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Transport justice has two essential dimensions: (1) compensating for inequalities in access to mobility, and (2) mitigating the disproportionately burdensome negative consequences of transport. In light of the urgently needed action regarding climate change especially in the transport sector, measures reducing carbon emissions to mitigate the impact are inevitable. However, policy measures for reaching climate targets should avoid increasing unequal mobility chances. Therefore, there is a need for concepts striving to mitigate both climate impacts and transport injustice. The paper addresses the potential of introducing individual mobility budgets to achieve transport-related climate goals while reducing inequalities in mobility. The concept proposed in this contribution is based on a combination of qualitative and quantitative impact assessment methods including a stakeholder involvement process and transport modelling based on different data sources. The results provide policy recommendations as well as further research requirements, which are already partly addressed iin follow-up projects.

Frank Ebel, Franziska Gürtler, Bastian Schmidt und Gerald Richter: 50 historische Wirtshäuser Schwäbische Alb und Mittleres Neckartal. Verlag Friedrich Pustet Regensburg 2017. 192 Seiten mit farbigen Abbildungen. Fest gebunden € 24,95. ISBN 978-3-7917-2932-9

Refitting public spaces with light-emitting diode (LED) technology in lieu of conventional luminaires bears the risk of compromising lighting quality that road users have already adapted to; this is because the LED technology has been well tested indoors, but it has not been necessarily evaluated outdoors. Further insight into the effects of street lighting on road users is necessary to resolve potential deficiencies of available technologies. This study compares the effects of three different lighting scenarios (conventional lighting, state-of-the-art LED, optimized LED) on road users via surveys (N = 598 persons) and observations (N = 1341 persons) in the city of Vienna. In terms of the uniformity of street illumination and the comfort it provides, the results show the positive effects of LED street lighting both on surveyed pedestrians and vehicle drivers. The observations of pedestrian walking behavior revealed an unexpected result—no significant differences were noted apart from a more centric walking path along the sidewalk under LED illumination, particularly with the optimized LED luminaire.

MATSim is an open-source simulation framework for mesoscopic traffic simulations that has gained popularity in recent years. In this paper, we present a MATSim model for the city of Vienna, with a particular emphasis on the intermodal routing framework used to create agent trips, and the development of a utility function to specify different agents’ mode preferences. To create agent activity chains, we use mobility diaries from the national transportation survey in Austria and disaggregate the available geospatial information to best fit the reported travel times. The novelty of the intermodal framework is the ability to create trips that do not consist of only one mode of transportation, but to also include bicycle, car, and demand-responsive transport (e.g., cab, car sharing) trips in combination with public transportation. To represent the different mobility behaviors of agents, we divide the population into groups and assign them different utility functions for transportation modes according to their socio-demographic characteristics. After presenting the validation of the model, we discuss ways to improve the model.

Light Emitting Diode (LED) technology promises a great energy saving potential and is therefore increasingly used for traffic areas or urban spaces, respectively. While many LED luminaires for street lighting meet the current standards, many light experts as well as the general public judge the lightings as glaring and visually uncomfortable. This leaves room for technological improvement which should be based on shortcomings of the currently available street lighting installations. To address them, we concentrate on road user perception and behavioral impact of conventional luminaire and LED lighting. To identify the influence of different lighting conditions in streets on road users, their perception and behavior had to be surveyed before and after refitting to LED technology in a selected test area. Surveys (n = 386) and participant observation (n = 873) were chosen as methods to provide a reliable basis for the construction of improved LED luminaires by an industrial partner.

This paper proposes a fully specified statistical model for the quasi-dynamic estimation of origin–destination (OD) flows from traffic counts for highway stretches and networks or for urban areas where the path choice is of minor importance. Hereby, the approach (E. Cascetta et al. , Transp. Res. B, Methodol. , vol. 55, pp. 171–187, 2013) is extended by eliminating the need for supplying a historic OD matrix. This is done by a combination of least squares estimation for replicating measured link flows with maximum entropy methods to fill in the non-observable part of the distribution across paths. Additionally, it is stressed that the quasi-dynamic assumption of constant path choice proportions over time-of-day-intervals for days of the same day category can be used in order to enhance estimation by including multi-day observations. Jointly one obtains a statistical framework with an explicit estimation algorithm that can be used to test the quasi-dynamic assumption. The approach is demonstrated to provide accurate results in a small-scale simulation study as well as two real-world case studies, one dealing with a highway segment where taxi floating car data provides the true OD flows for the taxis, and the other one dealing with an urban area with a very limited number of alternative paths allowing for explicit path enumeration.

The cofactor forms of riboflavin (FMN and FAD) play a crucial role in the mediation of both enzymatic processes and light perception by photo-sensitive proteins, and thus structural analogues of this chromophore are highly useful tools to assist in the elucidation of enzymatic mechanisms. 1-Deazariboflavin has been rarely utilised for this purpose, due in part to its previously difficult and inefficient synthesis. Recent examination has enabled a remarkable improvement in the overall synthetic yield from 11.0 to 61.3%, allowing reliable production of 1-deazariboflavin for use as a tool in enzymatic mechanistic determination.

The plant blue light receptor phototropin comprises a protein kinase domain and two FMN-binding LOV domains (LOV1 and LOV2). Blue light irradiation of recombinant LOV domains is conducive to the addition of a cysteinyl thiolate group to carbon 4a of the FMN chromophore, and spontaneous cleavage of that photoadduct completes the photocycle of the receptor. The present study is based on (13)C NMR signal modulation observed after reconstitution of LOV domains of different origins with random libraries of (13)C-labeled FMN isotopologues. Using this approach, all (13)C signals of FMN bound to LOV1 and LOV2 domains of Avena sativa and to the LOV2 domain of the fern, Adiantum capillus-veneris, could be unequivocally assigned under dark and under blue light irradiation conditions. (13)C Chemical shifts of FMN are shown to be differently modulated by complexation with the LOV domains under study, indicating slight differences in the binding interactions of FMN and the apoproteins.

Theg-tensors of two chemically different flavin mononucleotide (FMN) radicals, one of which is covalently bound via N(5) of its 7,8-dimethyl isoalloxazine moiety, and the other one non-covalently bound to mutant LOV domains of the blue-light receptor phototropin, LOV1 C57M and LOV2 C450A, respectively, have been determined by very high microwave frequency and high magnetic field electron paramagnetic resonance (EPR) performed at 360 GHz and 12.8 T. Due to the high spectral resolution of the frozen-solution continuous-wave EPR spectra, the anisotropy of theg-tensors could be fully resolved. By least-squares fittings of spectral simulations to expermental data, the principal values ofg have been established:g X=2.00554(5),g Y=2.00391(5), andg Z=2.00247(7) for the N(5)-alkyl-chain-linked FMN radical in LOV1 C57M-675, andg X=2.00427(5),g Y=2.00360(5), andg Z=2.00220(7) for the noncovalently bound FMN radical in LOV2 C450A-605. By a comparison of these values to the ones from the flavin adenine dinucleotide radicals in two photolyases, the radical in LOV2 C450A-605 could be clearly identified as a neutral FMN radical, FMNH. In contrast, LOV1 C57M-675 exhibits significantly shifted principal components ofg, the differences being caused by spin-orbit coupling of the nearby sulfur from the reactive methionine residue, and the modified chemical structure due to the covalent attachment at N(5) of the radical to the apoprotein. The results clearly show the potential of using theg-tensor as probe of the global electronic and chemical structure of protein-bound flavin radicals.

Cyclobutane-type pyrimidine dimers generated by ultraviolet irradiation of DNA can be cleaved by DNA photolyase. The enzyme-catalysed reaction is believed to be initiated by the light-induced transfer of an electron from the anionic FADH− chromophore of the enzyme to the pyrimidine dimer. In this contribution, first infrared experiments using a novel E109A mutant of Escherichia coli DNA photolyase, which is catalytically active but unable to bind the second cofactor methenyltetrahydrofolate, are described. A stable blue-coloured form of the enzyme carrying a neutral FADH radical cofactor can be interpreted as an intermediate analogue of the light-driven DNA repair reaction and can be reduced to the enzymatically active FADH− form by red-light irradiation. Difference Fourier transform infrared (FT-IR) spectroscopy was used to monitor vibronic bands of the blue radical form and of the fully reduced FADH− form of the enzyme. Preliminary band assignments are based on experiments with 15N-labelled enzyme and on experiments with D2O as solvent. Difference FT-IR measurements were also used to observe the formation of thymidine dimers by ultraviolet irradiation and their repair by light-driven photolyase catalysis. This study provides the basis for future time-resolved FT-IR studies which are aimed at an elucidation of a detailed molecular picture of the light-driven DNA repair process.

NusB is a prokaryotic transcription factor involved in antitermination processes, during which it interacts with the boxA portion of the mRNA nut site. Previous studies have shown that NusB exhibits an all-helical fold, and that the protein from Escherichia coli forms monomers, while Mycobacterium tuberculosis NusB is a dimer. The functional significance of NusB dimerization is unknown. We have determined five crystal structures of NusB from Thermotoga maritima. In three crystal forms the protein appeared monomeric, whereas the two other crystal forms contained assemblies, which resembled the M. tuberculosis dimers. In solution, T. maritima NusB could be cross-linked as dimers, but it migrated as a monomer in gel-filtration analyses, suggesting a monomer/dimer equilibrium with a preference for the monomer. Binding to boxA-like RNA sequences could be detected by gel-shift analyses and UV-induced cross-linking. An N-terminal arginine-rich sequence is a probable RNA binding site of the protein, exhibiting aromatic residues as potential stacking partners for the RNA bases. Anions located in various structures support the assignment of this RNA binding site. The proposed RNA binding region is hidden in the subunit interface of dimeric NusB proteins, such as NusB from M. tuberculosis, suggesting that such dimers have to undergo a considerable conformational change or dissociate for engagement with RNA. Therefore, in certain organisms, dimerization may be employed to package NusB in an inactive form until recruitment into antitermination complexes.

The C terminus of transcription factor NusA from Escherichia coli comprises two repeat units, which bind during antitermination to protein N from phage λ. To delineate the structural basis of the NusA–λN interaction, we attempted to crystallize the NusA C-terminal repeats in complex with a λN peptide (residues 34–47). The two NusA domains became proteolytically separated during crystallization, and crystals contained two copies of the first repeat unit in contact with a single λN fragment. The NusA modules employ identical regions to contact the peptide but approach the ligand from opposite sides. In contrast to the α-helical conformation of the λN N terminus in complex with boxB RNA, residues 34–40 of λN remain extended upon interaction with NusA. Mutational analyses indicated that only one of the observed NusA–λN interaction modes is biologically significant, supporting an equimolar ratio of NusA and λN in antitermination complexes. Solution studies indicated that additional interactions are fostered by the second NusA repeat unit, consistent with known compensatory mutations in NusA and λN. Contrary to the RNA polymerase α subunit, λN binding does not stimulate RNA interaction of NusA. The results demonstrate that λN serves as a scaffold to closely oppose NusA and the mRNA in antitermination complexes.

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 μmol 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 13C NMR spectroscopy. The protein contains 1 eq of Zn2+/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.

An open reading frame (Acc. no. P50740) on the Bacillus subtilis chromosome extending from bp 184 997-186 043 with similarity to the idi-2 gene of Streptomyces sp. CL190 specifying type II isopentenyl diphosphate isomerase was expressed in a recombinant Escherichia coli strain. The recombinant protein with a subunit mass of 39 kDa was purified to apparent homogeneity by column chromatography. The protein was shown to catalyse the conversion of dimethylallyl diphosphate into isopentenyl diphosphate and vice versa at rates of 0.23 and 0.63 mumol.mg(-1).min(-1), respectively, as diagnosed by H-1 spectroscopy. FMN and divalent cations are required for catalytic activity; the highest rates were found with Ca2+. NADPH is required under aerobic but not under anaerobic assay conditions. The enzyme is related to a widespread family of (S)-alpha-hydroxyacid oxidizing enzymes including flavocytochrome b(2) and L-lactate dehydrogenase and was shown to catalyse the formation of [2,3-C-13(2)]lactate from [2,3-C-13(2)]pyruvate, albeit at a low rate of 1 nmol.mg(-1).min(-1). Putative genes specifying type II isopentenyl diphosphate isomerases were found in the genomes of Archaea and of certain eubacteria but not in the genomes of fungi, animals and plants. The analysis of the occurrence of idi-1 and idi-2 genes in conjunction with the mevalonate and nonmevalonate pathway in 283 completed and unfinished prokaryotic genomes revealed 10 different classes. Type II isomerase is essential in some important human pathogens including Staphylococcus aureus and Enterococcus faecalis where it may represent a novel target for anti-infective therapy.

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.

The regulation of ribosomal RNA biosynthesis in Escherichia coli by antitermination requires binding of NusB protein to a dodecamer sequence designated boxA on the nascent RNA. The affinity of NusB protein for boxA RNA exceeds that for the homologous DNA segment by more than three orders of magnitude as shown by surface plasmon resonance measurements. DNA RNA discrimination by NusB protein was shown to involve methyl groups (i.e. discrimination of uracil versus thymine) and 2′ hydroxyl groups (i.e. discrimination of ribose versus deoxyribose side-chains) in the RNA motif. Ligand perturbation experiments monitored by 1H15N correlation NMR experiments identified amide NH groups whose chemical shifts are affected selectively by ribose/deoxyribose exchange in the 5′ and the central part of the dodecameric boxA motif respectively. The impact of structural modification of the boxA motif on the affinity for NusB protein as observed by 1H15N heterocorrelation was analysed by a generic algorithm.

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.

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.

The flavin cofactor of Escherichia coli DNA photolyase in its neutral radical form, FADH•, was investigated by high-frequency/high-field continuous-wave electron paramagnetic resonance at 360 GHz. The data presented are the first flavin radical spectra where the full rhombic symmetry of the g-tensor is resolved. A fit of the spectrum yields accurate principal values of g, which show only a small anisotropy: gX = 2.004 31(5), gY = 2.003 60(5) and gZ = 2.002 17(7). The hyperfine splitting observed in the gY region could be assigned to a hyperfine tensor component of the H(5) proton in the 7,8-dimethyl isoalloxazine moiety of FADH•. From a comparison of this effective hyperfine coupling with the principal value obtained from pulsed (Davies) electron−nuclear double resonance, the orientation of the g-tensor principal axes with respect to the H(5) hyperfine principal axes could be derived. Remaining ambiguities in the sign of the angle between the principal axes of g and the molecular axes are discussed by ta...

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.

Recent developments in NMR have extended the size range of proteins amenable to structural and functional characterization to include many larger proteins involved in important cellular processes. By applying a combination of residue-specific isotope labeling and protein deuteration strategies tailored to yield specific information, we were able to determine the solution structure and study structure–activity relationships of 3,4-dihydroxy-2-butanone-4-phosphate synthase, a 47-kDa enzyme from the Escherichia coli riboflavin biosynthesis pathway and an attractive target for novel antibiotics. Our investigations of the enzyme's ligand binding by NMR and site-directed mutagenesis yields a conclusive picture of the location and identity of residues directly involved in substrate binding and catalysis. Our studies illustrate the power of state-of-the-art NMR techniques for the structural characterization and investigation of ligand binding in protein complexes approaching the 50-kDa range in solution.

The PHOT1 ( NPH1 ) gene from Avena sativa specifies the blue light receptor for phototropism, phototropin, which comprises two FMN-binding LOV domains and a serine/threonine protein kinase domain. Light exposure is conducive to autophosphorylation of the protein kinase domain. We have reconstituted a recombinant LOV2 domain of A. sativa phototropin with various 13 C/ 15 N-labeled isotopomers of the cofactor, FMN. The reconstituted protein samples were analyzed by NMR spectroscopy under dark and light conditions. Blue light irradiation is shown to result in the addition of a thiol group (cysteine 450) to the 4a position of the FMN chromophore. The adduct reverts spontaneously in the dark by elimination. The light-driven flavin adduct formation results in conformational modification, which was diagnosed by 1 H and 31 P NMR spectroscopy. This conformational change is proposed to initiate the transmission of the light signal via conformational modulation of the protein kinase domain conducive to autophosphorylation of NPH1 .

This paper describes the recombinant expression of the ispC gene of Escherichia coli specifying 2C-methyl-D-erythritol 4-phosphate synthase in a modified form that can be purified efficiently by metal-chelating chromatography. The enzyme was used for the preparation of isotope-labeled 2C-methyl-D-erythritol 4-phosphate employing isotope-labeled glucose and pyruvate as starting materials. The simple one-pot methods described afford numerous isotopomers of 2C-methyl-D-erythritol 4-phosphate carrying (3)H, (13)C, or (14)C from commercially available precursors. The overall yield based on the respective isotope-labeled starting material is approximately 50%.

Riboflavin synthase of Escherichia coli is a homotrimer of 23.4 kDa subunits catalyzing the formation of the carbocyclic ring of the vitamin, riboflavin, by dismutation of 6,7-dimethyl-8-ribityllumazine. Intramolecular sequence similarity suggested that each subunit folds into two topologically similar domains. In order to test this hypothesis, sequence segments comprising amino-acid residues 1–97 or 101–213 were expressed in recombinant E. coli strains. The recombinant N-terminal domain forms a homodimer that can bind riboflavin, 6,7-dimethyl-8-ribityllumazine and trifluoromethyl-substituted 8-ribityllumazine derivatives as shown by absorbance, circular dichroism, and NMR spectroscopy. Most notably, the recombinant domain dimer displays the same diastereoselectivity for ligands as the full length protein. The minimum N-terminal peptide segment required for ligand binding comprises amino-acid residues 1–87. The recombinant C-terminal domain comprising amino-acid residues 101–213 is relatively unstable and was shown not to bind riboflavin but to differentiate between certain diastereomeric trifluoromethyl-8-ribityllumazine derivatives. The data show that a single domain comprises the intact binding site for one substrate molecule. The enzyme-catalyzed dismutation requires two substrate molecules to be bound in close proximity, and each active site of the enzyme appears to be located at the interface of an N-terminal and C-terminal domain.

GTP cyclohydrolase II catalyzes the first committed reaction in the biosynthesis of the vitamin riboflavin. The recombinant enzyme from Escherichia coli is shown to produce 2,5-diamino-6-β-ribosylamino-4(3H)-pyrimidinone 5′-phosphate and GMP at an approximate molar ratio of 10:1. The main product is subject to spontaneous isomerization affording the α-anomer. 18O from solvent water is incorporated by the enzyme into the phosphate group of the 5-aminopyrimidine derivative as well as GMP. These data are consistent with the transient formation of a covalent phosphoguanosyl derivative of the enzyme. Subsequent ring opening of the covalently bound nucleotide followed by hydrolysis of the phosphodiester bond could then afford the pyrimidine type product. The hydrolysis of the phosphodiester bond without prior ring opening could afford GMP. The enzyme reaction is cooperative with a Hill coefficient of 1.3. Inhibition by pyrophosphate is competitive. Inhibition by orthophosphate is partially uncompetitive at low concentration and competitive at concentrations above 6 mm.

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. (19)F 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.

Density functional theory is used to calculate the electronic structure of the neutral flavin radical, FADH(*), formed in the light-induced electron-transfer reaction of DNA repair in cis,syn-cyclobutane pyrimidine dimer photolyases. Using the hybrid B3LYP functional together with the double-zeta basis set EPR-II, (1)H, (13)C, (15)N, and (17)O isotropic and anisotropic hyperfine couplings are calculated and explained by reference to the electron densities of the highest occupied molecular orbital and of the unpaired spin distribution on the radical. Comparison of calculated and experimental hyperfine couplings obtained from EPR and ENDOR/TRIPLE resonance leads to a refined structure for the FAD cofactor in Escherichia coli DNA photolyase. Hydrogen bonding at N3H, O4, and N5H results in significant changes in the unpaired spin density distribution and hyperfine coupling constants. The calculated electronic structure of FADH(*) provides evidence for a superexchange-mediated electron transfer between the cyclobutane pyrimidine dimer lesion and the 7,8-dimethyl isoalloxazine moiety of the flavin cofactor via the adenine moiety.

Elastic electron backscattering coefficients have been measured for 24 elemental solids (Ag, Al, Au, Be, Bi, C, Co, Cu, Fe, Ge, Mg, Mn, Mo, Ni, Ta, Te, Ti, Pb, Pd, Pt, Si, V, W and Zn) for energies between 50 and 3400 eV. Values for the electron inelastic mean free path (IMFP) were derived from the experimental intensities on the basis of a simple physical model that accounts for bulk elastic and inelastic scattering only. The internal consistency of the data set and the evaluation procedure is satisfactory for energies >150 eV. For energies ≥200 eV the IMFP values were found to be well described by the Bethe equation for inelastic scattering, as found earlier by analysis of optical scattering data with the aid of the linear response theory [Surf. Interface Anal. 21 (1994) 165]. We also compare the parameters β (being related to the total dipole matrix elements for inelastic scattering) and γ (determining the details of the energy dependence of the IMFP) that appear in the Bethe equation with corresponding results based on optical data. Both parameters were found to agree well with the earlier analysis by Tanuma et al. [Surf. Interface Anal. 21 (1994) 165] within the statistical accuracy of the present analysis. Experimental data on the recoil energy in elastic scattering were also analyzed and compared with detailed results of Monte Carlo simulations of this phenomenon. The recoil energies are correctly predicted to within 10% by the single deflection model based on the Rutherford cross section.

The neutral flavin radical complex of DNA photolyase from Escherichia coli was studied by resonance Raman spectroscopy for the first time. The experiments were carried out with enzyme variants that lack the light-harvesting methenyltetrahydrofolate cofactor so that the resonance Raman spectra exclusively display the vibrational bands of the flavin radical in the catalytic site. Spectral changes induced upon H–D exchange and upon substituting the natural flavin by its 15N-labelled isotopomer allow a tentative assignment of the prominent bands in the region between 1200 and 1650 cm−1. It is shown that most of the bands observed with 568 nm excitation originate from modes localized in the isoalloxazine ring. In conjunction with previous data on flavin radicals, the present results allow the identification of the marker bands that appear to be particularly sensitive towards changes in the electron density distribution of this ring and, hence, reflect the specific intermolecular interactions in the catalytic site. Copyright © 2001 John Wiley & Sons, Ltd.

Elastic electron backscattering coefficients have been measured for 24 elemental solids (Ag, Al, Au, Be, Bi, C, Co, Cu, Fe, Ge, Mg, Mn, Mo, Ni, Ta, Te, Ti, Pb, Pd, Pt, Si, V, W, Zn) for energies between 50 and 3400 eV. Values for the electron inelastic mean free path (IMFP) were derived from the experimental intensities on the basis of a simple physical model for elastic backscattering that accounts for bulk elastic and inelastic scattering only. For energies ⩾200 eV the IMFP values were found to be well described by the Bethe equation for inelastic scattering, as found earlier by analysis of optical scattering data with the aid of linear response theory (Surf. Interf. Anal. 21 (1994) 165). The parameters β and γ appearing in the Bethe equation are compared with corresponding results derived from optical data. Both, the parameter β , that is related to the total dipole matrix element for inelastic scattering, as well as the parameter γ , that describes the energy dependence of the IMFP, were found to coincide with the results based on optical data within the experimental accuracy of our analysis. Since the present analysis of electron scattering data is entirely free of linear response theory, the present results may be regarded as an independent test of the previous analysis based on optical scattering data.

A hypothetical gene with similarity to the ispD gene of Escherichia coli was cloned from Arabidopsis thaliana cDNA. The ORF of 909 bp specifies a protein of 302 amino acid residues. The cognate chromosomal gene consists of 2,071 bp and comprises 11 introns with a size range of 78–202 bp. A fragment comprising amino acid residues 76–302 was expressed in a recombinant E. coli strain. The protein was purified to homogeneity and was shown to catalyze the formation of 4-diphosphocytidyl-2C-methyl- d -erythritol from 2C-methyl- d -erythritol 4-phosphate with a specific activity of 67 μmol⋅min −1 mg −1 . The Michaelis constants for 4-diphosphocytidyl-2C-methyl- d -erythritol and CTP were 500 μM and 114 μM, respectively.

NMR investigations of larger macromolecules (>20 kDa) are severely hindered by rapid 1H and 13C transverse relaxation. Replacement of non-exchangeable protons with deuterium removes many efficient 1H-1H and 1H-13C relaxation pathways. The main disadvantage of deuteration is that many of the protons which would normally be the source of NOE-based distance restraints are removed. We report the development of a novel labeling strategy which is based on specific protonation and 14N-labeling of the residues phenylalanine, tyrosine, threonine, isoleucine and valine in a fully deuterated, 15N-labeled background. This allows the application of heteronuclear half-filters, 15N-editing and 1H-TOCSY experiments to select for particular magnetization transfer pathways. Results from investigations of a 47 kDa dimeric protein labeled in this way demonstrated that the method provides useful information for the structure determination of large proteins.

One of the proteins encoded by the riboflavin operon of Bacillus subtilis, RibA, was identified as the rate limiting enzyme in an industrial riboflavin producing strain. An additional single copy of the ribA gene was introduced into the sacB locus of the riboflavin production strain and was expressed constitutively from the medium strength vegI promoter. This led to improved riboflavin titers and yields of riboflavin on glucose of up to 25%. Both enzymatic activities of RibA, the 3,4-dihydroxy-2-butanone 4-phosphate synthase activity located in the N-terminal half of the protein and the GTP cyclohydrolase II activity of the C-terminal domain, are necessary for the improved riboflavin productivity.

An open reading frame located at 69.0 kilobases on the Escherichia coli chromosome was shown to code for dihydroneopterin aldolase, catalyzing the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin in the biosynthetic pathway of tetrahydrofolate. The gene was subsequently designated folB. The FolB protein shows 30% identity to the paralogous dihydroneopterin-triphosphate epimerase, which is specified by the folX gene located at 2427 kilobases on the E. coli chromosome. The folX and folB gene products were both expressed to high yield in recombinant E. coli strains, and the recombinant proteins were purified to homogeneity. Both enzymes form homo-octamers. Aldolase can use L-threo-dihydroneopterin and D-erythro-dihydroneopterin as substrates for the formation of 6-hydroxymethyldihydropterin, but it can also catalyze the epimerization of carbon 2' of dihydroneopterin and dihydromonapterin at appreciable velocity. Epimerase catalyzes the epimerization of carbon 2' in the triphosphates of dihydroneopterin and dihydromonapterin. However, the enzyme can also catalyze the cleavage of the position 6 side chain of several pteridine derivatives at a slow rate. Steady-state kinetic parameters are reported for the various enzyme-catalyzed reactions. We propose that the polarization of the 2'-hydroxy group of the substrate could serve as the initial reaction step for the aldolase as well as for the epimerase activity. A deletion mutant obtained by targeting the folX gene of E. coli has normal growth properties on complete medium as well as on minimal medium. Thus, the physiological role of the E. coli epimerase remains unknown. The open reading frame ygiG of Hemophilus influenzae specifies a protein with the catalytic properties of an aldolase. However, the genome of H. influenzae does not specify a dihydroneopterin-triphosphate epimerase.

Flowing enzymes: Continuous extraction of products enhances the enzymatic productivity of sesquiterpenes (see figure). Even unnatural substrates are tolerated leading to valuable unnatural target molecules in superior yields compared with batch protocols.

The heterologous expression of mammalian membrane proteins in lower eukaryotes is often hampered by aberrant protein localization, structure, and function, leading to enhanced degradation and, thus, low expression levels. Substantial quantities of functional membrane proteins are necessary to elucidate their structure-function relationships. Na,K-ATPases are integral, human membrane proteins that specifically interact with cholesterol and phospholipids, ensuring protein stability and enhancing ion transport activity. In this study, we present a Pichia pastoris strain which was engineered in its sterol pathway towards the synthesis of cholesterol instead of ergosterol to foster the functional expression of human membrane proteins. Western blot analyses revealed that cholesterol-producing yeast formed enhanced and stable levels of human Na,K-ATPase α3β1 isoform. ATPase activity assays suggested that this Na,K-ATPase isoform was functionally expressed in the plasma membrane. Moreover, [(3)H]-ouabain cell surface-binding studies underscored that the Na,K-ATPase was present in high numbers at the cell surface, surpassing reported expression strains severalfold. This provides evidence that the humanized sterol composition positively influenced Na,K-ATPase α3β1 stability, activity, and localization to the yeast plasma membrane. Prospectively, cholesterol-producing yeast will have high potential for functional expression of many mammalian membrane proteins.

The gene coding for riboflavin synthase of Escherichia coli has been cloned by marker rescue on a 6-kb fragment that has been sequenced. The riboflavin synthase gene is identical to the ribC locus and codes for a protein of 213 amino acids with a miss of 23.4 kDa. It was mapped to a position at 37.5 min on the physical map of the E. coli chromosome. The 3′ end of the ribC gene is directly adjacent to the cfa gene, which codes for cyclopropane-fatty-acid synthase. This gene is followed by two open reading frames designated ydhC and ydhB, which are predicted to code for putative proteins with 403 amino acids and 310 amino acids, respectively. The gene ydhC is similar to genes coding for resistance against various antibiotics (cmlA, bcr) and probably codes for a transmembrane protein. The protein specified by ydhB shows sequence similarity to a large family of DNA-binding proteins and probably represents a helix-turn-helix protein. The ydhB gene is directly adjacent to the regulatory gene purR. A 288-bp segment of the cfa gene has earlier been mapped incorrectly to a position adjacent to greA at 67 min. The ribC gene was hyperexpressed in recombinant E. coli strains to a level of about 30 % of cellular protein. The protein was purified to homogeneity by chromatography. The specific activity was 26000 nmol · mg−1· h−1. The protein sediments at a velocity of s20= 3.8 S. Sedimentation-equilibrium centrifugation indicated a molecular mass of 70 kDa, consistent with a trimer structure. The primary structure of riboflavin synthase is characterized by internal sequence similarity (25 identical amino acids in the C-terminal and N-terminal parts) suggesting two structurally similar folding domains.

A gene located at 443 kilobases on the Escherichia coli chromosome (subsequently designated ribE) was expressed in a recombinant E. coli strain and was shown to code for the enzyme 6,7-dimethyl-8-ribityllumazine synthase. The recombinant enzyme was purified to homogeneity. The protein is an icosahedral capsid of 60 subunits with a mass of about 1 MDa as shown by hydrodynamic studies and by electron microscopy. In contrast to the icosahedral lumazine synthase-riboflavin synthase complex of Bacillus subtilis, the lumazine synthase of E. coli is not physically associated with another enzyme of the riboflavin pathway, and the core of the icosahedral capsid is empty. The RIB4 gene of Saccharomyces cerevisiae was also expressed to a high level (about 40% of cellular protein) in E. coli. The recombinant protein is a pentamer of 90 kDa. An insertion of 4 amino acids into helix α4 is likely to hinder the formation of an icosahedral capsid by the yeast protein. The kinetic properties of lumazine synthase of E. coli, B. subtilis, and S. cerevisiae are similar.

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits enclosing a triplet of alpha subunits. An X-ray structure of 0.32 nm resolution has been obtained for the icosahedral capsid of the native alpha 3 beta 60 complex [Ladenstein, R., Schneider, M., Huber, R., Bartunik, H. D., Wilson, K., Schott, K.Bacher, A. (1988) J. Mol. Biol. 203, 1045-1070]. beta subunits were isolated after denaturation of the alpha 3 beta 60 complex and were subsequently reconstituted in a ligand-driven reaction yielding artifactual, hollow beta 60 capsids with icosahedral symmetry. Hexagonal crystals (space group P6(3)22) of the reconstituted capsids diffracted X-rays to a resolution of 0.32 nm. Crystallographic intensity data were obtained using synchrotron radiation. Freeze-etched electron-microscopic images and rotation function calculations showed that the hexagonal crystal forms of the artifactual beta 60 capsids and the native alpha 3 beta 60 complex are isomorphous. Orientation and translation parameters of the beta-subunit model were refined by XPLOR rigid-body refinement. The electron-density map was improved by cyclic icosahedral averaging and phase extension from 0.5-0.32 nm resolution. The beta-subunit structure was partially refined by energy minimization and crystallographic refinement (XPLOR) assuming strict icosahedral symmetry (final R factor 30.9% for data at 0.8-0.32 nm resolution). The topology and chain folding of the beta subunits in the artifactual beta 60 capsid are similar to the native alpha 3 beta 60 enzyme. Structural features of the substrate-binding site and the binding of the substrate-analogous ligand 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione are discussed. Ligand binding occurs at the pentamer interfaces and includes van der Waals' interactions and hydrogen bonding. The binding pocket shows a hydrophobic region which accomodates the pyrimidinedione ring and a hydrophilic region to which the ribityl side chain binds. Most amino acid residues involved in the active site are conserved as shown by sequence comparisons with the putative lumazine-synthase genes of Escherichia coli and Photobacterium leiognathi. In the final electron-density map, a residual density feature was tentatively assigned to a bound phosphate ion which mimics the binding of the second substrate, 3,4-dihydroxy-2-butanone 4-phosphate. This putative phosphate-binding site involves a highly conserved amino acid sequence containing three basic residues.

GTP cyclohydrolase II catalyzes the first committed step in the biosynthesis of riboflavin. The gene coding for this enzyme in Escherichia coli has been cloned by marker rescue. Sequencing indicated an open reading frame of 588 bp coding for a 21.8-kDa peptide of 196 amino acids. The gene was mapped to a position at 28.2 min on the E. coli chromosome and is identical with ribA. GTP cyclohydrolase II was overexpressed in a recombinant strain carrying a plasmid with the cloned gene. The enzyme was purified to homogeneity from the recombinant strain. The N-terminal sequence determined by Edman degradation was identical to the predicted sequence. The sequence is homologous to the 3' part of the central open reading frame in the riboflavin operon of Bacillus subtilis.

3,4-Dihydroxy-2-butanone 4-phosphate is biosynthesized from ribulose 5-phosphate and serves as the biosynthetic precursor for the xylene ring of riboflavin. The gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli has been cloned and sequenced. The gene codes for a protein of 217 amino acid residues with a calculated molecular mass of 23,349.6 Da. The enzyme was purified to near homogeneity from a recombinant E. coli strain and had a specific activity of 1,700 nmol mg-1 h-1. The N-terminal amino acid sequence and the amino acid composition of the protein were in agreement with the deduced sequence. The molecular mass as determined by ion spray mass spectrometry was 23,351 +/- 2 Da, which is in agreement with the predicted mass. The previously reported loci htrP, "luxH-like," and ribB at 66 min of the E. coli chromosome are all identical to the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase, but their role had not been hitherto determined. Sequence homology indicates that gene luxH of Vibrio harveyi and the central open reading frame of the Bacillus subtilis riboflavin operon code for 3,4-dihydroxy-2-butanone 4-phosphate synthase.

13C-NMR experiments were performed on photo-excited fully and partially 13C-labelled LOV2 domains of the blue-light receptor phototropin. In the present paper, we report on nuclear-spin polarized tryptophan resonances that are generated by light-induced intraprotein electron transfer to the FMN cofactor. The spectra are discussed with respect to earlier data obtained from 13C-NMR experiments on unlabelled LOV2 domains that have been reconstituted with FMN 13C isotopologues.

The plant blue light receptor phototropin comprises a protein kinase domain and two FMN-binding LOV domains (LOV1 and LOV2). Blue light irradiation of recombinant LOV domains is conducive to the addition of a cysteinyl thiolate group to carbon 4a of the FMN chromophore, and spontaneous cleavage of that photoadduct completes the photocycle of the receptor. The present study is based on (13)C NMR signal modulation observed after reconstitution of LOV domains of different origins with random libraries of (13)C-labeled FMN isotopologues. Using this approach, all (13)C signals of FMN bound to LOV1 and LOV2 domains of Avena sativa and to the LOV2 domain of the fern, Adiantum capillus-veneris, could be unequivocally assigned under dark and under blue light irradiation conditions. (13)C Chemical shifts of FMN are shown to be differently modulated by complexation with the LOV domains under study, indicating slight differences in the binding interactions of FMN and the apoproteins.

In 1949 an enzyme was discovered that could repair UV-light-induced DNA lesions, by effectively reversing their formation with the aid of blue light. The class of enzymes has subsequently been named photolyases and the process, photoreactivation. All photolyases utilize a non-covalently bound cofactor, flavin adenine dinucleotide (FAD). With the exception of cryptochromes, with which photolyases share a high degree of structural homology in the N-terminal domain, photolyases show no similarity to other blue-light sensing flavoproteins, such as the phototropins or the recently discovered BLUF domains. Although, flavoproteins are ubiquitous, redox-active catalysts for one- and two-electron transfer reactions, it is the properties of the photoexcited states of the different redox states of FAD that are also important in photolyases. Two milestones mark the progress of photolyase research in recent years. The first was the elucidation of the three-dimensional structure of the enzyme in 1995 that revealed remarkable details, such as the FAD-cofactor arrangement in an unusual U-shaped configuration, and in late 2004 a structure was presented that included a bound fragment of repaired DNA. In the 10 years between, research on photolyases continued at a frenetic pace using all available biochemical and biophysical tools. This chapter focuses on recent biophysical studies and shows how different approaches have yielded details on the fundamental aspects of the modus operandi of this unique class of flavoenzymes.

Phototropin is a blue-light receptor involved in the phototropic response of higher plants. The photoreceptor comprises a protein kinase domain and two structurally similar flavin-mononucleotide (FMN) binding domains designated LOV1 and LOV2. Blue-light irradiation of recombinant LOV2 domains induces the formation of a covalent adduct of the thiol group of a functional cysteine in the cofactor-binding pocket to C(4a) of the FMN. Cysteine-to-alanine mutants of LOV domains are unable to form that adduct but generate an FMN radical upon illumination. The recombinant C450A mutant of the LOV2 domain of Avena sativa phototropin was reconstituted with universally and site-selectively (13)C-labeled FMN and the (13)C NMR signals were unequivocally assigned. (13)C NMR spectra were acquired in darkness and under blue-light irradiation. The chemical shifts and the coupling patterns of the signals were not affected by irradiation. However, under blue-light exposure, exceptionally strong nuclear-spin polarization was developed in the resonances belonging to certain carbons of the FMN's isoalloxazine moiety. An enhancement of the NMR absorption was observed for the signals of C(5a), C(7), and C(9). NMR lines in emission were detected for the signals belonging to C(2), C(4), C(4a), C(6), C(8), and C(9a). The signal of C(10a) remained in absorption but was slightly attenuated. In contrast, the intensities of the NMR signals belonging to the carbons of the ribityl side chain of FMN were not affected by light. The observation of spin-polarized (13)C-nuclei in the NMR spectra of the mutant LOV2 domain is clear evidence for radical-pair intermediates in the reaction steps following optical sample excitation.

The recent observation of photoinduced radical pairs comprising a flavin radical and an oxidized amino acid residue in various blue-light-sensitive proteins has highlighted the need to gain a more complete understanding of the electronic structure of flavin radicals. In particular, precise knowledge of the anisotropy of the Zeeman interaction quantified by the g-tensor is necessary for attaining an unambiguous identification of flavin radicals by electron paramagnetic resonance (EPR). In a recent study of a protein-bound neutral flavin radical, we have determined the principal values of the g-tensor using high-frequency/high magnetic field EPR performed at 360 GHz/12.8 T. However, in those experiments, the orientation of the principal axes of g could not be unambiguously established with respect to the molecular frame of the isoalloxazine moiety. In this contribution we resolve this ambiguity by pulsed electron-nuclear double resonance (ENDOR) at 95 GHz/3.5 T (W-band). At such high values of the microwave frequency and the magnetic field, the g anisotropy provides improved spectral resolution compared to an ENDOR experiment performed at conventional 9.5 GHz/0.35 mT (X-band). This enables one to utilize Zeeman magnetoselection to obtain single-crystal-like data from disordered samples in frozen solution. Experiments exploiting this orientation selection have allowed us to use the hyperfine coupling of the methyl protons at C(8alpha) of the isoalloxazine ring to determine the angle between the molecular frame and the principal axes of g. Quite surprisingly, the g-tensor in FADH* is not oriented as one would have expected for a 1,3-semibenzoquinone radical. For the latter, the X-axis of g commonly bisects the smaller angle between the two axes along the C=O bonds. In FADH*, the large spin density on N(5) and C(4a) apparently contributes to a significant (44 degrees ) reorientation of the g-tensor axes.

Cyclobutane-type pyrimidine dimers generated by ultraviolet irradiation of DNA can be cleaved by DNA photolyase. The enzyme-catalysed reaction is believed to be initiated by the light-induced transfer of an electron from the anionic FADH- chromophore of the enzyme to the pyrimidine dimer. In this contribution, first infrared experiments using a novel E109A mutant of Escherichia coli DNA photolyase, which is catalytically active but unable to bind the second cofactor methenyltetrahydrofolate, are described. A stable blue-coloured form of the enzyme carrying a neutral FADH radical cofactor can be interpreted as an intermediate analogue of the light-driven DNA repair reaction and can be reduced to the enzymatically active FADH- form by red-light irradiation. Difference Fourier transform infrared (FT-IR) spectroscopy was used to monitor vibronic bands of the blue radical form and of the fully reduced FADH- form of the enzyme. Preliminary band assignments are based on experiments with 15N-labelled enzyme and on experiments with D2O as solvent. Difference FT-IR measurements were also used to observe the formation of thymidine dimers by ultraviolet irradiation and their repair by light-driven photolyase catalysis. This study provides the basis for future time-resolved FT-IR studies which are aimed at an elucidation of a detailed molecular picture of the light-driven DNA repair process.

An X- (9.7 GHz) and W-bond (94 GHz) pulsed electron-nuclear double resonance (ENDOR) study of the flavin cofactor of Escherichia coli DNA photolyose in its neutral radical form is presented. Through proton and deuteron ENDOR measurements at T=80 K, we detect and characterize the full anisotropy of the hyperfine coupling (hfc) tensor of the proton or deuteron bound to N(5) of the isoalloxazine ring. Scaling of the anisotropic proton hfc components by multiplication with the quotient of the magnetogyric ratio of a deuteron and a proton, chi(D)/chi(H), reveals subtle differences compared to the respective deuteron couplings obtained by 95-GHz deuterium ENDOR spectroscopy on an H-->D buffer-ex-changed sample. These differences can be attributed to the different lengths of N(5)-H and N(5)-D bonds arising from the different masses of protons and deuterons. From the R-3 dependence of the dipolar hyperfine splitting, we estimated that the N(5)-D bond is about 2.5% shorter than the respective N(5)-H bond. That such subtle bond-length differences con be resolved by pulsed ENDOR spectroscopy suggests that this method may be favorably used to probe the geometry of hydrogen bonds between the H(5) of the paramagnetic flavin and the protein backbone. Such information is only obtained with difficulty by other types of spectroscopy.