Preparation of polyfunctional arynes via 2-magnesiated diaryl sulfonates.
Explore the source record for details and available documents.
Journal Article↗
Biomedical subjects
Markus Fischer
Publications and source records attributed to Markus Fischer.
Potential anti-infective targets in pathogenic yeasts: structure and properties of 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans.
A synthetic gene specifying a putative 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans directed the synthesis of a 22.5 kDa peptide in a recombinant Escherichia coli strain. The recombinant protein was purified to apparent homogeneity by two chromatographic steps and was shown to catalyze the formation of L-3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 332 nmol mg(-1) min(-1). Hydrodynamic studies indicated a native molecular mass of 41 kDa in line with a homodimer structure. The protein was crystallized in its apoform. Soaking yielded crystals in complex with the substrate ribulose 5-phosphate. The structures were solved at resolutions of 1.6 and 1.7 angstroms, respectively, using 3,4-dihydroxy-2-butanone 4-phosphate synthase of E. coli for molecular replacement. Structural comparison with the orthologs of Magnaporthe grisea and Methanococcus jannaschii revealed a hitherto unknown conformation of the essential acidic active-site loop.
Amino Acid Sequence↗
Rapid preparation of isotopolog libraries by in vivo transformation of 1)C-glucose. Studies on 6,7-dimethyl-8-ribityllumazine, a biosynthetic precursor of vitamin B2.
An Escherichia coli strain engineered for expression of the ribABGH genes of Bacillus subtilis was shown to produce 100 mg of the riboflavin precursor 6,7-dimethyl-8-ribityllumazine per liter of minimal medium. Growth of the recombinant strain in medium supplemented with [U-13C6]glucose and/or 15NH4Cl as single sources of carbon and/or nitrogen afforded 6,7-dimethyl-8-ribityllumazine universally labeled with 13C and/or 15N. The yield of [U-13C13]-6,7-dimethyl-8-ribityllumazine based on [U-13C6]glucose was 25 mg/g. Fermentation with [1-13C1]-, [2-13C1]-, or [3-13C1]glucose afforded mixtures of 6,7-dimethyl-8-ribityllumazine isotopologs, predominantly with 13C enrichment of single carbon atoms. The isotope-labeled samples enabled a comprehensive NMR analysis of 6,7-dimethyl-8-ribityllumazine. Isotopolog libraries of a wide variety of microbial metabolites can be produced by the same experimental approach.
Bacillus subtilis↗
Metal sites in 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with the substrate ribulose 5-phosphate.
The crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal centre has recently been determined at 1.7 A resolution. The enzyme converts ribulose 5-phosphate into 3,4-dihydroxy-2-butanone 4-phosphate, while its C4 atom is released as formate. The resulting four-carbon body supplies all eight C atoms for the xylene moiety of riboflavin. Three of the four hydroxyl groups of ribulose 5-phosphate were coordinated by the metal ions. Based on crystallographic refinement, the metals were assigned as zinc and calcium, which were present in the crystallization buffer. Neither metal supports the enzymatic reaction. In the present study, the correctness of this assignment is assessed using anomalous diffraction data collected at the high-energy side of the zinc absorption edge (lambda = 1.2823 A). Only the three tentative zinc ions give strong peaks in an anomalous difference Fourier map (>20sigma), whereas the four tentative calcium ions do not show anomalous signals above the noise level. These results confirm the initial assignment. In addition, the resolution was improved to 1.55 A.
Binding Sites↗
Evolution of vitamin B2 biosynthesis: structural and functional similarity between pyrimidine deaminases of eubacterial and plant origin.
The Arabidopsis thaliana open reading frame At4g20960 predicts a protein whose N-terminal part is similar to the eubacterial 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate deaminase domain. A synthetic open reading frame specifying a pseudomature form of the plant enzyme directed the synthesis of a recombinant protein which was purified to apparent homogeneity and was shown by NMR spectroscopy to convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate at a rate of 0.9 micromol mg(-1) min(-1). The substrate and product of the enzyme are both subject to spontaneous anomerization of the ribosyl side chain as shown by (13)C NMR spectroscopy. The protein contains 1 eq of Zn(2+)/subunit. The deaminase activity could be assigned to the N-terminal section of the plant protein. The deaminase domains of plants and eubacteria share a high degree of similarity, in contrast to deaminases from fungi. These data show that the riboflavin biosynthesis in plants proceeds by the same reaction steps as in eubacteria, whereas fungi use a different pathway.
Amino Acid Sequence↗
Biosynthesis of tetrahydrofolate in plants: crystal structure of 7,8-dihydroneopterin aldolase from Arabidopsis thaliana reveals a novel adolase class.
Dihydroneopterin aldolase (DHNA) catalyses a retroaldol reaction yielding 6-hydroxymethyl-7,8-dihydropterin, a biosynthetic precursor of the vitamin, tetrahydrofolate. The enzyme is a potential target for antimicrobial and anti-parasite chemotherapy. A gene specifying a dihydroneopterin aldolase from Arabidopsis thaliana was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified to apparent homogeneity and crystallised using polyethylenglycol as the precipitating agent. The crystal structure was solved by X-ray diffraction analysis at 2.2A resolution. The enzyme forms a D(4)-symmetric homooctamer. Each polypeptide chain is folded into a single domain comprising an antiparallel four-stranded beta-sheet and two long alpha-helices. Four monomers are arranged in a tetrameric ring, and two of these rings form a hollow cylinder. Well defined purine derivatives are found at all eight topologically equivalent active sites. The subunit fold of the enzyme is related to substructures of dihydroneopterin triphosphate epimerase, GTP cyclohydrolase I, and pyruvoyltetrahydropterin synthase, which are all involved in the biosynthesis of pteridine type cofactors, and to urate oxidase, although some members of that superfamily have no detectable sequence similarity. Due to structural and mechanistical differences of DHNA in comparison with class I and class II aldolases, a new aldolase class is proposed.
Amino Acid Sequence↗
Sequence and structure of epoxide hydrolases: a systematic analysis.
Epoxide hydrolases (EC 3.3.2.3) are ubiquitous enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. More than 100 epoxide hydrolases (EH) have been identified or predicted, and 3 structures are available. Although they catalyze the same chemical reaction, sequence similarity is low. To identify conserved regions, all EHs were aligned. Phylogenetic analysis identified 12 homologous families, which were grouped into 2 major superfamilies: the microsomal EH superfamily, which includes the homologous families of Mammalian, Insect, Fungal, and Bacterial EHs, and the cytosolic EH superfamily, which includes Mammalian, Plant, and Bacterial EHs. Bacterial EHs show a high sequence diversity. Based on structure comparison of three known structures from Agrobacterium radiobacter AD1 (cytosolic EH), Aspergillus niger (microsomal EH), Mus musculus (cytosolic EH), and multisequence alignment and phylogenetic analysis of 95 EHs, the modular architecture of this enzyme family was analyzed. Although core and cap domain are highly conserved, the structural differences between the EHs are restricted to only two loops: the NC-loop connecting the core and the cap and the cap-loop, which is inserted into the cap domain. EHs were assigned to either of three clusters based on loop length. By using this classification, core and cap region of all EHs, NC-loops and cap-loops of 78% and 89% of all EHs, respectively, could be modeled. Representative models are available from the Lipase Engineering Database, http://www.led.uni-stuttgart.de.
Amino Acid Sequence↗
Structure of the molybdenum-cofactor biosynthesis protein MoaB of Escherichia coli.
The moaABC operon of Escherichia coli is involved in early steps of the biosynthesis of the molybdenum-binding cofactor molybdopterin, but the precise functions of the cognate proteins are not known. The crystal structure of the MoaB protein from E. coli was determined by multiple anomalous dispersion at 2.1 angstroms A resolution and refined to an R factor of 20.4% (Rfree = 25.0%). The protein is a 32-symmetric hexamer, with the monomers consisting of a central beta-sheet flanked by helices on both sides. The overall fold of the monomer is similar to those of the MogA protein of E. coli, the G-domains of rat and human gephyrin and the G-domains of Cnx1 protein from A. thaliana, all of which are involved in the insertion of an unknown molybdenum species into molybdopterin to form the molybdenum cofactor. Furthermore, the MoaB protein shows significant sequence similarity to the cinnamon protein from Drosophila melanogaster. In addition to other functions, all these proteins are involved in the biosynthesis of the molybdenum cofactor and have been shown to bind molybdopterin. The close structural homology to MogA and the gephyrin and Cnx1 domains suggests that MoaB may bind a hitherto unidentified pterin compound, possibly an intermediate in molybdopterin biosynthesis.
Amino Acid Sequence↗
The database of epoxide hydrolases and haloalkane dehalogenases: one structure, many functions.
UNLABELLED: The epoxide hydrolases and haloalkane dehalogenases database (EH/HD) integrates sequence and structure of a highly diverse protein family, including mainly the Asp-hydrolases of EHs and HDs but also proteins, such as Ser-hydrolases non-heme peroxidases, prolyl iminopetidases and 2-hydroxymuconic semialdehyde hydrolases. These proteins have a highly conserved structure, but display a remarkable diversity in sequence and function. A total of 305 protein entries were assigned to 14 homologous families, forming two superfamilies. Annotated multisequence alignments and phylogenetic trees are provided for each homologous family and superfamily. Experimentally derived structures of 19 proteins are superposed and consistently annotated. Sequence and structure of all 305 proteins were systematically analysed. Thus, deeper insight is gained into the role of a highly conserved sequence motifs and structural elements. AVAILABILITY: The EH/HD database is available at http://www.led.uni-stuttgart.de
Amino Acid Motifs↗
Folate biosynthesis in higher plants. cDNA cloning, heterologous expression, and characterization of dihydroneopterin aldolases.
Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to be cloned and characterized from plants. This enzyme catalyzes conversion of 7,8-dihydroneopterin (DHN) to 6-hydroxymethyl-7,8-dihydropterin, and is encoded by the folB gene in Escherichia coli. The E. coli FolB protein also mediates epimerization of DHN to 7,8-dihydromonapterin. Searches of the Arabidopsis genome detected three genes encoding substantially diverged FolB homologs (AtFolB1-3, sharing 57%-73% identity), for which cDNAs were isolated. A fourth cDNA specifying a FolB-like protein (LeFolB1) was obtained from tomato (Lycopersicon esculentum) by reverse transcription-PCR. When overproduced in E. coli, recombinant AtFolB1, AtFolB2, and LeFolB1 proteins all had both dihydroneopterin aldolase and epimerase activities, and carried out the aldol cleavage reaction on the epimerization product, 7,8-dihydromonapterin, as well as on DHN. AtFolB3, however, could not be expressed in active form. Size exclusion chromatography indicated that the plant enzyme is an octamer, like the bacterial enzyme. Quantifying expression of the Arabidopsis genes by real-time reverse transcription-PCR showed that AtFolB1 and AtFolB2 messages occur at low levels throughout the plant, whereas the AtFolB3 mRNA was detected only in siliques and only with an extremely low abundance. Sequence comparisons and phylogenetic analysis of FolB homologs from 16 plants indicated that their N-terminal regions are highly variable, and that most species have a small number of FolB genes that diverged after separation of the lineages leading to families. The substantial divergence of FolB homologs in Arabidopsis and other plants suggests that some of them may act on substrates other than DHN.
Aldehyde-Lyases↗
Design, synthesis, and evaluation of 9-D-ribitylamino-1,3,7,9-tetrahydro-2,6,8-purinetriones bearing alkyl phosphate and alpha,alpha-difluorophosphonate substituents as inhibitors of tiboflavin synthase and lumazine synthase.
Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin, an essential metabolite that is involved in electron transport processes. To obtain structural probes of these two enzymes, as well as inhibitors of potential value as antibiotics, a series of ribitylpurinetriones bearing alkyl phosphate and alpha,alpha-difluorophosphonate substituents were synthesized. Since the purinetrione ring system and the ribityl hydroxyl groups can be alkylated, the synthesis required the generation of these two moieties in protected form before the desired alkylation reaction could be carried out. These substances were designed as intermediate analogue inhibitors of lumazine synthase that would bind to its phosphate-binding site. All of the compounds were found to be effective inhibitors of both Bacillus subtilis lumazine synthase as well as Escherichia coli riboflavin synthase. Molecular modeling of the binding of 3-(1,3,7,9-tetrahydro-9-D-ribityl-2,6,8-trioxopurin-7-yl)propane 1-phosphate provided a structural explanation for how these compounds are able to effectively inhibit both enzymes. Interestingly, the enzyme kinetics of these new compounds in comparison with the parent purinetrione demonstrated unexpectedly that the phosphate and phosphonate substituents contributed negatively to the binding. A possible explanation for these effects on lumazine synthase would be that the inorganic phosphate in the assay buffer competes with the substituted purinetriones for binding to the enzyme. This would be consistent with the observed increase in K(m) of the 3,4-dihydroxybutanone-4-phosphate substrate from 5.2 microM in Tris buffer or from 6.7 microM in MOPS buffer to 50 microM in phosphate buffer when tested on Bacillus subtilis lumazine synthase. However, when tested in Tris buffer vs Mycobacterium tuberculosis lumazine synthase, three of the phosphate inhibitors displayed inhibition constants in the 4-5 nM range, indicating that they are much more potent than the parent purinetrione. Under these conditions, the phosphate moieties of the inhibitors do contribute positively to their binding. The alpha,alpha-difluorophosphonate analogue, which is expected to have enhanced metabolic stability relative to the phosphates, was also found to be an inhibitor of Mycobacterium tuberculosis lumazine synthase with a K(i) of 60 nM.
Bacillus subtilis↗
Molecular modeling of family GH16 glycoside hydrolases: potential roles for xyloglucan transglucosylases/hydrolases in cell wall modification in the poaceae.
Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)-beta-galactanases, (1,4)-beta-galactanases/kappa-carrageenases, "nonspecific" (1,3/1,3;1,4)-beta-D-glucan endohydrolases, and (1,3;1,4)-beta-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)-beta-D-glucan endohydrolases and a kappa-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional (3D) dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-beta-d-glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos x tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)-beta-D-xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos x tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)-beta-D-glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-beta-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-beta-D-glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.
Amino Acid Sequence↗
Structural basis of charge transfer complex formation by riboflavin bound to 6,7-dimethyl-8-ribityllumazine synthase.
The amino acid residue tryptophan 27 of 6,7-dimethyl-8-ribityllumazine synthase of the yeast Schizosaccharomyces pombe was replaced by tyrosine. The structures of the W27Y mutant protein in complex with riboflavin, the substrate analogue 5-nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, and the product analogue 6-carboxyethyl-7-oxo-8-ribityllumazine, were determined by X-ray crystallography at resolutions of 2.7-2.8 A. Whereas the indole system of W27 forms a coplanar pi-complex with riboflavin, the corresponding phenyl ring in the W27Y mutant establishes only peripheral contact with the heterocyclic ring system of the bound riboflavin. These findings provide an explanation for the absence of the long wavelength shift in optical absorption spectra of riboflavin bound to the mutant enzyme. The structures of the mutants are important tools for the interpretation of the unusual physical properties of riboflavin in complex with lumazine synthase.
Binding Sites↗
Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments.
BACKGROUND: Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs. RESULTS: A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20 degrees C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg(-1) min(-1) with an apparent KM of 5.7 microM. 19F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. 19F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins. CONCLUSION: Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The 19F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism.
Amino Acid Sequence↗
Presteady state kinetic analysis of riboflavin synthase.
Riboflavin synthase catalyzes a mechanistically complex dismutation affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione from 6,7-dimethyl-8-ribityllumazine. The kinetics of the enzyme from Escherichia coli were studied under single turnover conditions. Stopped flow as well as quenched flow experiments documented the transient formation of a pentacyclic reaction intermediate. No other transient species were sufficiently populated to allow detection. The data are best described by a sequence of one second order and one first order reaction.
Catalysis↗
Structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with divalent metal ions and the substrate ribulose 5-phosphate: implications for the catalytic mechanism.
Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-A resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.
Binding Sites↗
Temperature-dependent presteady state kinetics of lumazine synthase from the hyperthermophilic eubacterium Aquifex aeolicus.
6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Presteady state kinetic experiments using the enzyme from the hyperthermophilic bacterium Aquifex aeolicus were monitored by multiwavelength photometry. An early optical transient absorbing around 330 nm is interpreted as a Schiff base intermediate obtained by reaction of the position 5 amino group of the heterocyclic substrate with the carbonyl group of 3,4-dihydroxy-2-butanone 4-phosphate. A second transient with an absorption maximum at 445 nm represents an intermediate resulting from the elimination of orthophosphate from the Schiff base. The rate-determining step is the subsequent formation of the 7-exomethylene type anion of 6,7-dimethyl-8-ribityllumazine. The rate constants for the three partial reactions identified by the stopped flow experiments show linear Arrhenius relations in the temperature range of 15-70 degrees C.
Eubacterium↗
A structure-based model of the reaction catalyzed by lumazine synthase from Aquifex aeolicus.
6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of riboflavin, which, as a coenzyme, plays a vital role in the electron transfer process for energy production in all cellular organisms. The enzymes involved in lumazine biosynthesis have been studied in considerable detail. However, the conclusive mechanism of the reaction catalyzed by lumazine synthase has remained unclear. Here, we report four crystal structures of the enzyme from the hyperthermophilic bacterium Aquifex aeolicus in complex with different inhibitor compounds. The structures were refined at resolutions of 1.72 A, 1.85 A, 2.05 A and 2.2 A, respectively. The inhibitors have been designed in order to mimic the substrate, the putative reaction intermediates and the final product. Structural comparisons of the native enzyme and the inhibitor complexes as well as the kinetic data of single-site mutants of lumazine synthase from Bacillus subtilis showed that several highly conserved residues at the active site, namely Phe22, His88, Arg127, Lys135 and Glu138 are most likely involved in catalysis. A structural model of the catalytic process, which illustrates binding of substrates, enantiomer specificity, proton abstraction/donation, inorganic phosphate elimination, formation of the Schiff base and cyclization is proposed.
Bacteria↗