Adelbert Bacher

Heavy riboflavin synthase from Bacillus subtilis: Crystal structure analysis of the icosahedral β60 capsid at 3·3 Å resolution☆

Geometric features as well as possible functional properties of the substrate binding sites at the pentamer interfaces are described. Ligand binding at the pentamer interface regions increases the stability of the beta 60 capsid considerably and influences the reassembly of isolated beta-subunits.

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 sativaphototropin 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.

Synthesis of nanophase iron oxide in lumazine synthase capsids.

An enzyme-based bio-nanoreactor: By acting as a mineralization template, lumazine synthase, a 60-subunit enzyme complex which has a hollow porous shell, can fabricate nanocrystalline iron oxide. Fe ions can permeate the capsid through hydrophilic funnel-shaped channels lined with glutamic acid residues and become encapsulated in the cavity as FeIII oxide. The capsid increases in size from 15 to 30 nm in diameter through formation of a higher order structure as the concentration of FeIII increases.

Riboflavin Synthase of Escherichia coli EFFECT OF SINGLE AMINO ACID SUBSTITUTIONS ON REACTION RATE AND LIGAND BINDING PROPERTIES

Conserved amino acid residues of riboflavin synthase from Escherichia coli were modified by site-directed mutagenesis. Replacement or deletion of phenylalanine 2 afforded catalytically inactive proteins. S41A and H102Q mutants had substantially reduced reaction velocities. Replacements of various other conserved polar residues had little impact on catalytic activity.19F NMR protein perturbation experiments using a fluorinated intermediate analog suggest that the N-terminal sequence motif MFTG is part of one of the substrate-binding sites of the protein.

The lumazine synthase/riboflavin synthase complex: shapes and functions of a highly variable enzyme system

The xylene ring of riboflavin (vitamin B2) is assembled from two molecules of 3,4‐dihydroxy‐2‐butanone 4‐phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X‐ray crystallography, X‐ray small‐angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi‐spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.

Pseudilins: Halogenated, Allosteric Inhibitors of the Non‐Mevalonate Pathway Enzyme IspD

The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis have been identified as attractive targets with novel modes of action for the development of herbicides for crop protection and agents against infectious diseases. This pathway is present in many pathogenic organisms and plants, but absent in mammals. By using high-throughput screening, we identified highly halogenated marine natural products, the pseudilins, to be inhibitors of the third enzyme, IspD, in the pathway. Their activity against the IspD enzymes from Arabidopsis thaliana and Plasmodium vivax was determined in photometric and NMR-based assays. Cocrystal structures revealed that pseudilins bind to an allosteric pocket by using both divalent metal ion coordination and halogen bonding. The allosteric mode of action for preventing cosubstrate (CTP) binding at the active site was elucidated. Pseudilins show herbicidal activity in plant assays and antiplasmodial activity in cell-based assays.

Deorphaning Pyrrolopyrazines as Potent Multi‐Target Antimalarial Agents

The discovery of pyrrolopyrazines as potent antimalarial agents is presented, with the most effective compounds exhibiting EC50 values in the low nanomolar range against asexual blood stages of Plasmodium falciparum in human red blood cells, and Plasmodium berghei liver schizonts, with negligible HepG2 cytotoxicity. Their potential mode of action is uncovered by predicting macromolecular targets through avant-garde computer modeling. The consensus prediction method suggested a functional resemblance between ligand binding sites in non-homologous target proteins, linking the observed parasite elimination to IspD, an enzyme from the non-mevalonate pathway of isoprenoid biosynthesis, and multi-kinase inhibition. Further computational analysis suggested essential P. falciparum kinases as likely targets of our lead compound. The results obtained validate our methodology for ligand- and structure-based target prediction, expand the bioinformatics toolbox for proteome mining, and provide unique access to deciphering polypharmacological effects of bioactive chemical agents.

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.

Biosynthesis of riboflavin : 13C-NMR techniques for the analysis of multiply 13C-labeled riboflavins

Abstract Several recently developed NMR techniques were used to analyze samples of biosynthetically labeled riboflavin tetraacetate. The xylene ring of riboflavin consists of two biogenetically identical halves arising from dismutation of a lumazine precursor. The origin of the four carbons which eventually form this xylene ring eas investigated. Carbon-13 labeled precursors were fed to growing cultures of Ashbya gossypii , and the riboflavin produced was isolated and acetylated. A sample of riboflavin tetraacetate derived from [U- 13 C 6 ]glucose gave a complex 13 C- NMR spectrum due to the presence of many 13 C- 13 C couplings and overlapping multiplets. 13 C-homonuclear-J-spectroscopy, which was performed analogously to the proton experiment simplified the spectral analysis of this material. Individual 13 C- multiplets could be viewed, and a projection of the data onto the 13 C-chemical shift axis yielded a 13 C- NMR spectrum which was both 13 C- and 1 H- broadband decoupled (normal 1 H-broadband decoupling was used throughout). A 13 C-homonuclear-2D-chemical shift correlation spectrum was employed to determine coupling partners in the ribityl side chain. However, 2D-INADEQUATE was better suited for determining the carbon-carbon connectivities of the entire spectrum. The combination of these three techniques permitted unambiguous 13 C- NMR assignments to be made, as well as elucidation of the biochemically maintained 13 C- 13 C connectivities in the conversion of the labeled glucose to riboflavin. A riboflavin tetraacetate sample derived from [2, 3- 13 C 2 ]-succinate posed another analytical problem; the ovserved amounts of 13 C- 13 C coupling and the calculated 13 C-enrichments were not the same for the corresponding carbons of the biogenetically equal halves of the xylene ring. This was found to be due to the effect of a neighboring 13 C-atom on the relaxation characterists of an observed 13 C-atom. Remeasurement of the sample in the presence of an electronic relaxation reagent and using an NOE suppressive 1 H-decoupling scheme, yielded equal enrichments and amounts of coupling for the biogenetically equivalent sites. The results suggest that the four carbon atoms in question arise from a pentose or its biochemical equivalent, by excision of a C-4 of a pentose rather loss of a terminal carbon atom (C-1 or C-5).

Catalysis of an Essential Step in Vitamin B2 Biosynthesis by a Consortium of Broad Spectrum Hydrolases.

An enzyme catalysing the essential dephosphorylation of the riboflavin precursor, 5‐amino‐6‐ribitylamino‐2,4(1H,3H)‐pyrimidinedione 5′‐phosphate (6), was purified about 800‐fold from a riboflavin‐producing Bacillus subtilis strain, and was assigned as the translation product of the ycsE gene by mass spectrometry. YcsE is a member of the large haloacid dehalogenase (HAD) superfamily. The recombinant protein was expressed in Escherichia coli. It catalyses the hydrolysis of 6 (vmax, 12 μmol mg−1 min−1; KM, 54 μm) and of FMN (vmax, 25 μmol mg−1 min−1; KM, 135 μm). A ycsE deletion mutant of B. subtilis was not riboflavin dependent. Two additional proteins (YwtE, YitU) that catalyse the hydrolysis of 6 at appreciable rates were identified by screening 13 putative HAD superfamily members from B. subtilis. The evolutionary processes that have resulted in the handling of an essential step in the biosynthesis of an essential cofactor by a consortium of promiscuous enzymes require further analysis.