Biomedical subjects

Markus Fischer

Publications and source records attributed to Markus Fischer.

At least 73 records · Page 4Linked to original sources

PubMed2003

Biosynthesis of riboflavin. Single turnover kinetic analysis of 6,7-dimethyl-8-ribityllumazine synthase.

6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate, affording the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine. Single turnover experiments monitored by multiwavelength photometry were performed with the recombinant lumazine synthase of Bacillus subtilis. Mixing of the enzyme with the pyrimidine type substrate is conducive to a hypsochromic shift as well as a decrease in absorbance of the heterocyclic substrate; the rate constant for that reaction is 0.02 s(-1) microM(-1). Rapid mixing of the complex between enzyme and pyrimidine type substrate with the second substrate, 3,4-dihydroxy-2-butanone 4-phosphate, is followed by the appearance of an early optical transient characterized by an absorption maxima at 330 nm of low intensity which was tentatively assigned as a Schiff base intermediate. The subsequent elimination of phosphate affords a transient with intense absorption maxima at 455 and 282 nm, suggesting an intermediate with an extended system of conjugated double bonds. The subsequent formation of the enzyme product, 6,7-dimethyl-8-ribityllumazine, is the rate-determining step.

Bacillus subtilis↗

PubMed2003

Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold.

The essential redox cofactors riboflavin monophosphate (FMN) and flavin adenine dinucleotide (FAD) are synthesised from their precursor, riboflavin, in sequential reactions by the metal-dependent riboflavin kinase and FAD synthetase. Here, we describe the 1.6A crystal structure of the Schizosaccharomyces pombe riboflavin kinase. The enzyme represents a novel family of phosphoryl transferring enzymes. It is a monomer comprising a central beta-barrel clasped on one side by two C-terminal helices that display an L-like shape. The opposite side of the beta-barrel serves as a platform for substrate binding as demonstrated by complexes with ADP and FMN. Formation of the ATP-binding site requires significant rearrangements in a short alpha-helix as compared to the substrate free form. The diphosphate moiety of ADP is covered by the glycine-rich flap I formed from parts of this alpha-helix. In contrast, no significant changes are observed upon binding of riboflavin. The ribityl side-chain might be covered by a rather flexible flap II. The unusual metal-binding site involves, in addition to the ADP phosphates, only the strictly conserved Thr45. This may explain the preference for zinc observed in vitro.

Adenosine Diphosphate↗

PubMed2003

Enzyme catalysis via control of activation entropy: site-directed mutagenesis of 6,7-dimethyl-8-ribityllumazine synthase.

6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyses the penultimate step in the biosynthesis of riboflavin. In Bacillus subtilis, 60 lumazine synthase subunits form an icosahedral capsid enclosing a homotrimeric riboflavin synthase unit. The ribH gene specifying the lumazine synthase subunit can be expressed in high yield. All amino acid residues exposed at the surface of the active site cavity were modified by PCR assisted mutagenesis. Polar amino acid residues in direct contact with the enzyme substrates, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, could be replaced with relative impunity with regard to the catalytic properties. Only the replacement of Arg127, which forms a salt bridge with the phosphate group of 3,4-dihydroxy-2-butanone 4-phosphate, reduced the catalytic rate by more than one order of magnitude. Replacement of His88, which is believed to assist in proton transfer reactions, reduced the catalytic activity by about one order of magnitude. Surprisingly, the activation enthalpy deltaH of the lumazine synthase reaction exceeds that of the uncatalysed reaction. On the other hand, the free energy of activation deltaG of the uncatalysed reaction is characterised by a large entropic term (TdeltaS) of -37.8 kJmol(-1), whereas the entropy of activation (TdeltaS) of the enzyme-catalysed reaction is -6.7 kJmol(-1). This suggests that the rate enhancement by the enzyme is predominantly achieved by establishing a favourable topological relation of the two substrates, whereas acid/base catalysis may play a secondary role.

Bacillus subtilis↗

PubMed2003

Biosynthesis of pteridines. Reaction mechanism of GTP cyclohydrolase I.

GTP cyclohydrolase I catalyses the hydrolytic release of formate from GTP followed by cyclization to dihydroneopterin triphosphate. The enzymes from bacteria and animals are homodecamers containing one zinc ion per subunit. Replacement of Cys110, Cys181, His112 or His113 of the enzyme from Escherichia coli by serine affords catalytically inactive mutant proteins with reduced capacity to bind zinc. These mutant proteins are unable to convert GTP or the committed reaction intermediate, 2-amino-5-formylamino-6-(beta-ribosylamino)-4(3H)-pyrimidinone 5'-triphosphate, to dihydroneopterin triphosphate. The crystal structures of GTP complexes of the His113Ser, His112Ser and Cys181Ser mutant proteins determined at resolutions of 2.5A, 2.8A and 3.2A, respectively, revealed the conformation of substrate GTP in the active site cavity. The carboxylic group of the highly conserved residue Glu152 anchors the substrate GTP, by hydrogen bonding to N-3 and to the position 2 amino group. Several basic amino acid residues interact with the triphosphate moiety of the substrate. The structure of the His112Ser mutant in complex with an undefined mixture of nucleotides determined at a resolution of 2.1A afforded additional details of the peptide folding. Comparison between the wild-type and mutant enzyme structures indicates that the catalytically active zinc ion is directly coordinated to Cys110, Cys181 and His113. Moreover, the zinc ion is complexed to a water molecule, which is in close hydrogen bond contact to His112. In close analogy to zinc proteases, the zinc-coordinated water molecule is suggested to attack C-8 of the substrate affording a zinc-bound 8R hydrate of GTP. Opening of the hydrated imidazole ring affords a formamide derivative, which remains coordinated to zinc. The subsequent hydrolysis of the formamide motif has an absolute requirement for zinc ion catalysis. The hydrolysis of the formamide bond shows close mechanistic similarity with peptide hydrolysis by zinc proteases.

Amino Acid Sequence↗

PubMed2003

Blue light perception in plants. Detection and characterization of a light-induced neutral flavin radical in a C450A mutant of phototropin.

The LOV2 domain of Avena sativa phototropin and its C450A mutant were expressed as recombinant fusion proteins and were examined by optical spectroscopy, electron paramagnetic resonance, and electron-nuclear double resonance. Upon irradiation (420-480 nm), the LOV2 C450A mutant protein gave an optical absorption spectrum characteristic of a flavin radical even in the absence of exogenous electron donors, thus demonstrating that the flavin mononucleotide (FMN) cofactor in its photogenerated triplet state is a potent oxidant for redox-active amino acid residues within the LOV2 domain. The FMN radical in the LOV2 C450A mutant is N(5)-protonated, suggesting that the local pH close to the FMN is acidic enough so that the cysteine residue in the wild-type protein is likely to be also protonated. An electron paramagnetic resonance analysis of the photogenerated FMN radical gave information on the geometrical and electronic structure and the environment of the FMN cofactor. The experimentally determined hyperfine couplings of the FMN radical point to a highly restricted delocalization of the unpaired electron spin in the isoalloxazine moiety. In the light of these results a possible radical-pair mechanism for the formation of the FMN-C(4a)-cysteinyl adduct in LOV domains is discussed.

Avena↗

PubMed2003

The Lipase Engineering Database: a navigation and analysis tool for protein families.

The Lipase Engineering Database (LED) (http://www.led.uni-stuttgart.de) integrates information on sequence, structure, and function of lipases, esterases, and related proteins. Sequence data on 806 protein entries are assigned to 38 homologous families, which are grouped into 16 superfamilies with no global sequence similarity between each other. For each family, multisequence alignments are provided with functionally relevant residues annotated. Pre-calculated phylogenetic trees allow navigation inside superfamilies. Experimental structures of 45 proteins are superposed and consistently annotated. The LED has been applied to systematically analyze sequence-structure-function relationships of this vast and diverse enzyme class. It is a useful tool to identify functionally relevant residues apart from the active site residues, and to design mutants with desired substrate specificity.

Amino Acid Sequence↗

PubMed2003

Biosynthesis of riboflavin in archaea. 6,7-dimethyl-8-ribityllumazine synthase of Methanococcus jannaschii.

Heterologous expression of the putative open reading frame MJ0303 of Methanococcus jannaschii provided a recombinant protein catalysing the formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Steady state kinetic analysis at 37 degrees C and pH 7.0 indicated a catalytic rate of 11 nmol.mg-1.min-1; Km values for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxybutanone 4-phosphate were 12.5 and 52 micro m, respectively. The enzyme sediments at an apparent velocity of about 12 S. Sedimentation equilibrium analysis indicated a molecular mass around 1 MDa but was hampered by nonideal solute behaviour. Negative-stained electron micrographs showed predominantly spherical particles with a diameter of about 150 A. The data suggest that the enzyme from M. jannaschii can form capsids with icosahedral 532 symmetry consisting of 60 subunits.

Amino Acid Sequence↗

PubMed2003

Evaluation and application of a broad-spectrum polymerase chain reaction assay for human papillomaviruses in the screening of squamous cell tumours of the head and neck.

OBJECTIVE: The results of studies analysing the prevalence of human papillomaviruses (HPVs) in squamous cell carcinomas of the head and neck region often vary depending on the different molecular biological methods applied. Focusing on the histomorphological criteria of HPV infections, the percentage of HPV-positive cancers should be higher than has been found in most studies. The aim of this study was to increase the detection spectrum of HPV polymerase chain reaction (PCR) screening using degenerate consensus primers. MATERIAL AND METHODS: To increase the sensitivity of the assay to one copy of HPV DNA per cell, a two-step PCR was carried out. The products were directly sequenced by means of a cycle sequencing approach. Seventy biopsies from squamous cell carcinomas of the upper aerodigestive tract were screened using the primer system. RESULTS: According to the PCR results, 0/2 carcinomas of the floor of the mouth or tongue, 7/16 biopsies of oropharyngeal cancers, 3/13 hypopharyngeal cancers, 13/34 squamous cell carcinomas of the larynx, 4/4 biopsies from carcinomas of the nasal cavity or paranasal sinuses and 1/1 squamous cell carcinoma of the parotid gland were positive. CONCLUSION: This broad-spectrum PCR can effectively detect HPV in head and neck cancers.

Base Sequence↗

PubMed2003

Structure and properties of an engineered transketolase from maize.

The gene specifying plastid transketolase (TK) of maize (Zea mays) was cloned from a cDNA library by southern blotting using a heterologous probe from sorghum (Sorghum bicolor). A recombinant fusion protein comprising thioredoxin of Escherichia coli and mature TK of maize was expressed at a high level in E. coli and cleaved with thrombin, affording plastid TK. The protein in complex with thiamine pyrophoshate was crystallized, and its structure was solved by molecular replacement. The enzyme is a C2 symmetric homodimer closely similar to the enzyme from yeast (Saccharomyces cerevisiae). Each subunit is folded into three domains. The two topologically equivalent active sites are located in the subunit interface region and resemble those of the yeast enzyme.

Amino Acid Sequence↗

PubMed2002

Incorporation of an amide into 5-phosphonoalkyl-6-D-ribitylaminopyrimidinedione lumazine synthase inhibitors results in an unexpected reversal of selectivity for riboflavin synthase vs lumazine synthase.

Several analogues of a hypothetical intermediate in the reaction catalyzed by lumazine synthase were synthesized and tested as inhibitors of both Bacillus subtilis lumazine synthase and Escherichia coli riboflavin synthase. The new compounds were designed by replacement of a two-carbon fragment of several 5-phosphonoalkyl-6-D-ribitylaminopyrimidinedione lumazine synthase inhibitors with an amide linkage that was envisioned as an analogue of a Schiff base moiety of a hypothetical intermediate in the enzyme-catalyzed reaction. The incorporation of the amide group led to an unexpected reversal in selectivity for inhibition of lumazine synthase vs riboflavin synthase. Whereas the parent 5-phosphonoalkyl-6-D-ribitylaminopyrimidinediones were lumazine synthase inhibitors and did not inhibit riboflavin synthase, the amide-containing derivatives inhibited riboflavin synthase and were only very weak or inactive as lumazine synthase inhibitors. Molecular modeling of inhibitor-lumazine synthase complexes did not reveal a structural basis for these unexpected findings. However, molecular modeling of one of the inhibitors with E. coli riboflavin synthase demonstrated that the active site of the enzyme could readily accommodate two ligand molecules.

Amides↗

PubMed2002

Folate synthesis in plants: the first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I.

GTP cyclohydrolase I (GCHI) mediates the first and committing step of the pterin branch of the folate-synthesis pathway. In microorganisms and mammals, GCHI is a homodecamer of approximately 26-kDa subunits. Genomic approaches identified tomato and Arabidopsis cDNAs specifying approximately 50-kDa proteins containing two GCHI-like domains in tandem and indicated that such bimodular proteins occur in other plants. Neither domain of these proteins has a full set of the residues involved in substrate binding and catalysis in other GCHIs. The tomato and Arabidopsis cDNAs nevertheless encode functional enzymes, as shown by complementation of a yeast fol2 mutant and by assaying GCHI activity in extracts of complemented yeast cells. Neither domain expressed separately had GCHI activity. Recombinant tomato GCHI formed dihydroneopterin triphosphate as reaction product, as do other GCHIs, but unlike these enzymes it did not show cooperative behavior and was inhibited by its substrate. Denaturing gel electrophoresis verified that the bimodular GCHI polypeptide is not cleaved in vivo into its component domains, and size-exclusion chromatography indicated that the active enzyme is a dimer. The deduced tomato and Arabidopsis GCHI polypeptides lack overt targeting sequences and thus are presumably cytosolic, in contrast to other plant folate-synthesis enzymes, which are mitochondrial proteins with typical signal peptides. GCHI mRNA and protein are strongly in expressed unripe tomato fruits, implying that fruit folate is made in situ rather than imported. As ripening advances, GCHI expression declines sharply, and folate content drops, suggesting that folate synthesis fails to keep pace with turnover.

Amino Acid Sequence↗

PubMed2002

Biosynthesis of riboflavin in archaea studies on the mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase of Methanococcus jannaschii.

The hypothetical protein predicted by the open reading frame MJ0055 of Methanococcus jannaschii was expressed in a recombinant Escherichia coli strain under the control of a synthetic gene optimized for translation in an eubacterial host. The recombinant protein catalyzes the formation of the riboflavin precursor 3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 174 nmol mg(-1) min(-1) at 37 degrees C. The homodimeric 51.6-kDa protein requires divalent metal ions, preferentially magnesium, for activity. The reaction involves an intramolecular skeletal rearrangement as shown by (13)C NMR spectroscopy using [U-(13)C(5)]ribulose 5-phosphate as substrate. A cluster of charged amino acid residues comprising arginine 25, glutamates 26 and 28, and aspartates 21 and 30 is essential for catalytic activity. Histidine 164 and glutamate 185 were also shown to be essential for catalytic activity.

Amino Acid Sequence↗

PubMed2002

Formation of metal nanoclusters on specific surface sites of protein molecules.

During vacuum condensation of metals on frozen proteins, nanoclusters are preferentially formed at specific surface sites (decoration). Understanding the nature of metal/protein interaction is of interest for structure analysis and is also important in the fields of biocompatibility and sensor development. Studies on the interaction between metal and distinct areas on the protein which enhance or impede the probability for cluster formation require information on the structural details of the protein's surface underlying the metal clusters. On three enzyme complexes, lumazine synthase from Bacillus subtilis, proteasome from Thermoplasma acidophilum and GTP cyclohydrolase I from Escherichia coli, the decoration sites as determined by electron microscopy (EM) were correlated with their atomic surface structures as obtained by X-ray crystallography. In all three cases, decoration of the same protein results in different cluster distributions for gold and silver. Gold decorates surface areas consisting of polar but uncharged residues and with rough relief whereas silver clusters are preferentially formed on top of protein pores outlined by charged and hydrophilic residues and filled with frozen buffer under the experimental conditions. A common quality of both metals is that they strictly avoid condensation on hydrophobic sites lacking polar and charged residues. The results open ways to analyse the binding mechanism of nanoclusters to small specific sites on the surface of hydrated biomacromolecules by non-microscopic, physical-chemical methods. Understanding the mechanism may lead to advanced decoration techniques resulting in fewer background clusters. This would improve the analysis of single molecules with regard to their symmetries and their orientation in the adsorbed state and in precrystalline assemblies as well as facilitate the detection of point defects in crystals caused by misorientation or by impurities.

Bacillus subtilis↗

PubMed2002

Design, synthesis, and evaluation of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-ribitylaminolumazines as inhibitors of riboflavin synthase and lumazine synthase.

A series of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-D-ribityllumazine were synthesized as inhibitors of both Escherichia coli riboflavin synthase and Bacillus subtilis lumazine synthase. The compounds were designed to bind to both the ribitylpurine binding site and the phosphate binding site of lumazine synthase. In the carboxyalkyl series, maximum activity against both enzymes was observed with the 3'-carboxypropyl compound 22. Lengthening or shortening the chain linking the carboxyl group to the lumazine by one carbon resulted in decreased activity. In the phosphonoxyalkyl series, the 3'-phosphonoxypropyl compound 33 was more potent than the 4'-phosphonoxybutyl derivative 39 against lumazine synthase, but it was less potent against riboflavin synthase. Molecular modeling suggested that the terminal carboxyl group of 6-(3'-carboxypropyl)-7-oxo-8-D-ribityllumazine (22) may bind to the side chains of Arg127 and Lys135 of the enzyme. A hypothetical molecular model was also constructed for the binding of 6-(2'-carboxyethyl)-7-oxolumazine (15) in the active site of E. coli riboflavin synthase, which demonstrated that the active site could readily accommodate two molecules of the inhibitor.

Binding Sites↗

PubMed2002

Biosynthesis of tetrahydrofolate. Stereochemistry of dihydroneopterin aldolase.

7,8-Dihydroneopterin aldolase catalyzes the formation of the tetrahydrofolate precursor, 6-hydroxymethyl-7,8-dihydropterin, and is a potential target for antimicrobial and anti-parasite chemotherapy. The last step of the enzyme-catalyzed reaction is believed to involve the protonation of an enol type intermediate. In order to study the stereochemical course of that reaction step, [1',2',3',6,7-13C5]dihydroneopterin was treated with aldolase in deuterated buffer. The resulting, partially deuterated [6alpha,6,7-13C3]6-hydroxymethyl-7,8-dihydropterin was converted to partially deuterated 6-(R)-[6,7,9,11-13C4]5,10-methylenetetrahydropteroate by a sequence of three enzyme-catalyzed reactions followed by treatment with [13C]formaldehyde. The product was analyzed by multinuclear NMR spectroscopy. The data show that the carbinol group of enzymatically formed 6-hydroxymethyl-dihydropterin contained 2H predominantly in the pro-S position.

Aldehyde-Lyases↗

PubMed2002

The structural basis of riboflavin binding to Schizosaccharomyces pombe 6,7-dimethyl-8-ribityllumazine synthase.

Riboflavin is an essential cofactor in all organisms. Its direct biosynthetic precursor, 6,7-dimethyl-8-ribityllumazine, is synthesised by the enzyme 6,7-dimethyl-8-ribityllumazine synthase. Recently, we have found that the enzyme from Schizosaccharomyces pombe binds riboflavin, the final product of the pathway with a relatively high affinity with a KD of 1.2 microM. Here, we report on the crystal structure of lumazine synthase from S. pombe with bound riboflavin and compare the binding mode with those of the substrate analogue inhibitor 5-nitro-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione and of the product analogue 6-carboxyethyl-7-oxo-8-ribityllumazine. In all complexes the pyrimidinedione moieties of each respective ligand bind in a very similar orientation. Binding of riboflavin additionally involves a stacking interaction of the dimethylbenzene moiety with the side-chain of His94, a highly conserved residue in all lumazine synthases. The enzyme from Bacillus subtilis showed a KD of at least 1 mM whereas the very homologous enzyme from Saccharomyces cerevisiae had a comparable KD of 3.9 microM. Structural comparison of the S. cerevisiae, the S. pombe, and the mutant enzymes suggests that fine tuning of affinity is achieved by influencing this stacking interaction.

Amino Acid Sequence↗

PubMed2002

Investigation of the binding of epimer A of the covalent hydrate of 6,7-bis(trifluoromethyl)-8-D-ribityllumazine to a recombinant F22W Bacillus subtilis lumazine synthase mutant by (15)N[(19)F] REDOR NMR.

The two epimeric covalent hydrates A and B of 6,7-bis(trifluoromethyl)-8-D-ribityllumazine are metabolically stable analogues of hypothetical intermediates proposed in the reactions catalyzed by riboflavin synthase and lumazine synthase. To confirm the stereochemical assignments previously based solely on results for epimer B, a (15)N[(19)F] REDOR NMR study was performed on the complex formed from epimer A and a recombinant, uniformly (15)N-labeled F22W mutant of Bacillus subtilis lumazine synthase. The results indicate that the fluorines of the ligands are closer to the side chain nitrogens of Arg127 and farther away from the side chain nitrogens of Lys135 in epimer B than in epimer A. These results are consistent with the assignment of the earlier 7R configuration of epimer A and the 7S configuration of epimer B.

Bacillus subtilis↗

PubMed2002

Reaction mechanism of GTP cyclohydrolase I: single turnover experiments using a kinetically competent reaction intermediate.

GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopterin 3'-triphosphate, which is the first committed precursor of tetrahydrofolate and tetrahydrobiopterin. The kinetically competent reaction intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone, was used as substrate for single turnover experiments monitored by multiwavelength photometry. The early reaction phase is characterized by the rapid appearance of an optical transient with an absorption maximum centred at 320. This species is likely to represent a Schiff base intermediate at the initial stage of the Amadori rearrangement of the carbohydrate side-chain. Deconvolution of the optical spectra suggested four linearly independent processes. A fifth reaction step was attributed to photodecomposition of the enzyme product. Pre-steady state experiments were also performed with the H179A mutant which can catalyse a reversible conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone but is unable to form the final product, dihydroneopterin triphosphate. Optical spectroscopy failed to detect any intermediate in the reversible reaction sequence catalysed by the mutant protein. The data obtained with the wild-type and mutant protein in conjunction with earlier quenched flow studies show that the enzyme-catalysed opening of the imidazole ring of GTP and the hydrolytic release of formate from the resulting formamide type intermediate are both rapid reactions by comparison with the subsequent rearrangement of the carbohydrate side-chain which precedes the formation of the dihydropyrazine ring of dihydroneopterin triphosphate.

Catalysis↗