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Lyase
Kinds of Lyase Terms modified by Lyase Selected AbstractsCutting Long Syntheses Short: Access to Non-Natural Tyrosine Derivatives Employing an Engineered Tyrosine Phenol LyaseADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 4 2010Birgit Seisser Abstract The chemical synthesis of 3-substituted tyrosine derivatives requires a minimum of four steps to access optically enriched material starting from commercial precursors. Attempting to short-cut the cumbersome chemical synthesis of 3-substituted tyrosine derivatives, a single step biocatalytic approach was identified employing the tyrosine phenol lyase from Citrobacter freundii. The enzyme catalyses the hydrolysis of tyrosine to phenol, pyruvate and ammonium as well as the reverse reaction, thus the formation of tyrosine from phenol, pyruvate and ammonium. Since the wild-type enzyme possessed a very narrow substrate spectrum, structure-guided, site-directed mutagenesis was required to change the substrate specificity of this CC bond forming enzyme. The best variant M379V transformed, for example, o -cresol, o -methoxyphenol and o -chlorophenol efficiently to the corresponding tyrosine derivatives without any detectable side-product. In contrast, all three phenol compounds were non-substrates for the wild-type enzyme. Employing the mutant, various L -tyrosine derivatives (3-Me, 3-OMe, 3-F, 3-Cl) were obtained with complete conversion and excellent enantiomeric excess (>97%) in just a single ,green' step starting from pyruvate and commercially available phenol derivatives. [source] Mixed Aromatic Acyloin Condensations with Recombinant Benzaldehyde Lyase: Synthesis of ,-Hydroxydihydrochalcones and Related ,-Hydroxy KetonesADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6-7 2003Monica Sanchez-Gonzalez Abstract Recombinant benzaldehyde lyase (BAL), expressed and purified from E.,coli strain JM-109, was used to catalyze the condensation of a series of methoxybenzaldehydes and phenylacetaldehyde in the synthesis of ,-(R)-hydroxydihydrochalcones. Enantiomerically pure 1-hydroxy-1,3-diphenylpropan-2-ones and o -anisoin were also obtained as products of the BAL reaction. The R absolute configurations of chiral centers were determined by CD spectroscopy. ,-(R)-Hydroxydihydrochalcones and 1-hydroxy-1,3-diphenylpropan-2-ones are valuable synthons for chemoenzymatic syntheses of flavonoids. This is the first synthesis of ,-(R)-hydroxydihydrochalcones by a microbial enzyme. [source] Pyruvate Formate Lyase (PFL) and PFL Activating Enzyme in the Chytrid Fungus Neocallimastix frontalis: A Free-Radical Enzyme System Conserved Across Divergent Eukaryotic Lineages,THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 4 2004GABRIEL GELIUS-DIETRICH ABSTRACT Fermentative formate production involves the activity of pyruvate formate lyase, an oxygen-sensitive enzyme that employs a glycyl radical in its reaction mechanism. While common among anaerobic prokaryotes, this enzyme has so far been found in only two distantly related eukaryotic lineages, anaerobic chytridiomycetes and chlorophytes. Sequence comparisons of homologues from the chytridiomycetes Piromyces and Neocallimastix, the chlorophyte Chlamydomonas, and numerous prokaryotes suggest a single, eubacterial origin of eukaryotic pyruvate formate lyases. Pyruvate formate lyase activating enzyme introduces the glycyl radical into the pyruvate formate lyase protein chain. We discovered this enzyme, which had not previously been reported from eukaryotes, in the same two eukaryotic lineages and show that it shares a similar evolutionary history to pyruvate formate lyase. Sequences with high homology to pyruvate formate lyase activating enzyme were identified in the genomes of the anaerobic protozoan parasites Trichomonas vaginalis, Entamoeba histolytica, and Giardia intestinalis. While the occurrence of pyruvate formate lyase activating enzyme together with pyruvate formate lyase in fungi and chlorophytes was to be expected, the target protein of a glycyl radical enzyme-activating enzyme in these protozoa remains to be identified. [source] Alteration of the Diastereoselectivity of 3-Methylaspartate Ammonia Lyase by Using Structure-Based MutagenesisCHEMBIOCHEM, Issue 13 2009Hans Raj Abstract 3-Methylaspartate ammonia-lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)-3-methylaspartic acid and (2S,3R)-3-methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3 proton of the respective stereoisomer to generate an enolate anion intermediate that is stabilized by coordination to the essential active-site MgII ion. The crystal structure of MAL in complex with (2S,3S)-3-methylaspartic acid suggests that Lys331 is the only candidate in the vicinity that can function as a general base catalyst. The structure of the complex further suggests that two other residues, His194 and Gln329, are responsible for binding the C4 carboxylate group of (2S,3S)-3-methylaspartic acid, and hence are likely candidates to assist the MgII ion in stabilizing the enolate anion intermediate. In this study, the importance of Lys331, His194, and Gln329 for the activity and stereoselectivity of MAL was investigated by site-directed mutagenesis. His194 and Gln329 were replaced with either an alanine or arginine, whereas Lys331 was mutated to a glycine, alanine, glutamine, arginine, or histidine. The properties of the mutant proteins were investigated by circular dichroism (CD) spectroscopy, kinetic analysis, and 1H NMR spectroscopy. The CD spectra of all mutants were comparable to that of wild-type MAL, and this indicates that these mutations did not result in any major conformational changes. Kinetic studies demonstrated that the mutations have a profound effect on the values of kcat and kcat/KM; this implicates Lys331, His194 and Gln329 as mechanistically important. The 1H NMR spectra of the amination and deamination reactions catalyzed by the mutant enzymes K331A, H194A, and Q329A showed that these mutants have strongly enhanced diastereoselectivities. In the amination direction, they catalyze the conversion of mesaconate to yield only (2S,3S)-3-methylaspartic acid, with no detectable formation of (2S,3R)-3-methylaspartic acid. The results are discussed in terms of a mechanism in which Lys331, His194, and Gln329 are involved in positioning the substrate and in formation and stabilization of the enolate anion intermediate. [source] Factors Mediating Activity, Selectivity, and Substrate Specificity for the Thiamin Diphosphate-Dependent Enzymes Benzaldehyde Lyase and Benzoylformate DecarboxylaseCHEMBIOCHEM, Issue 12 2006Michael Knoll Abstract Benzaldehyde lyase from Pseudomonas fluorescens and benzoylformate decarboxylase from Pseudomonas putida are homologous thiamin diphosphate-dependent enzymes that catalyze carboligase and carbolyase reactions. Both enzymes catalyze the formation of chiral 2-hydroxy ketones from aldehydes. However, the reverse reaction has only been observed with benzaldehyde lyase. Whereas benzaldehyde lyase is strictly R specific, the stereoselectivity of benzoylformate decarboxylase from P. putida is dependent on the structure and orientation of the substrate aldehydes. In this study, the binding sites of both enzymes were investigated by using molecular modelling studies to explain the experimentally observed differences in the activity, stereo- and enantioselectivity and substrate specificity of both enzymes. We designed a detailed illustration that describes the shape of the binding site of both enzymes and sufficiently explains the experimental effects observed with the wild-type enzymes and different variants. These findings demonstrate that steric reasons are predominantly responsible for the differences observed in the (R)-benzoin cleavage and in the formation of chiral 2-hydroxy ketones. [source] A New (R)-Hydroxynitrile Lyase from Prunus mume: Asymmetric Synthesis of Cyanohydrins.CHEMINFORM, Issue 6 2006Samik Nanda Abstract For Abstract see ChemInform Abstract in Full Text. [source] ChemInform Abstract: Novel Steroidal Vinyl Fluorides as Inhibitors of Steroid C17(20) Lyase.CHEMINFORM, Issue 20 2002Joseph P. Burkhart Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Inhibition of Histidine Ammonia Lyase by Heteroaryl-alanines and AcrylatesCHEMISTRY & BIODIVERSITY, Issue 5 2006Adrian Katona Abstract Histidine ammonia lyase (HAL) catalyzes the elimination of ammonia from the substrate to form (E)-urocanate. The interaction between HAL and acrylic acids or alanines substituted with heteroaryl groups in the , -position was investigated. These proved to be strong competitive inhibitors when the heteroaryl groups were furanyl, thiophenyl, benzofuranyl, and benzothiophenyl, carrying the alanyl or acrylic side chains either in 2 or 3 positions, with Ki values between 18 and 139,,M. The exception was (furan-3-yl)alanine which was found to be inert. Tryptophan and 1-methyltryptophan, as well as the corresponding acrylates (=prop-2-enoates), are strong mixed inhibitors of HAL. Theoretically, L -histidine can be dissected into 4-methyl-1H -imidazole and glycine. Whereas these two compounds separately are only very weak inhibitors of HAL, equimolar amounts of both show a Ki value of 1.7±0.09,mM which is to be compared with the Km value of 15.6,mM for the normal reaction. We conclude that 5-methyl-1H -imidazole and glycine mimic the substrate and occupy the active site of HAL in a similar orientation. [source] Proteomic analysis of liver cancer cells treated with 5-Aza-2,-deoxycytidine (AZA),DRUG DEVELOPMENT RESEARCH, Issue 1 2009Shujun Bai Abstract 5-Aza-2,-deoxycytidine (AZA) is a potent inhibitor of DNA methylation that exhibits anti-tumor activity in a variety of tumor cells via reactivation of tumor suppressor genes. However, few studies have been done on the biological and clinical significance of AZA in human hepatocellular carcinoma. To identify potential genes that may be aberrantly methylated and confer growth advantage to neoplastic cells and to better understand the molecular mechanism(s) underlying AZA anti-tumor activity, a proteomics approach was used to annotate global gene expression changes of HepG2 cell line pre- and post-treatment with AZA. A total of 56 differentially expressed proteins were identified by 2D gel analysis, 48 of which were up-regulated while the remaining 8 were down regulated. Among the identified proteins, eight of these showed marked changed proteins, including seven up-regulated proteins: glutathione S-transferase P, protein DJ-1, peroxiredoxin-2, UMP-CMP kinase, cytochrome c-type heme lyase, enhancer of rudimentary homolog, profilin-1, and one down-regulated protein, heat-shock protein ,,1. The possible implication of these proteins in hepatocarcinogenesis is discussed. We tested two up-regulated proteins, glutathione S-transferase P and peroxiredoxin-2, using RT-PCR and their expression was consistent with the results obtained in the protein level. Both of these genes were methylated when methylation-specific PCR was used against their promoter regions. Following treatment with AZA, the gene promoter regions were found to be unmethylated, concomitant with overexpression of the proteins compared to HepG2 cells without treatment. These data provide useful information in evaluating the therapeutic potential of AZA for the treatment of HCC. Drug Dev Res 69, 2009. © 2009 Wiley-Liss, Inc. [source] Pseudomonas fluorescens orchestrates a fine metabolic-balancing act to counter aluminium toxicityENVIRONMENTAL MICROBIOLOGY, Issue 6 2010Joseph Lemire Summary Aluminium (Al), an environmental toxin, is known to disrupt cellular functions by perturbing iron (Fe) homeostasis. However, Fe is essential for such metabolic processes as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, the two pivotal networks that mediate ATP production during aerobiosis. To counter the Fe conundrum induced by Al toxicity, Pseudomonas fluorescens utilizes isocitrate lyase and isocitrate dehydrogenase-NADP dependent to metabolize citrate when confronted with an ineffective aconitase provoked by Al stress. By invoking fumarase C, a hydratase devoid of Fe, this microbe is able to generate essential metabolites. To compensate for the severely diminished enzymes like Complex I, Complex II and Complex IV, the upregulation of a H2O-generating NADH oxidase enables the metabolism of citrate, the sole carbon source via a modified TCA cycle. The overexpression of succinyl-CoA synthetase affords an effective route to ATP production by substrate-level phosphorylation in the absence of O2. This fine metabolic balance enables P. fluorescens to survive the dearth of bioavailable Fe triggered by an Al environment, a feature that may have potential applications in bioremediation technologies. [source] Completing the hypusine pathway in PlasmodiumFEBS JOURNAL, Issue 20 2009Deoxyhypusine hydroxylase is an E-Z type HEAT repeat protein In searching for new targets for antimalarials we investigated the biosynthesis of hypusine present in eukaryotic initiation factor-5A (eIF-5A) in Plasmodium. Here, we describe the cloning and expression of deoxyhypusine hydroxylase (DOHH), which completes the modification of eIF-5A through hydroxylation of deoxyhypusine. The dohh cDNA sequence revealed an ORF of 1236 bp encoding a protein of 412 amino acids with a calculated molecular mass of 46.45 kDa and an isoelectric point of 4.96. Interestingly, DOHH from Plasmodium has a FASTA SCORE of only 27 compared with its human ortholog and contains several matches similar to E-Z-type HEAT-like repeat proteins (IPR004155 (InterPro), PF03130 (Pfam), SM00567 (SMART) present in the phycocyanin lyase subunits of cyanobacteria. Purified DOHH protein displayed hydroxylase activity in a novel in vitro DOHH assay, but phycocyanin lyase activity was absent. dohh is present as a single-copy gene and is transcribed in the asexual blood stages of the parasite. A signal peptide at the N-terminus might direct the protein to a different cellular compartment. During evolution, Plasmodium falciparum acquired an apicoplast that lost its photosynthetic function. It is possible that plasmodial DOHH arose from an E/F-type phycobilin lyase that gained a new role in hydroxylation. Structured digital abstract ,,MINT-7255047: DHS (uniprotkb:P49366) enzymaticly reacts (MI:0414) with eIF-5A (uniprotkb:Q710D1) by enzymatic studies (MI:0415) ,,MINT-7255326: DOHH (uniprotkb:Q8I701) enzymaticly reacts (MI:0414) with eIF-5A (uniprotkb:Q710D1) by enzymatic studies (MI:0415) [source] Two beta-alanyl-CoA:ammonia lyases in Clostridium propionicumFEBS JOURNAL, Issue 3 2005Gloria Herrmann The fermentation of ,-alanine by Clostridium propionicum proceeds via activation to the CoA-thiol ester, followed by deamination to acryloyl-CoA, which is also an intermediate in the fermentation of l -alanine. By shifting the organism from the carbon and energy source ,-alanine to ,-alanine, the enzyme ,-alanyl-CoA:ammonia lyase is induced 300-fold (, 30% of the soluble protein). The low basal lyase activity is encoded by the acl1 gene, whereas the almost identical acl2 gene (six amino acid substitutions) is responsible for the high activity after growth on ,-alanine. The deduced ,-alanyl-CoA:ammonia lyase proteins are related to putative ,-aminobutyryl-CoA ammonia lyases involved in lysine fermentation and found in the genomes of several anaerobic bacteria. ,-Alanyl-CoA:ammonia lyase 2 was purified to homogeneity and characterized as a heteropentamer composed of 16 kDa subunits. The apparent Km value for acryloyl-CoA was measured as 23 ± 4 µm, independent of the concentration of the second substrate ammonia; kcat/Km was calculated as 107 m,1·s,1. The apparent Km for ammonia was much higher, 70 ± 5 mm at 150 µm acryloyl-CoA with a much lower kcat/Km of 4 × 103 m,1·s,1. In the reverse reaction, a Km of 210 ± 30 µM was obtained for ,-alanyl-CoA. The elimination of ammonia was inhibited by 70% at 100 mm ammonium chloride. The content of ,-alanyl-CoA:ammonia lyase in ,-alanine grown cells is about 100 times higher than that required to sustain the growth rate of the organism. It is therefore suggested that the enzyme is needed to bind acryloyl-CoA, in order to keep the toxic free form at a very low level. A formula was derived for the calculation of isomerization equilibra between l -alanine/,-alanine or d -lactate/3-hydroxypropionate. [source] Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyaseFEBS JOURNAL, Issue 14 2002Tadayoshi Kanao The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl -ACL), an ,4,4 heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent Km value of 0.21 ± 0.04 mm. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (nH) of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 ± 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a Ki value of 0.037 ± 0.006 mm. Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl -ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl -ACL. His273 of the , subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme. However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA. [source] Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesisFEBS JOURNAL, Issue 12 2002Emine Mattern-Dogru In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the ,/, hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By site-directed mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the ,/, hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the ,/, hydrolase enzyme superfamily. [source] 2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulansFEBS JOURNAL, Issue 12 2001Characterization, comparison of both enzymes In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo -2-methylisocitrate; the erythro -diastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources (, 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mm pyruvate, 150 mm succinate and 10 µm PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved. [source] Identification of the aceA gene encoding isocitrate lyase required for the growth of Pseudomonas aeruginosa on acetate, acyclic terpenes and leucineFEMS MICROBIOLOGY LETTERS, Issue 2 2007Alma Laura Díaz-Pérez Abstract Pseudomonas aeruginosa PAO1 mutants affected in acyclic monoterpenes, n-octanol, and acetate assimilation were isolated using transposon mutagenesis. The isocitrate lyase gene (aceA) corresponding to ORF PA2634 of the PAO1 strain genome was identified in one of these mutants. The aceA gene encodes a protein that is 72% identical to the isocitrate lyase (ICL) characterized from Colwellia maris, but is less than 30% identical to their homologues from pseudomonads. The genetic arrangement of aceA suggests that it is a monocistronic gene, and no adjacent related genes were found. The ICL protein was detected as a 60-kDa band in sodium dodecyl sulfate polyacrylamide gel electrophoresis from cultures grown on acetate, but not in glucose-grown PAO1 cultures. Genetic complementation further confirmed that the aceA gene encodes the ICL enzyme. The ICL enzyme activity in crude extracts from cultures of the PAO1 strain was induced by acetate, citronellol and leucine, and repressed by growth on glucose or citrate. These results suggest that ICL is involved in the assimilation of acetate, acyclic monoterpenes of the citronellol family, alkanols, and leucine, in which the final intermediary acetyl-coenzyme A may be channelled to the glyoxylate shunt. [source] Two phenotypically compensating isocitrate dehydrogenases in Ralstonia eutrophaFEMS MICROBIOLOGY LETTERS, Issue 1 2003Zheng-Xiang Wang Abstract The tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase (IDH) and the glyoxylate bypass enzyme isocitrate lyase are involved in catabolism of isocitrate and play a key role in controlling the metabolic flux between the TCA cycle and the glyoxylate shunt. Two IDH genes icd1 and icd2 of Ralstonia eutropha HF39, encoding isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2), were identified and characterized. Icd1 was functionally expressed in Escherichia coli, whereas icd2 was expressed in E. coli but no activity was obtained. Interposon-mutants of icd1 (HF39,icd1) and icd2 (HF39,icd2) of R. eutropha HF39 were constructed and their phenotypes were investigated. HF39,icd1 retained 43% of IDH activity, which was not induced by acetate, and HF39,icd2 expressed 74% of acetate-induced IDH activity. Both HF39,icd1and HF39,icd2 kept the same growth rate on acetate as the wild-type. These data suggested that IDH1 is induced by acetate. The interposon-mutants HF39,icd1 and HF39,icd2 accumulated the same amount of poly(3-hydroxybutyric acid) as the wild-type. [source] Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plantsFEMS MICROBIOLOGY REVIEWS, Issue 4 2005David Mendoza-Cózatl Abstract Glutathione (,-glu-cys-gly; GSH) is usually present at high concentrations in most living cells, being the major reservoir of non-protein reduced sulfur. Because of its unique redox and nucleophilic properties, GSH serves in bio-reductive reactions as an important line of defense against reactive oxygen species, xenobiotics and heavy metals. GSH is synthesized from its constituent amino acids by two ATP-dependent reactions catalyzed by ,-glutamylcysteine synthetase and glutathione synthetase. In yeast, these enzymes are found in the cytosol, whereas in plants they are located in the cytosol and chloroplast. In protists, their location is not well established. In turn, the sulfur assimilation pathway, which leads to cysteine biosynthesis, involves high and low affinity sulfate transporters, and the enzymes ATP sulfurylase, APS kinase, PAPS reductase or APS reductase, sulfite reductase, serine acetyl transferase, O -acetylserine/O -acetylhomoserine sulfhydrylase and, in some organisms, also cystathionine ,-synthase and cystathionine ,-lyase. The biochemical and genetic regulation of these pathways is affected by oxidative stress, sulfur deficiency and heavy metal exposure. Cells cope with heavy metal stress using different mechanisms, such as complexation and compartmentation. One of these mechanisms in some yeast, plants and protists is the enhanced synthesis of the heavy metal-chelating molecules GSH and phytochelatins, which are formed from GSH by phytochelatin synthase (PCS) in a heavy metal-dependent reaction; Cd2+ is the most potent activator of PCS. In this work, we review the biochemical and genetic mechanisms involved in the regulation of sulfate assimilation-reduction and GSH metabolism when yeast, plants and protists are challenged by Cd2+. [source] Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl geneFEMS YEAST RESEARCH, Issue 7 2007Loknath Gidijala Abstract We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host,vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine ,-lyase. With this new host,vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein,PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL,SKL produced in H. polymorpha displays normal enzymatic activities. [source] Gluconeogenesis in Candida albicansFEMS YEAST RESEARCH, Issue 3 2002D. Eschrich Abstract According to different metabolic situations in various stages of Candida albicans pathogenesis the regulation of carbohydrate metabolism was investigated. We report the genetic characterization of all major C. albicans gluconeogenic and glyoxylate cycle genes (fructose-1,6-bisphosphatase, PEP carboxykinase, malate synthase and isocitrate lyase) which were isolated after functional complementation of the corresponding Saccharomyces cerevisiae deletion mutants. Remarkably, the regulation of the heterologously expressed C. albicans gluconeogenic and glyoxylate cycle genes was similar to that of the homologous S. cerevisiae genes. A C. albicans,Cafbp1 deletion strain failed to utilize non-fermentable carbon sources but hyphal growth was not affected. Our results show that regulation of gluconeogenesis in C. albicans is similar to that of S. cerevisiae and that the current knowledge on how gluconeogenesis is regulated will facilitate the physiological understanding of C. albicans. [source] Analysing scots pine defence-related transcripts and fungal DNA levels in seedlings single- or dual-inoculated with endophytic and pathogenic Rhizoctonia speciesFOREST PATHOLOGY, Issue 6 2009H. Grönberg Summary Fungal DNA and induction of host defence-related transcripts were monitored by real-time PCR in young Scots pine seedlings inoculated with pathogenic uninucleate (UNR) and endophytic binucleate (BNR) Rhizoctonia species. The UNR (teleomorph Ceratobasidium bicorne) causes root dieback in conifer seedlings following invasion of the vascular cylinder via root apex and destroying apical meristems whilst the BNR, representing anastomosis group AG-I of genus Ceratobasidium, is primarily restricted to the cortex in basal root regions. In the experiment 1 the fungi were simultaneously inoculated on roots, while in experiment 2, BNR was pre-inoculated 168 h before inoculation with UNR. Nucleic acids were extracted from infected roots at intervals up to 192 h post-infection (hpi), and the genomic DNA levels of the host and fungi and the transcript levels of a house-keeping gene (glyceraldehyde-3-phosphate dehydrogenase) and nine putative defence genes were quantified. In simultaneous inoculation UNR was more competitive than BNR whereas pre-inoculation of BNR suppressed but did not completely prevent root colonization by UNR. Stilbene synthase (STS) transcription was significantly up-regulated in single-inoculations with both fungi and in dual inoculation in both experiments. Maximum STS transcript levels were observed in roots single-inoculated with UNR; the peak level at 48 hpi in experiment 2 was significantly higher than in seedlings single-inoculated with BNR or co-inoculated with both fungi, the latter two treatments showing relatively similar STS transcript levels. Similarly, transcript levels of phenylalanine ammonia lyase at 48 hpi in experiment 2 were significantly higher in roots single-inoculated with UNR compared with BNR or in UNR+BNR co-inoculations. The other seven putative defence genes monitored did not show any clear-cut up-regulation following fungal inoculation. We conclude that BNR suppresses UNR in Scots pine roots via direct competition for infection sites, since the studied transcripts showed no evidence of BNR induced resistance against UNR. [source] Argininosuccinate lyase deficiency: mutational spectrum in Italian patients and identification of a novel ASL pseudogene,HUMAN MUTATION, Issue 7 2007Eva Trevisson Abstract Argininosuccinic aciduria (ASAuria) is an inborn error of metabolism caused by mutations in the argininosuccinate lyase (ASL) gene, which leads to the accumulation of argininosuccinic acid (ASA) in body fluids and severe hyperammonemia. A severe neonatal form and a milder late-onset variant are described. We report a novel ASL pseudogene located in the centromeric region of chromosome 7, 14 novel mutations in the ASL gene, and a novel intronic polymorphism found in a cohort of Italian patients. Our approach relied exclusively on genomic DNA analysis. We found seven missense mutations, two nonsense, three small insertions/deletions, and two splicing mutations. Only two patients harbored previously described mutations, and among the novel variants only two were present in more than one kindred. The pathogenicity of the splicing mutations was demonstrated by a functional splicing assay that employed a hybrid minigene. We also performed molecular modeling using the reported three-dimensional structure of ASL to predict the functional consequences of the missense mutations. There was no genotype,phenotype correlation. Patients with neonatal onset display developmental delay and seizures despite adequate metabolic control. Moreover, hepatomegaly, fibrosis, and abnormal liver function tests are common complications in these patients, but not in patients with the late infancy form. We stress the importance of mutation analysis in patients with ASAuria, to confirm the clinical diagnosis, and to perform DNA-based prenatal diagnosis in future pregnancies of these families. Hum Mutat 28(7), 694,702, 2007. © 2007 Wiley-Liss, Inc. [source] Deletion hotspot in the argininosuccinate lyase gene: association with topoisomerase II and DNA polymerase , sites ,HUMAN MUTATION, Issue 11 2006John Christodoulou Abstract Molecular analysis of argininosuccinate lyase (ASAL) deficiency has led to the identification of a deletion hotspot in the ASL gene. Six individuals with ASAL deficiency had alleles that led to a complete absence of exon 13 from the ASL mRNA; each had a partial deletion of exon 13 in the genomic DNA. In all six patients, the deletions begin 18 bp upstream of the 3, end of exon 13. In four cases, the deletions were 13 bp in length, and ended within exon 13, whereas in two other patients the deletions were 25 bp and extended into intron 13. The sequence at which these deletions begin overlaps both a putative topoisomerase II recognition site and a DNA polymerase , mutation/frameshift site. Moreover, the topoisomerase II cut site is situated precisely at the beginning of the deletions, which are flanked by small (2- and 3-bp) direct repeats. We note that a similar concurrence of these two putative enzyme sites can be found in a number of other deletion sites in the human genome, most notably the ,F508 deletion in the CFTR gene. These findings suggest that the joint presence of these two enzyme sites represents a DNA sequence context that may favor the occurrence of small deletions. Hum Mutat 27(11), 1065,1071, 2006. © 2006 Wiley-Liss, Inc. [source] Accelerated repair and reduced mutagenicity of oxidative DNA damage in human bladder cells expressing the E. coli FPG proteinINTERNATIONAL JOURNAL OF CANCER, Issue 7 2006Monica Ropolo Abstract Repair of some oxidized purines such as 8-oxo-7,8-dihydroguanine (8-oxoG) is inefficient in human cells in comparison to repair of other major endogenous lesions (e.g. uracil, abasic sites or oxidized pyrimidines). This is due to the poor catalytic properties of hOGG1, the major DNA glycosylase involved in 8-oxoG removal. The formamidopyrimidine DNA glycosylase (FPG) protein from E. coli is endowed with a potent 8-oxoG glycolytic activity coupled with a ,,,-AP lyase. In this study, we have expressed FPG fused to the enhanced green fluorescent protein (EGFP) in human bladder cells to accelerate the repair of oxidative DNA damage. Cells expressing the fusion protein EGFP,FPG repaired 8-oxoG and AP sites at accelerated rates, in particular via the single-nucleotide insertion base excision repair (BER) pathway and were resistant to mutagenicity of the oxidizing carcinogen potassium bromate. FPG may stably protect human cells from some harmful effects of oxidative DNA damage. © 2005 Wiley-Liss, Inc. [source] Age-related changes in human meniscal glycosaminoglycansINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2004Gareth Blackburn Introduction With an increased human lifespan, a major challenge is now to ensure a concomitant increase in healthspan. Meniscal damage and degradation are common and are strongly correlated with subsequent osteoarthritis. Indeed, meniscal damage has been identified in about 60% of people over 60. Markers of pathology will facilitate intervention but first require normal age-related changes to be established. Methods Undamaged vascular and avascular regions of medial and lateral human menisci were comminuted and the tissue extracted into 4- m GuHCl and subject to associative CsCl density gradient centrifugation. Aggrecan and the small leucine rich PGs (SLRPs) were isolated and their GAG profiles examined by HPAEC fingerprinting, following enzyme depolymerization, and by an NMR spectroscopy. Results and discussion Analysis of aggrecan and the SLRPs show that there is a complex and dynamic pattern of KS, CS and DS abundance and distribution within human menisci, which changes with age. The abundance of SLRPs is higher in the avascular than vascular tissues, however, this is not reflected in the abundance of aggrecan which is present at similar levels in both tissue regions. The data show no other significant differences between medial and lateral and between vascular and avascular tissue regions. Analysis of the sulfation pattern of CS following digestion by ACII lyase, shows that in both aggrecan and SLRPs the 4-sulfation level falls with age from 20 to 35% in young tissues to 10,20% in older. Subsequent analyses following ABC lyase depolymerization, to include DS, shows very significant change with age from CS + DS 4-sulfation levels of ca. 40,55% in young tissue to ca. 15,30% in older. The difference between these datasets represents the contribution made by 4-sulfated DS. Thus, analysis of the difference suggests that DS makes a decreasing contribution to the CS/DS profile with age. Indeed, this is confirmed by an NMR analysis of these samples. Analysis of the resonances in the region 1.95,2.2 p.p.m. (ref to TSP) allows the estimation of the contribution made by DS, CS and KS. These data show that, in aggrecan, the contribution made by DS chains falls from ca. 10% in younger tissues to ca. 2,4% in older tissues. NMR analysis also shows that KS levels fall with age from ca. 15,20% in younger tissues to 5,10% in older tissues. Analysis of the structure of the KS chains shows chains with a structure similar to that of in articular cartilage but that at all ages there are very low levels of fucosylation (ca. 1,5%). Previous studies of age-related changes in CS/DS and KS structures have shown significant changes in the first 17 years of life, with only modest nonpathological changes after that time. These data from meniscal tissues do not show such a dramatic halting of normal age-related changes. Indeed, the data show gradual age-related changes in DS, CS and KS abundance and structure throughout life. These baseline age-related changes data will now allow the analysis of pathology-related changes. [source] Effects of heat treatment on the quality of fresh-cut Chinese water chestnutINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 2 2004Litao Peng Summary A heat treatment to inhibit browning and maintain the quality of fresh-cut Chinese water chestnut was developed. Slices of Chinese water chestnut, cv. Guilin, were immersed in boiling water for 30 s, placed into film-wrapped trays and then stored at 4 °C for up to 12 days. Changes in browning, eating quality and disease incidence were measured. The effect of heat treatment on the content of total phenolics and activities of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD) was also evaluated. The heat treatment effectively prevented browning associated with PAL, PPO and POD activities and total phenolic content and delayed the decrease in eating quality, which is associated with reduced total soluble solids, titratable acidity and ascorbic acid, compared with fresh-cut Chinese water chestnut. Inhibition of browning by heat treatment without microbial growth was achieved for 12 days of storage at 4 °C. These results showed that heat treatment effectively maintained the quality of fresh-cut Chinese water chestnut. [source] Efficient Tandem Biocatalytic Process for the Kinetic Resolution of Aromatic ,-Amino AcidsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 9 2010Bian Wu Abstract We describe a simple and efficient enzymatic tandem reaction for the preparation of enantiomerically pure , -phenylalanine and its analogues from the corresponding racemates. In this process, phenylalanine aminomutase (PAM) catalyzes the stereoselective isomerization of (R)- , -phenylalanines to (S)- , -phenylalanines, which are in situ transformed to cinnamic acids by phenylalanine ammonia lyase (PAL). Preparative scale conversions are done with a mutated PAM with enhanced catalytic activity. [source] Cutting Long Syntheses Short: Access to Non-Natural Tyrosine Derivatives Employing an Engineered Tyrosine Phenol LyaseADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 4 2010Birgit Seisser Abstract The chemical synthesis of 3-substituted tyrosine derivatives requires a minimum of four steps to access optically enriched material starting from commercial precursors. Attempting to short-cut the cumbersome chemical synthesis of 3-substituted tyrosine derivatives, a single step biocatalytic approach was identified employing the tyrosine phenol lyase from Citrobacter freundii. The enzyme catalyses the hydrolysis of tyrosine to phenol, pyruvate and ammonium as well as the reverse reaction, thus the formation of tyrosine from phenol, pyruvate and ammonium. Since the wild-type enzyme possessed a very narrow substrate spectrum, structure-guided, site-directed mutagenesis was required to change the substrate specificity of this CC bond forming enzyme. The best variant M379V transformed, for example, o -cresol, o -methoxyphenol and o -chlorophenol efficiently to the corresponding tyrosine derivatives without any detectable side-product. In contrast, all three phenol compounds were non-substrates for the wild-type enzyme. Employing the mutant, various L -tyrosine derivatives (3-Me, 3-OMe, 3-F, 3-Cl) were obtained with complete conversion and excellent enantiomeric excess (>97%) in just a single ,green' step starting from pyruvate and commercially available phenol derivatives. [source] Synthesis of Aliphatic (S)-,-Hydroxycarboxylic Amides using a One-Pot Bienzymatic Cascade of Immobilised Oxynitrilase and Nitrile HydrataseADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 3 2009Sander van Pelt Abstract A one-pot bienzymatic cascade combining a hydroxynitrile lyase (Manihot esculenta, E.C. 4.1.2.10) and a nitrile hydratase (Nitriliruptor alkaliphilus, E.C. 4.2.1.84) for the synthesis of enantiopure aliphatic ,-hydroxycarboxylic amides from aldehydes is described. Both enzymes were immobilised as cross-linked enzyme aggregates (CLEAs). Stability tests show that the nitrile hydratase CLEAs are sensitive to water-immiscible organic solvents as well as to aldehydes and hydrogen cyanide (HCN), but are remarkably stable and show useful activity in acidic aqueous environments of pH,4,5. The cascade reactions are consequently carried out by using a portionwise feed of HCN and moderate concentrations of aldehyde in acidic aqueous buffer to suppress the uncatalysed hydrocyanation background reaction. After optimisation, this method was used to synthesise five different kinds of aliphatic ,-hydroxycarboxylic amides from the corresponding aldehydes with good yields and with enantiomeric purities comparable to those obtained for the ,-hydroxynitriles in the microaqueous hydrocyanation using hydroxynitrile lyase and an excess of HCN. [source] Mixed Aromatic Acyloin Condensations with Recombinant Benzaldehyde Lyase: Synthesis of ,-Hydroxydihydrochalcones and Related ,-Hydroxy KetonesADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6-7 2003Monica Sanchez-Gonzalez Abstract Recombinant benzaldehyde lyase (BAL), expressed and purified from E.,coli strain JM-109, was used to catalyze the condensation of a series of methoxybenzaldehydes and phenylacetaldehyde in the synthesis of ,-(R)-hydroxydihydrochalcones. Enantiomerically pure 1-hydroxy-1,3-diphenylpropan-2-ones and o -anisoin were also obtained as products of the BAL reaction. The R absolute configurations of chiral centers were determined by CD spectroscopy. ,-(R)-Hydroxydihydrochalcones and 1-hydroxy-1,3-diphenylpropan-2-ones are valuable synthons for chemoenzymatic syntheses of flavonoids. This is the first synthesis of ,-(R)-hydroxydihydrochalcones by a microbial enzyme. [source] | |||||||||||||||||||||||||||||||||||||