of YN2 over time during which the carbon source switched from citrate to dioxane. The results indicate that strain YN2 was a methylotroph, which provides YN2 a major advantage as a pollutant degrader. A large number of genes involved in dioxane metabolism were constitutively expressed prior to dioxane exposure. Multiple genes related to the catabolism of each intermediate were upregulated by treatment in response to dioxane. Glyoxylate metabolism was essential during dioxane degradation by YN2, and the key intermediate glyoxylate was metabolized through three routes: glyoxylate carboligase pathway, malate synthase pathway, and anaplerotic ethylmalonyl–CoA pathway. Genes related to quorum sensing and transporters were significantly upregulated during the early stages of degradation (T0, T24) prior to dioxane depletion, while the expression of genes encoding two-component systems was significantly increased at late degradation stages (T48) when total organic carbon in the culture was exhausted. This study is the first to report the participation of genes encoding glyoxalase, as well as methylotrophic genes xoxF and mox, in dioxane metabolism. The present study reveals multiple genetic and transcriptional strategies used by YN2 to rapidly increase biomass during growth on dioxane, achieve high degradation efficiency and tolerance, and adapt to dioxane exposure quickly, which provides useful information regarding the molecular basis for efficient dioxane biodegradation.

Background Emergence of Candida glabrata, which causes potential life-threatening invasive candidiasis, has been widely associated with high morbidity and mortality. In order to cause disease in vivo, a robust and highly efficient metabolic adaptation is crucial for the survival of this fungal pathogen in human host. In fact, reprogramming of the carbon metabolism is believed to be indispensable for phagocytosed C. glabrata within glucose deprivation condition during infection. Methods In this study, the metabolic responses of C. glabrata under acetate growth condition was explored using high-throughput transcriptomic and proteomic approaches. Results Collectively, a total of 1482 transcripts (26.96%) and 242 proteins (24.69%) were significantly up- or down-regulated. Both transcriptome and proteome data revealed that the regulation of alternative carbon metabolism in C. glabrata resembled other fungal pathogens such as Candida albicans and Cryptococcus neoformans, with up-regulation of many proteins and transcripts from the glyoxylate cycle and gluconeogenesis, namely isocitrate lyase (ICL1), malate synthase (MLS1), phosphoenolpyruvate carboxykinase (PCK1) and fructose 1,6-biphosphatase (FBP1). In the absence of glucose, C. glabrata shifted its metabolism from glucose catabolism to anabolism of glucose intermediates from the available carbon source. This observation essentially suggests that the glyoxylate cycle and gluconeogenesis are potentially critical for the survival of phagocytosed C. glabrata within the glucose-deficient macrophages. Conclusion Here, we presented the first global metabolic responses of C. glabrata to alternative carbon source using transcriptomic and proteomic approaches. These findings implicated that reprogramming of the alternative carbon metabolism during glucose deprivation could enhance the survival and persistence of C. glabrata within the host.

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.

Mycobacterium tuberculosis remains one of the most successful bacterial pathogens worldwide. The development of drug-resistant strains and the ability of the bacteria to persist in a latent form in the host are major problems for tuberculosis (TB) control. Glyoxylate shunt is a metabolic bypass of the Krebs cycle and is the key for M. tuberculosis to survive under latent conditions. Malate synthase (MtbMS) catalyzes the second step of the glyoxylate cycle and converts glyoxylate into malate. Phenyl-diketo acid (PDKA) is a potent inhibitor of MtbMS, and its efficacy is validated in a mouse model of TB. To identify novel PDKA analogs as anti-TB compounds, PDKA analogs that obeyed the Lipinski rules (n = 5473) were analyzed and docked with MtbMS structure in three sequential modes. These compounds were then assessed for ADMET parameters. Of the compounds examined, 19 were found to fit well for redocking studies. After optimization, four prospective inhibitors were identified, that along with the reference compound PDKA were subjected to 50 ns molecular dynamics simulation and binding-free energy analyses to evaluate the complex dynamics after ligand binding, the stability of the bound complexes, and the intermolecular interactions between the complexes. The MtbMS-PDKA complex showed the binding free energy of –57.16 kJ·mol−1. After a thorough analysis, our results suggested that three compounds which had binding-free energy of –127.96, −97.60, and –83.98 kJ·mol−1, with PubChem IDs 91937661, 14016246, and 126487337, respectively, have the potential to inhibit MtbMS and can be taken as lead compounds for drug discovery against TB. Communicated by Ramaswamy H. Sarma

Persistent infection by Mycobacterium tuberculosis requires the glyoxylate shunt. This is a bypass to the tricarboxylic acid cycle in which isocitrate lyase (ICL) and malate synthase (MS) catalyze the net incorporation of carbon during mycobacterial growth on acetate or fatty acids as the primary carbon source. To identify a potential antitubercular compound, we performed a structure-based screening of natural compounds from the ZINC database (n = 1 67 740) against the M tuberculosis MS (MtbMS) structure. The ligands were screened against MtbMS, and 354 ligands were found to have better docking score. These compounds were assessed for Lipinski and absorption, distribution, metabolism, excretion, and toxicity prediction where 15 compounds were found to fit well for redocking studies. After refinement by molecular docking and drug-likeness analysis, four potential inhibitors (ZINC1483899, ZINC1754310, ZINC2269664, and ZINC15729522) were identified. These four ligands with phenyl-diketo acid were further subjected to molecular dynamics simulation to compare the dynamics and stability of the protein structure after ligand binding. The binding energy analysis was calculated to determine the intermolecular interactions. Our results suggested that the four compounds had a binding free energy of -201.96, -242.02, -187.03, and -169.02 kJ·mol-1 , for compounds with IDs ZINC1483899, ZINC1754310, ZINC2269664, and ZINC15729522, respectively. We concluded that two compounds (ZINC1483899 and ZINC1754310) displayed considerable structural and pharmacological properties and could be probable drug candidates to fight against M tuberculosis parasites.

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.

Bacteria have remarkable mechanisms to survive severe external stresses, and one of the most enigmatic is the nonreplicative persistent (NRP) state. Practically, NRP bacteria are difficult to treat, and so inhibiting the proteins underlying this survival state may render such bacteria more susceptible to external stresses, including antibiotics. Unfortunately, we know little about the proteins and mechanisms conferring survival through the NRP state. Here, we report that a universal stress protein (Usp) is a primary regulator of bacterial survival through the NRP state in Micrococcus luteus NCTC 2665, a biosafety level 1 (BSL1) mycobacterial relative. Usps are widely conserved, and bacteria, including Mycobacterium tuberculosis, Mycobacterium smegmatis, and Escherichia coli, have multiple paralogs with overlapping functions that have obscured their functional roles. A kanamycin resistance cassette inserted into the M. luteus universal stress protein A 616 gene (ΔuspA616::kanM. luteus) ablates the UspA616 protein and drastically impairs M. luteus survival under even short-term starvation (survival, 83% wild type versus 32% ΔuspA616::kanM. luteus) and hypoxia (survival, 96% wild type versus 48% ΔuspA616::kanM. luteus). We observed no detrimental UspA616 knockout phenotype in logarithmic growth. Proteomics demonstrated statistically significant log-phase upregulation of glyoxylate pathway enzymes isocitrate lyase and malate synthase in ΔuspA616::kanM. luteus. We note that these enzymes and the M. tuberculosis UspA616 homolog (Rv2623) are important in M. tuberculosis virulence and chronic infection, suggesting that Usps are important stress proteins across diverse bacterial species. We propose that UspA616 is a metabolic switch that controls survival by regulating the glyoxylate shunt. IMPORTANCE Bacteria tolerate severe external stresses, including antibiotics, through a nonreplicative persistent (NRP) survival state, yet the proteins regulating this survival state are largely unknown. We show a specific universal stress protein (UspA616) controls the NRP state in Micrococcus luteus. Usps are widely conserved across bacteria, but their biological function(s) has remained elusive. UspA616 inactivation renders M. luteus susceptible to stress: bacteria die instead of adapting through the NRP state. UspA616 regulates malate synthase and isocitrate lyase, glyoxylate pathway enzymes important for chronic Mycobacterium tuberculosis infection. These data show that UspA616 regulates NRP stress survival in M. luteus and suggest a function for homologous proteins in other bacteria. Importantly, inhibitors of UspA616 and homologs may render NRP bacteria more susceptible to stresses, including current antibiotics.

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.

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

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

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

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.

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.

Trans -aconitate methyltransferase regulator (TamR) is a member of the ligand-responsive multiple antibiotic resistance regulator (MarR) family of transcription factors. In Streptomyces coelicolor , TamR regulates transcription of tamR (encoding TamR), tam (encoding trans -aconitate methyltransferase) and sacA (encoding aconitase); up-regulation of these genes promotes metabolic flux through the citric acid cycle. DNA binding by TamR is attenuated and transcriptional derepression is achieved on binding of ligands such as citrate and trans -aconitate to TamR. In the present study, we show that three additional genes are regulated by S. coelicolor TamR. Genes encoding malate synthase ( aceB1 ; SCO6243 ), malate dehydrogenase ( mdh ; SCO4827 ) and isocitrate dehydrogenase ( idh ; SCO7000 ) are up-regulated in vivo when citrate and trans -aconitate accumulate, and TamR binds the corresponding gene promoters in vitro , a DNA binding that is attenuated by cognate ligands. Mutations to the TamR binding site attenuate DNA binding in vitro and result in constitutive promoter activity in vivo . The predicted TamR binding sites are highly conserved in the promoters of these genes in Streptomyce s species that encode divergent tam–tamR gene pairs, suggesting evolutionary conservation. Like aconitase and trans -aconitate methyltransferase, malate dehydrogenase, isocitrate dehydrogenase and malate synthase are closely related to the citric acid cycle, either catalysing individual reaction steps or, in the case of malate synthase, participating in the glyoxylate cycle to produce malate that enters the citric acid cycle to replenish the intermediate pool. Taken together, our data suggest that TamR plays an important and conserved role in promoting metabolic flux through the citric acid cycle. Abbreviations: EGFP, enhanced green fluorescent protein; EMSA, electrophoretic mobility shift assay(s); MarR, multiple antibiotic resistance regulator; qRT-PCR, quantitative reverse transcription–PCR; TamR, trans-aconitate methyltransferase regulator; TBE, Tris/borate/EDTA

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.

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

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.

International audience; Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to the systemic disease candidiasis. Its ability to adopt various morphological forms, such as unicellular yeasts, filamentous pseudohyphae and hyphae, contributes to its ability to survive within the host. It has been suggested that the antioxidant glutathione is involved in the filamentation process. We investigated S-glutathionylation, the reversible binding of glutathione to proteins, and the functional consequences on C. albicans metabolic remodeling during the yeast-to-hyphae transition. Our work provided evidence for the specific glutathionylation of mitochondrial proteins involved in bioenergetics pathways in filamentous forms and a regulation of the main enzyme of the glyoxylate cycle, isocitrate lyase, by glutathionylation. Isocitrate lyase inactivation in the hyphal forms was reversed by glutaredoxin treatment, in agreement with a glutathionylation process, which was confirmed by proteomic data showing the binding of one glutathione molecule to the enzyme (data are available via ProteomeXchange with identifier PXD003685). We also assessed the effect of alternative carbon sources on glutathione levels and isocitrate lyase activity. Changes in nutrient availability led to morphological flexibility and were related to perturbations in glutathione levels and isocitrate lyase activity, confirming the key role of the maintenance of intracellular redox status in the adaptive metabolic strategy of the pathogen.

Mycobacterium tuberculosis can maintain a nonreplicating persistent state in the host for decades, but must maintain the ability to efficiently reactivate and produce active disease to survive and spread in a population. Among the enzymes expressed during this dormancy is alanine dehydrogenase, which converts pyruvate to alanine, and glyoxylate to glycine concurrent with the oxidation of NADH to NAD. It is involved in the metabolic remodeling of M. tuberculosis through its possible interactions with both the glyoxylate and methylcitrate cycle. Both mRNA levels and enzymatic activities of isocitrate lyase, the first enzyme of the glyoxylate cycle, and alanine dehydrogenase increased during entry into nonreplicating persistence, while the gene and activity for the second enzyme of the glyoxylate cycle, malate synthase were not. This could suggest a shift in carbon flow away from the glyoxylate cycle and instead through alanine dehydrogenase. Expression of ald was also induced in vitro by other persistence-inducing stresses such as nitric oxide, and was expressed at high levels in vivo during the initial lung infection in mice. Enzyme activity was maintained during extended hypoxia even after transcription levels decreased. An ald knockout mutant of M. tuberculosis showed no reduction in anaerobic survival in vitro, but resulted in a significant lag in the resumption of growth after reoxygenation. During reactivation the ald mutant had an altered NADH/NAD ratio, and alanine dehydrogenase is proposed to maintain the optimal NADH/NAD ratio during anaerobiosis in preparation of eventual regrowth, and during the initial response during reoxygenation.

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

The flow of carbon metabolites between cellular compartments is an essential feature of fungal metabolism. During growth on ethanol, acetate, or fatty acids, acetyl units must enter the mitochondrion for metabolism via the tricarboxylic acid cycle, and acetyl coenzyme A (acetyl-CoA) in the cytoplasm is essential for the biosynthetic reactions and for protein acetylation. Acetyl-CoA is produced in the cytoplasm by acetyl-CoA synthetase during growth on acetate and ethanol while β-oxidation of fatty acids generates acetyl-CoA in peroxisomes. The acetyl-carnitine shuttle in which acetyl-CoA is reversibly converted to acetyl-carnitine by carnitine acetyltransferase (CAT) enzymes is important for intracellular transport of acetyl units. In the filamentous ascomycete Aspergillus nidulans , a cytoplasmic CAT, encoded by facC , is essential for growth on sources of cytoplasmic acetyl-CoA while a second CAT, encoded by the acuJ gene, is essential for growth on fatty acids as well as acetate. We have shown that AcuJ contains an N-terminal mitochondrial targeting sequence and a C-terminal peroxisomal targeting sequence (PTS) and is localized to both peroxisomes and mitochondria, independent of the carbon source. Mislocalization of AcuJ to the cytoplasm does not result in loss of growth on acetate but prevents growth on fatty acids. Therefore, while mitochondrial AcuJ is essential for the transfer of acetyl units to mitochondria, peroxisomal localization is required only for transfer from peroxisomes to mitochondria. Peroxisomal AcuJ was not required for the import of acetyl-CoA into peroxisomes for conversion to malate by malate synthase (MLS), and export of acetyl-CoA from peroxisomes to the cytoplasm was found to be independent of FacC when MLS was mislocalized to the cytoplasm.

Pseudomonas aeruginosa causes chronic pulmonary infections, which can persist for decades, in patients with cystic fibrosis (CF). Current evidence suggests that the glyoxylate pathway is an important metabolic pathway for P. aeruginosa growing within the CF lung. In this study, we identified glcB, which encodes for the second key enzyme of the glyoxylate pathway, malate synthase, as a requirement for virulence of P. aeruginosa on alfalfa seedlings. While expression of glcB in PAO1, an acute isolate of P. aeruginosa, responds to some carbon sources that use the glyoxylate pathway, expression of glcB in FRD1, a CF isolate, is constitutively upregulated. Malate synthase activity is moderately affected by glcB expression and is nearly constitutive in both backgrounds, with slightly higher activity in FRD1 than in PAO1. In addition, RpoN negatively regulates glcB in PAO1 but not in FRD1. In summary, the genes encoding for the glyoxylate-specific enzymes appear to be coordinately regulated, even though they are not located within the same operon on the P. aeruginosa genome. Furthermore, both genes encoding for the glyoxylate enzymes can become deregulated during adaptation of the bacterium to the CF lung.

Assimilation of acetyl coenzyme A (acetyl-CoA) is an essential process in many bacteria that proceeds via the glyoxylate cycle or the ethylmalonyl-CoA pathway. In both assimilation strategies, one of the final products is malate that is formed by the condensation of acetyl-CoA with glyoxylate. In the glyoxylate cycle this reaction is catalyzed by malate synthase, whereas in the ethylmalonyl-CoA pathway the reaction is separated into two proteins: malyl-CoA lyase, a well-known enzyme catalyzing the Claisen condensation of acetyl-CoA with glyoxylate and yielding malyl-CoA, and an unidentified malyl-CoA thioesterase that hydrolyzes malyl-CoA into malate and CoA. In this study the roles of Mcl1 and Mcl2, two malyl-CoA lyase homologs in Rhodobacter sphaeroides , were investigated by gene inactivation and biochemical studies. Mcl1 is a true (3 S )-malyl-CoA lyase operating in the ethylmalonyl-CoA pathway. Notably, Mcl1 is a promiscuous enzyme and catalyzes not only the condensation of acetyl-CoA and glyoxylate but also the cleavage of β-methylmalyl-CoA into glyoxylate and propionyl-CoA during acetyl-CoA assimilation. In contrast, Mcl2 was shown to be the sought (3 S )-malyl-CoA thioesterase in the ethylmalonyl-CoA pathway, which specifically hydrolyzes (3 S )-malyl-CoA but does not use β-methylmalyl-CoA or catalyze a lyase or condensation reaction. The identification of Mcl2 as thioesterase extends the enzyme functions of malyl-CoA lyase homologs that have been known only as “Claisen condensation” enzymes so far. Mcl1 and Mcl2 are both related to malate synthase, an enzyme which catalyzes both a Claisen condensation and thioester hydrolysis reaction.

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

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.

Motivation: In recent years, several methods have been proposed for determining metabolic pathways in an automated way based on network topology. The aim of this work is to analyse these methods by tackling a concrete example relevant in biochemistry. It concerns the question whether even-chain fatty acids, being the most important constituents of lipids, can be converted into sugars at steady state. It was proved five decades ago that this conversion using the Krebs cycle is impossible unless the enzymes of the glyoxylate shunt (or alternative bypasses) are present in the system. Using this example, we can compare the various methods in pathway analysis. Results: Elementary modes analysis (EMA) of a set of enzymes corresponding to the Krebs cycle, glycolysis and gluconeogenesis supports the scientific evidence showing that there is no pathway capable of converting acetyl-CoA to glucose at steady state. This conversion is possible after the addition of isocitrate lyase and malate synthase (forming the glyoxylate shunt) to the system. Dealing with the same example, we compare EMA with two tools based on graph theory available online, PathFinding and Pathway Hunter Tool. These automated network generating tools do not succeed in predicting the conversions known from experiment. They sometimes generate unbalanced paths and reveal problems identifying side metabolites that are not responsible for the carbon net flux. This shows that, for metabolic pathway analysis, it is important to consider the topology (including bimolecular reactions) and stoichiometry of metabolic systems, as is done in EMA. Contact: ldpf@minet.uni-jena.de; schuster@minet.uni-jena.de Supplementary information: Supplementary data are available at Bioinformatics online.

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.

An efficient Agrobacterium tumefaciens -mediated gene-disruption method was developed for Sclerotinia sclerotiorum. As proof of principle, a transformation vector containing the full-length sequence of the glyoxylate cycle malate synthase gene (mls1) disrupted by gene cassette encoding resistance to hygromycin B was used as a test case for gene disruption. Gene disruption (knockout) mutants were generated by introducing the disruption construct into ascospores, selecting transformants on hygromycin-amended medium, and by rigorous hyphal tip purification of transformants. This procedure yielded stable, hygromycin-resistant transformants, and DNA blot hybridization analysis demonstrated a high frequency of gene disruption by homologous recombination. Defective function of the glyoxylate cycle and loss of pathogenicity in the knockout mutants was also demonstrated. Key words: Agrobacterium tumefaciens -mediated transformation, gene disruption, glyoxylate cycle, malate synthase, pathogenicity, Sclerotinia scl...

D-amino acid oxidases play an important role in converting D-amino acids to their corresponding α-keto acids. MRA_1916 of Mycobacterium tuberculosis H37Ra (Mtb-Ra) is annotated to be a D-amino acid oxidase (DAO). However, not much information is available about its physiological role during Mtb-Ra growth and survival. The present study was taken-up to understand the role of DAO during different stages of growth and effect of its down-regulation on growth. Recombinant Mtb-Ra strains with DAO and GlcB (malate synthase: MRA_1848) gene knockdown were developed and their growth was studied using Microtiter Alamar Blue Assay (MABA) with glycerol, acetate and glycine as a carbon source. Ethyl bromopyruvate (BrP) was used as an inhibitor of GlcB. MABA study showed inhibition of wild-type (WT) and knockdowns in the presence of BrP (2.5mM). However, growth inhibition of WT was less noticeable at lower concentrations of BrP. Mtb-Ra with DAO knockdown showed poor utilization of glycine in the presence of BrP. The DAO localization study showed its prominent distribution in cytosolic fraction and to some extent in cell wall and membrane fractions. Growth profile of WT under oxygen and nutritional stress showed changes in expression of DAO, GlcB, PckA (phosphoenolpyruvate carboxykinase: MRA_0219) and GlyA1 (serine hydroxymethyltransferase: MRA_1104).

Qualitative patterns of gene activation and repression are often conserved despite an abundance of quantitative variation in expression levels within and between species. A major challenge to interpreting patterns of expression divergence is knowing which changes in gene expression affect fitness. To characterize the fitness effects of gene expression divergence we placed orthologous promoters from eight yeast species upstream of malate synthase (MLS1) in Saccharomyces cerevisiae. As expected, we found these promoters varied in their expression level under activated and repressed conditions as well as in their dynamic response following loss of glucose repression. Despite these differences, only a single promoter driving near basal levels of expression caused a detectable loss of fitness. We conclude that the MLS1 promoter lies on a fitness plateau whereby even large changes in gene expression can be tolerated without a substantial loss of fitness.

Cyanobacteria are important photoautotrophic bacteria with extensive but variable metabolic capacities. The existence of the glyoxylate cycle, a variant of the TCA cycle, is still poorly documented in cyanobacteria. Previous studies reported the activities of isocitrate lyase and malate synthase, the key enzymes of the glyoxylate cycle in some cyanobacteria, but other studies concluded that these enzymes are missing. In this study the genes encoding isocitrate lyase and malate synthase from Chlorogloeopsis fritschii PCC 9212 were identified, and the recombinant enzymes were biochemically characterized. Consistent with the presence of the enzymes of the glyoxylate cycle, C. fritschii could assimilate acetate under both light and dark growth conditions. Transcript abundances for isocitrate lyase and malate synthase increased, and C. fritschii grew faster, when the growth medium was supplemented with acetate. Adding acetate to the growth medium also increased the yield of poly-3-hydroxybutyrate. When the genes encoding isocitrate lyase and malate synthase were expressed in Synechococcus sp. PCC 7002, the acetate assimilation capacity of the resulting strain was greater than that of wild type. Database searches showed that the genes for the glyoxylate cycle exist in only a few other cyanobacteria, all of which are able to fix nitrogen. This study demonstrates that the glyoxylate cycle exists in a few cyanobacteria, and that this pathway plays an important role in the assimilation of acetate for growth in one of those organisms. The glyoxylate cycle might play a role in coordinating carbon and nitrogen metabolism under conditions of nitrogen fixation. Background: Conflicting claims exist concerning the occurrence of the glyoxylate cycle in cyanobacteria. Results: The genes for isocitrate lyase and malate synthase were identified in Chlorogleopsis fritschii PCC 9212 and the purified enzymes were characterized. Conclusion: C. fritschii has a functional glyoxylate cycle and can grow in the dark on acetate. Significance: These results clarify the occurrence of the glyoxylate cycle in cyanobacteria.

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

Mycobacterium tuberculosis (M. tb) uses the glyoxalate bypass for intracellular survival in vivo. These studies provide evidence that the M. tb malate synthase (MS) has adapted to function as an adhesin which binds to laminin and fibronectin. This binding is achieved via the unique C-terminal region of the M. tb MS. The ability to function as an adhesin necessitates extracellular localization. We provide evidence that despite the absence of a Sec-translocation signal sequence the M. tb MS is secreted/excreted, and is anchored on the cell wall by an undefined mechanism. The MS of Mycobacterium smegmatis is cytoplasmic but the M. tb MS expressed in M. smegmatis localizes to the cell wall and enhances the adherence of the bacteria to lung epithelial A549 cells. Antibodies to the C-terminal laminin/fibronectin-binding domain interfere with the binding of the M. tb MS to laminin and fibronectin and reduce the adherence of M. tb to A549 cells. Coupled to the earlier evidence of in vivo expression of M. tb MS during active but not latent infection in humans, these studies show that a housekeeping enzyme of M. tb contributes to its armamentarium of virulence promoting factors.

In Corynebacterium glutamicum , the acetate-activating enzymes phosphotransacetylase and acetate kinase and the glyoxylate cycle enzymes isocitrate lyase and malate synthase are coordinately up-regulated in the presence of acetate in the growth medium. This regulation is due to transcriptional control of the respective pta-ack operon and the aceA and aceB genes, brought about at least partly by the action of the negative transcriptional regulator RamB. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, mass spectrometry, and peptide mass fingerprinting, we identified a LuxR-type transcriptional regulator, designated RamA, which binds to the pta-ack and aceA/aceB promoter regions. Inactivation of the ramA gene in the genome of C. glutamicum resulted in mutant RG2. This mutant was unable to grow on acetate as the sole carbon and energy source and, in comparison to the wild type of C. glutamicum , showed very low specific activities of phosphotransacetylase, acetate kinase, isocitrate lyase, and malate synthase, irrespective of the presence of acetate in the medium. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. By electrophoretic mobility shift analysis, purified His-tagged RamA protein was shown to bind specifically to the pta-ack and the aceA/aceB promoter regions, and deletion and mutation studies revealed in both regions two binding motifs each consisting of tandem A/C/TG 4-6 T/C or AC 4-5 A/G/T stretches separated by four or five arbitrary nucleotides. Our data indicate that RamA represents a novel LuxR-type transcriptional activator of genes involved in acetate metabolism of C. glutamicum .

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

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.

The relative levels of translatable messenger RNA for isocitrate lyase and malate synthase were determined in the dry seed and for the first seven days of development of cucumber cotyledons. After extraction and quantification of total and poly(A)-rich RNA each day, the RNA fractions were translated in an optimized wheat germ system and the specific polypeptides were immunoprecipitated quantitatively. The radiolabeled isocitrate lyase and malate synthase polypeptides were then fractionated on dodecylsulphate/polyacrylamide gels, visualized by exposure to X-ray film and quantified densitometrically. The relative levels of translatable messenger RNA for these enzymes rise and fall with a developmental program similar to the enzyme activities, but preceding the latter by about one day. This implies that the rise in enzyme activity is dependent upon a prior postgerminative increase in translatable messenger RNA for the enzymes. These studies also suggest that messenger RNA levels may be regulated, at least in part, by light.

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

A gene encoding malate synthase, a key enzyme of the glyoxylate cycle, has been cloned and characterized in the necrotrophic wheat pathogen Stagonospora nodorum. Expression studies of Mls1 showed high levels of transcript in ungerminated spores whereas malate synthase enzyme activities were low. Expression studies in planta found that Mls1 transcript levels decreased approximately 10-fold upon germination before slowly increasing throughout the remainder of the infection. To characterize Mls1 further, the gene was disrupted in S. nodorum by homologous recombination. In the absence of any supplied carbon source, the mls1 spores were unable to germinate and consequently the mutants were non-pathogenic. Germination and pathogenicity could be restored by the addition of either glucose or sucrose, implying that S. nodorum is reliant upon the catabolism of lipids for infection. Furthermore, analysis of lipid bodies in the mutant strain indicated that lipid mobilization and, consequently, peroxisomal beta-oxidation of fatty acids is delayed or inhibited by the disruption of the glyoxylate cycle. This study has demonstrated for the first time in a fungal phytopathogen the requirement of malate synthase for pathogenicity, suggesting that gluconeogenesis is both dependent on the glyoxylate cycle and required for infection.

A corynebacterial clone, previously isolated by scoring repression of lacZYA fused to the aceB promoter of Corynebacterium glutamicum , was analyzed further. In the clone, an open reading frame designated glxR , consisting of 681 nucleotides and encoding a 24,957-Da protein, was found. The molecular mass of a native GlxR protein was estimated by gel filtration column chromatography to be 44,000 Da, suggesting that the protein formed dimers. The predicted amino acid sequence contained both cyclic AMP (cAMP)- and DNA-binding motifs and was homologous with the cAMP receptor protein family of proteins. The aceB -repressing activity of the glxR clone was markedly relieved in an Escherichia coli cya mutant, but the activity was restored in growth medium containing cAMP. In glucose medium, the intracellular cAMP concentration of C. glutamicum reached 22 nmol/mg of protein in the early exponential phase and then decreased further; but in acetate medium, the intracellular cAMP concentration was only 5 nmol/mg of protein and remained low throughout the growth phase. The expression of glxR was not affected by the carbon source. Binding of purified GlxR to the promoter region of aceB could be demonstrated only in the presence of cAMP. These data suggest that GlxR may form dimers which bind to the aceB promoter region in the presence of cAMP and repress the glyoxylate bypass genes.

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

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

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