Gerald Richter

An optomechanical transducer in the blue light receptor phototropin from Avena sativa

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 13C/15N-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 1H and 31P 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.

Biosynthesis of riboflavin

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate. The imidazole ring of GTP is hydrolytically opened, yielding a 4,5-diaminopyrimidine that is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction, and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. Two reaction steps in the biosynthetic pathway catalyzed by 3,4-dihydroxy-2-butanone 4-phosphate synthase and riboflavin synthase are mechanistically very complex. The enzymes of the riboflavin pathway are potential targets for antibacterial agents.

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.

BIOSYNTHESIS OF RIBOFLAVIN : LUMAZINE SYNTHASE OF ESCHERICHIA COLI

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.

Biosynthesis of Flavins

The heterocyclic moieties of flavin and deazaflavin coenzymes are derived from a purine precursor by a common pathway with the intermediate 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione as the branching point. The formation of the xylene ring of riboflavin occurs by the stepwise condensation of this pyrimidine with two molecules of L-3,4-dihydroxy-2-butanone-4-phosphate. This novel carbohydrate is formed from a pentulose phosphate by a mechanistically complex rearrangement reaction. An enzyme complex from Bacillus subtilis catalyzing the last two steps in the riboflavin pathway has been studied in considerable detail. The unusual structure of the protein involves a capsid of 60 β subunits with icosahedral 532 symmetry. The core space of the capsid is occupied by 3 α subunits. All 5 genes coding for enzymes of riboflavin biosynthesis form a single operon in Bacillus subtilis. Five rib genes have been located at 4 positions of the Escherichia coli chromosome. In some luminescent bacteria, various rib genes form part of the luciferase operon.

Light-induced reactions of Escherichia coli DNA photolyase monitored by Fourier transform infrared spectroscopy

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.

Biosynthesis of Pteridines in Escherichia coli STRUCTURAL AND MECHANISTIC SIMILARITY OF DIHYDRONEOPTERIN-TRIPHOSPHATE EPIMERASE AND DIHYDRONEOPTERIN ALDOLASE

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 theE. coli chromosome. The folX andfolB 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 andd-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 frameygiG 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.

The NMR structure of the 47-kDa dimeric enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase and ligand binding studies reveal the location of the active site

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 enzymes 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.

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−1min−1 at 37 °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 13C NMR spectroscopy using [U-13C5]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.

Substrate binding to DNA photolyase studied by electron paramagnetic resonance spectroscopy.

Structural changes in Escherichia coli DNA photolyase induced by binding of a (cis,syn)-cyclobutane pyrimidine dimer (CPD) are studied by continuous-wave electron paramagnetic resonance and electron-nuclear double resonance spectroscopies, using the flavin adenine dinucleotide (FAD) cofactor in its neutral radical form as a naturally occurring electron spin probe. The electron paramagnetic resonance/electron-nuclear double resonance spectral changes are consistent with a large distance (> or =0.6 nm) between the CPD lesion and the 7,8-dimethyl isoalloxazine ring of FAD, as was predicted by recent model calculations on photolyase enzyme-substrate complexes. Small shifts of the isotropic proton hyperfine coupling constants within the FADs isoalloxazine moiety can be understood in terms of the cofactor binding site becoming more nonpolar because of the displacement of water molecules upon CPD docking to the enzyme. Molecular orbital calculations of hyperfine couplings using density functional theory, in conjunction with an isodensity polarized continuum model, are presented to rationalize these shifts in terms of the changed polarity of the medium surrounding the FAD cofactor.