Structure of 3,4-Dihydroxy-2-butanone 4-Phosphate Synthase from Methanococcus jannaschii in Complex with Divalent Metal Ions and the Substrate Ribulose 5-Phosphate: implications for the catalytic mechanism

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-Å 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

Structural Basis of Fosmidomycin Action Revealed by the Complex with 2-C-Methyl-D-erythritol 4-phosphate Synthase (IspC): implications for the catalytic mechanism and anti-malaria drug development

2-C-Methyl-D-erythritol 4-phosphate synthase (IspC) is the first enzyme committed to isoprenoid biosynthesis in the methylerythritol phosphate pathway, which represents an alternative route to the classical mevalonate pathway. As it is present in many pathogens and plants, but not in man, this pathway has attracted considerable interest as a target for novel antibiotics and herbicides. Fosmidomycin represents a specific high-affinity inhibitor of IspC. Very recently, its anti-malaria activity in man has been demonstrated in clinical trials. Here, we present the crystal structure of Escherichia coli IspC in complex with manganese and fosmidomycin at 2.5 Å resolution. The (N-formyl-N-hydroxy)amino group provides two oxygen ligands to manganese that is present in a distorted octahedral coordination, whereas the phosphonate group is anchored in a specific pocket by numerous hydrogen bonds. Both sites are connected by a spacer of three methylene groups. The substrate molecule, 1-D-deoxyxylulose 5-phosphate, can be superimposed onto fosmidomycin, explaining the stereochemical course of the reaction

Elucidation of novel biosynthetic pathways and metabolite flux patterns by retrobiosynthetic NMR analysis

The labelling patterns of metabolites from experiments with stable isotope-labelled precursors can be determined by NMR spectroscopy. Complex isotopomer mixtures are found when general metabolites such as glucose are used as stable isotope-labelled precursors which are diverted to all branches of intermediary metabolism. The complex results can be interpreted by a pattern recognition approach based on comparison between the labelling patterns of secondary metabolites and primary metabolites such as amino acids and ribonucleosides. The isotope labelling patterns of intermediates in central metabolic pools such as carbohydrate phosphates, dicarboxylic acids, and acetyl CoA can be obtained by biosynthetic retroanalysis. Biosynthetic pathways as well as metabolite flux patterns can be determined from these data. The method is illustrated using the classical mevalonate pathway and the more recently discovered deoxyxylulose pathway of terpenoid biosynthesis as examples. Applications of the retrobiosynthetic method of the biosynthesis of molybdopterin and of riboflavin are also discussed. Stable isotope experiments monitored by NMR spectroscopy have also been shown to be a powerful tool for the elucidation of metabolic flux in microorganisms with unusual lifestyles and in fermentation processe

Crystal structure of 7,8-dihydroneopterin triphosphate epimerase

AbstractBackground: Dihydroneopterin triphosphate (H2NTP) is the central substrate in the biosynthesis of folate and tetrahydrobiopterin. Folate serves as a cofactor in amino acid and purine biosynthesis and tetrahydrobiopterin is used as a cofactor in amino acid hydroxylation and nitric oxide synthesis. In bacteria, H2NTP enters the folate biosynthetic pathway after nonenzymatic dephosphorylation; in vertebrates, H2NTP is used to synthesize tetrahydrobiopterin. The dihydroneopterin triphosphate epimerase of Escherichia coli catalyzes the inversion of carbon 2′ of H2NTP.Results: The crystal structure of the homo-octameric protein has been solved by a combination of multiple isomorphous replacement, Patterson search techniques and cyclic averaging and has been refined to a crystallographic R factor of 18.8% at 2.9 Å resolution. The enzyme is a torus-shaped, D4 symmetric homo-octamer with approximate dimensions of 65 × 65 Å. Four epimerase monomers form an unusual 16-stranded antiparallel β barrel by tight association between the N- and C-terminal β strands of two adjacent subunits. Two tetramers associate in a head-to-head fashion to form the active enzyme complex.Conclusions: The folding topology, quaternary structure and amino acid sequence of epimerase is similar to that of the dihydroneopterin aldolase involved in the biosynthesis of the vitamin folic acid. The monomer fold of epimerase is also topologically similar to that of GTP cyclohydrolase I (GTP CH-1), 6-pyrovoyl tetrahydropterin synthase (PTPS) and uroate oxidase (UO). Despite a lack of significant sequence homology these proteins share a common subunit fold and oligomerize to form central β barrel structures employing different cyclic symmetry elements, D4, D5, D3 and D2, respectively. Moreover, these enzymes have a topologically equivalent acceptor site for the 2-amino-4-oxo pyrimidine (2-oxo-4-oxo pyrimidine in uroate oxidase) moiety of their respective substrates

Discovery and Development of a Small Molecule Library with Lumazine Synthase Inhibitory Activity

(E)-5-Nitro-6-(2-hydroxystyryl)pyrimidine-2,4(1H,3H)-dione (9) was identified as a novel inhibitor of Schizosaccharomyces pombe lumazine synthase by high-throughput screening of a 100,000 compound library. The Ki of 9 vs. Mycobacterium tuberculosis lumazine synthase was 95 μM. Compound 9 is a structural analog of the lumazine synthase substrate, 5-amino-6-(D-ribitylamino)-2,4-(1H,3H)pyrimidinedione (1). This indicates that the ribitylamino side chain of the substrate is not essential for binding to the enzyme. Optimization of the enzyme inhibitory activity through systematic structure modification of the lead compound 9 led to (E)-5-nitro-6-(4-nitrostyryl)pyrimidine-2,4(1H,3H)-dione (26), which has a Ki of 3.7 μM vs. M. tuberculosis lumazine synthase

Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by (19)F NMR protein perturbation experiments

BACKGROUND: Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs. RESULTS: A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20°C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg(-1 )min(-1 )with an apparent K(M )of 5.7 microM. (19)F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. (19)F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins. CONCLUSION: Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The (19)F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism

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

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

Femtosecond To Millisecond Dynamics Of Light Induced Allostery In The Avena Sativa LOV Domain

The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatio-temporal control of cell signalling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds timescale, which then modulate the activity of output domains responsible for biological signalling. Using time resolved vibrational spectroscopy coupled with isotope labelling we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 femtoseconds and one millisecond after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labelled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a sub-picosecond perturbation of the protein matrix occurs. In this perturbed environment the previously characterised reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the -sheet then -helix regions of the AsLOV2 domain, which ultimately gives rise to J-helix unfolding, yielding the signalling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513