Winfried Meining

Atomic structure of GTP cyclohydrolase I

BACKGROUND Tetrahydrobiopterin serves as the cofactor for enzymes involved in neurotransmitter biosynthesis and as regulatory factor in immune cell proliferation and the biosynthesis of melanin. The biosynthetic pathway to tetrahydrobiopterin consists of three steps starting from GTP. The initial reaction is catalyzed by GTP cyclohdrolase I (GTP-CH-I) and involves the chemically complex transformation of the purine into the pterin ring system. RESULTS The crystal structure of the Escherichia coli GTP-CH-I was solved by single isomorphous replacement and molecular averaging at 3.0 A resolution. The functional enzyme is a homodecameric complex with D5 symmetry, forming a torus with dimensions 65 A x 100 A. The pentameric subunits are constructed via an unprecedented cyclic arrangement of the four-stranded antiparallel beta-sheets of the five monomers to form a 20-stranded antiparallel beta-barrel of 35 A diameter. Two pentamers are tightly associated by intercalation of two antiparallel helix pairs positioned close to the subunit N termini. The C-terminal domain of the GTP-CH-I monomer is topologically identical to a subunit of the homohexameric 6-pyruvoyl tetrahydropterin synthase, the enzyme catalyzing the second step in tetrahydrobiopterin biosynthesis. CONCLUSIONS The active site of GTP-CH-I is located at the interface of three subunits. It represents a novel GTP-binding site, distinct from the one found in G proteins, with a catalytic apparatus that suggest involvement of histidines and, possibly, a cystine in the unusual reaction mechanism. Despite the lack of significant sequence homology between GTP-CH-I and 6-pyruvoyl tetrahydropterin synthase, the two proteins, which catalyze consecutive steps in tetrahydrobiopterin biosynthesis, share a common subunit fold and oligomerization mode. In addition, the active centres have an identical acceptor site for the 2-amino-4-oxo pyrimidine moiety of their substrates which suggests an evolutionarily conserved protein fold designed for pterin biosynthesis.

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.

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.

Structure of Methanocaldococcus Jannaschii Nucleoside Kinase: An Archaeal Member of the Ribokinase Family.

Nucleoside kinase from the hyperthermophilic archaeon Methanocaldococcus jannaschii (MjNK) is a member of the ribokinase family. In the presence of ATP and Mg(2+), MjNK is able to catalyze the phosphorylation of a variety of nucleosides, including inosine, cytidine, guanosine and adenosine. Here, the crystal structure of MjNK, the first structure of an archaeal representative of the ribokinase family, is presented. The structure was solved using the multiple-wavelength anomalous dispersion technique. Three-dimensional structures of the unliganded enzyme and a complex of MjNK, an ATP analogue and adenosine were determined to 1.7 and 1.9 A resolution, respectively. Each subunit comprises an alpha/beta-domain and a smaller lid domain and has an overall fold characteristic of the ribokinase superfamily. MjNK shares highest structural similarity to the ribokinases from Escherichia coli and Thermotoga maritima. Similar to ribokinase and other superfamily members, the lid domain of MjNK undergoes a significant conformational change upon substrate binding. In the crystal structure of the MjNK complex, subunit A adopts a closed conformation and subunit B an open conformation. In subunit A all substrates and Mg(2+) were observed, whereas in subunit B only the ATP analogue could be clearly identified in the electron density. The structures of MjNK and E. coli ribokinase (EcRK) were compared with respect to putative determinants of thermal stability. Relative to EcRK, MjNK shows an increased charged and a decreased hydrophobic accessible surface area, as well as a higher fraction of charged residues, ionic networks and large aromatic clusters, characteristics that are frequently observed in enzymes from hyperthermophiles.

Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. Alcohol dehydrogenase toward NADH cosubstrate specificity.

The NADP+‐dependent alcohol dehydrogenase from Ralstonia sp. (RasADH) belongs to the protein superfamily of short‐chain dehydrogenases/reductases (SDRs). As an enzyme that accepts different types of substrates—including bulky–bulky as well as small–bulky secondary alcohols or ketones—with high stereoselectivity, it offers potential as a biocatalyst for industrial biotechnology. To understand substrate and cosubstrate specificities of RasADH we determined the crystal structure of the apo‐enzyme as well as its NADP+‐bound state with resolutions down to 2.8 Å. RasADH displays a homotetrameric quaternary structure that can be described as a dimer of homodimers while in each subunit a seven‐stranded parallel β‐sheet, flanked by three α‐helices on each side, forms a Rossmann fold‐type dinucleotide binding domain. Docking of the well‐known substrate (S)‐1‐phenylethanol clearly revealed the structural determinants of stereospecificity. To favor practical RasADH application in the context of established cofactor recycling systems, for example, those involving an NADH‐dependent amino acid dehydrogenase, we attempted to rationally change its cosubstrate specificity from NADP+ to NAD+ utilizing the structural information that NADP+ specificity is largely governed by the residues Asn15, Gly37, Arg38, and Arg39. Furthermore, an extensive sequence alignment with homologous dehydrogenases that have different cosubstrate specificities revealed a modified general SDR motif ASNG (instead of NNAG) at positions 86–89 of RasADH. Consequently, we constructed mutant enzymes with one (G37D), four (N15G/G37D/R38V/R39S), and six (N15G/G37D/R38V/R39S/A86N/S88A) amino acid exchanges. RasADH (N15G/G37D/R38V/R39S) was better able to accept NAD+ while showing much reduced catalytic efficiency with NADP+, leading to a change in NADH/NADPH specificity by a factor of ∼3.6 million. Biotechnol. Biotechnol. Bioeng. 2013;110: 2803–2814.

Virtual Screening, Selection and Development of a Benzindolone Structural Scaffold for Inhibition of Lumazine Synthase

Virtual screening of a library of commercially available compounds versus the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a K(i) of 70microM. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (K(i) 38microM). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve pi-pi stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87.

Studies on GTP Cyclohydrolase I of Escherichia Coli

GTP cyclohydrolase I (EC 3.5.4.16) has been obtained from Escherichia coli wild type cells by affinity chromatography.1,2 The gene coding for the enzyme from E. coli has been cloned and sequenced3,4 and has been mapped at 2251 kb of the physical map of the E. coli chromosome.5 Strains carrying a plasmid with the gene under the control of its own promoter expressed about 100-fold increased enzyme levels. The protein has been crystallized from citrate buffer.6 GTP cyclohydrolase genes of rat,7 man,8,9 and Bacillus subtilis 10 have also been cloned, sequenced and expressed.

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 proteins 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.

METAMORPHOSIS OF AN ENZYME

ABSTRACT The protein shells of the bifunctional Lumazine/Riboflavin synthase complex found in bacteria, archaea and plants show some similarity to the assembly of small spherical viruses. Sixty lumazine synthase subunits form a T = 1 icosahedral capsid, which instead of nucleic acids in the central core, contains a trimer of riboflavin synthase. Lumazine synthases from fungi, yeasts and some bacteria, however, exist only in pentameric form. Capsid formation in icosahedral lumazine synthases is dependent on the presence of certain substrate-analogous ligands, on pH and phosphate concentration. The experimental background from X-ray crystallography, X-ray small angle scattering and electron microscopy will be discussed. Different active assemblies of the enzyme are observed in vivo and in vitro. There is experimental evidence for the formation of large capsids, obtained spontaneously or after certain mutations to the sequence of the lumazine synthase subunit. Those presumably metastable T = 3 capsids can be reassembled into T = 1 capsids by ligand-driven reassembly in vitro. Cryo electron microscopy of the IDEA mutant of Aquifex aeolicus LS surprisingly showed large icosahedral 180 subunit capsids (T = 3) with a diameter of ∼ 290 Å. The pentamers in this structure assumed an expanded conformation including a widened central channel. This feature led us to suggest a model for assembly-controlled catalysis, which relates the LS/RS complex, on a microscopic scale, to form and function of a biochemical reactor.

XtalBase - : a comprehensive data management system for macromolecular crystallography

The rate at which successful crystallization experiments and new structures of macromolecules are reported is growing at a fast pace. It is therefore desirable to collect data systematically, handle these data effectively and evaluate the accumulated knowledge in an automated fashion in order to optimize procedures for crystallization and data evaluation. A new web-based program, XtalBase, has been developed in order to aid the crystallographer in designing, preparing, documenting and evaluating crystallization experiments. XtalBase hosts a database of physicochemical data of compounds commonly used for the crystallization of macromolecules. Experimental conditions are constructed either from existing commercial screening sets, by random, by interpolation between start and end concentrations, or as sets of individually composed drops. Recipes needed for the preparation of solutions and drops are instantly calculated and displayed. The program allows for documenting observations in the form of scores, notes and pictures. The experimental data recorded within the program as well as data imported from the Biological Macromolecule Crystallization Database (BMCD) can be used to estimate a probability of success for crystallization conditions. The program enables the user statistically to correlate conditions with observations not only from their own experiments but from all experiments documented within the system. XtalBase provides facilities for storing a large variety of data, such as a sample history, collection and refinement parameters, coordinates, as well as publication data. Data can be imported and exported via customizable templates. The system can be configured to execute programs, which are fed by data from the database, and to extract data from the program output. The administrative interface allows the customization of all data tables and fields. All data can be searched in a unified search facility. The program presents the basis for a consistently defined database of crystallization data combined with tools to handle crystallization and crystallographic data via a web browser. The program home page is http://www.xtalbase.net.