Heidelberg, Univ., Diss., 2001. / Dateien im PDF-Format. Computerdatei im Fernzugriff.

Heidelberg, Universiẗat, Diss., 2001. / Dateien im PDF-Format.

All organisms must maintain an adequate level of thymidylate, which gets phosphorylated twice and then utilized by DNA polymerases for DNA replication that must precede cell division. Most organisms rely on classical thymidylate synthase (TSase) for this function. However, a subset of microorganisms – including a number of notable, widespread human pathogens – relies on an enzyme with a distinct structure and catalytic strategy. This enzyme is termed flavin-dependent thymidylate synthase (FDTS), as the flavin is required for thymidylate production. Because of this considerable orthogonality between FDTS and classical TSase, FDTS serves as a promising target for new therapeutics – one that could have only mild adverse effects on the host organism. FDTS catalyzes the reductive methylation of uridylate (2′-deoxyuridine-5′-monophosphate; dUMP) to yield thymidylate (2′-deoxythymidine-5′-monophosphate; dTMP). The methylene originally resides on CH2H4folate and is eventually transferred to the nucleotide. This methylene’s route to dUMP is unique in enzymology, and our experiments described herein strive to gain an understanding of the molecular details of its transfer. Compounds that mimic intermediates and transition states along this path are likely to bind FDTS tightly and could be leads for drugs, and our new insights could facilitate this. After methylene transfer is complete, a hydride transfer from flavin to the nucleotide occurs. We utilized rapid quench flow techniques in heavy water to follow the hydrogen transfers in FDTS; solvent isotope effects were measured and analyzed, furnishing evidence that the hydride transfer contributes to rate limitation. Reconstitution of the enzyme with unnatural flavins both reinforced these conclusions and suggested new hypotheses and experiments.

No description available.

O ácido fólico, a forma mais estável entre os folatos, é indicado como forma de prevenção sendo encontrado em forma de suplementação medicamentosa e alimentos fortificados, como leite e derivados. O folato pode reagir com a riboflavina singlete excitada, 1kq = 4,8·1010 L·mol-1·s-1, como determinado por desativação da fluorescência do estado estacionário, e com a riboflavina triplete excitada, com uma reação um pouco mais lenta, 3kq= 4,8·108 L·mol-1·s-1, como determinado por fotólise de pulso de laser e espectroscopia de absorção de transientes verificando-se que ambos os processos são competitivos e próximos ao limite de difusão. A preferência cinética de um a outro depende da matriz alimentar. O rendimento quântico para solução de riboflavina e de folato preparado em solvente aquoso e deuterado e em meio anaeróbico e aeróbico mostrou a prevalência do mecanismo fotoreacional do Tipo I. A voltametria cíclica apresentou um processo irreversível anódico de dois elétrons para o ácido fólico (E= 1,14 V vs. NHE). Os principais produtos da fotodegradação do folato sensibilizado pela riboflavina foram identificados por LC-IT-MS/MS como: 6-carboxipterina,p-aminobenzoil-L-ácido glutâmico e oxaziridina derivada do ácido fólico, como confirmando a desativação química do estado triplete excitado da riboflavina por transferência de elétrons com subseqüente clivagem oxidativa entre N(10) e C(9) no ácido fólico. / Folic acid, the most stable among folate, is recommended as prevention and it is found in supplementation and fortified foods such as milk and dairy products. The folate can react with singlet-excited state of riboflavin, 1kq= 4.8·1010 L·mol-1·s-1, as determined by steady-state fluorescence quenching, and with triplet-excited state of riboflavin in a slower reaction with 3kq= 4.8·108 L·mol-1·s-1, as determined by laser flash photolysis and transient absorption spectroscopy, verifying that both the processes are competitive and they are near limited diffusion. The kinetic preference depends on the matrix food. The quantum yield for the solution of riboflavin and folate prepared in aqueous and deuterated solvents and in anaerobic and aerobic medium showed the prevalence of the mechanism Type I. The cyclic voltammetry showed an irreversible two-electron anodic process for folate (E = 1.14 V vs. NHE). The main products of folate photodegradation sensitized by riboflavin were identified by LC-IT-MS/MS as: pterin-6-carboxylic acid, p-aminobenzoyl-L-glutamic acid and oxaziridine derivative of folic acid, as confirming chemical quenching of the triplet-excited state of riboflavin by electron transfer with subsequent oxidative cleavage between N(10) and C(9) in folic acid.

Despite a number of kinetic and spectroscopic studies, the chemical mechanisms of amine oxidation by flavoenzymes remain widely debated. The mechanisms of by Nmethyltryptophan oxidase (MTOX) and tryptophan 2-monooxygenase (TMO) were probed using a combination of pH and primary deuterium, solvent, and 15N kinetic isotope effects. Slow substrates were chosen for these studies; MTOX was characterized with N-methylglycine and TMO was characterized with L-alanine. Primary deuterium kinetic isotope effects of 7.2 and 5.3 were observed for sarcosine oxidation by MTOX and for alanine oxidation by TMO, respectively, independent of the substrate concentration and pH. Monitoring the reduction of flavin spectroscopically revealed no intermediate flavin species with both enzyme-substrate systems. Furthermore, the magnitudes of the 15N kinetic isotope effects observed with both systems suggest that nitrogen rehybridization and C-H bond cleavage are concerted. These results are consistent with both enzymes utilizing a hydride transfer mechanism for amine oxidation. The role of the iron ligands of tyrosine hydroxylase (TyrH) was also investigated. TyrH contains one iron per monomer, which is held by three conserved amino acid residues, two histidines and a glutamate. As a probe of the plasticity of the metal binding site, each of the metal ligands in TyrH was substituted with glutamine, glutamate, or histidine. The resulting proteins were characterized for metal content, catalytic activity, and dopamine binding. The H336E and H336Q enzymes retain substantial catalytic activity. In contrast, the E376Q enzyme retains about 0.4% of the wild-type catalytic activity, and the E376H enzyme has no significant activity. The H331E enzyme oxidizes tetrahydropterin in a tyrosine-independent manner. The position of the charge-transfer absorbance band for the H336E and H336Q enzyme-inhibitor complexes is shifted relative to that of the wild-type enzyme, consistent with the change in the metal ligand. In contrast, the E376H and E376Q enzymes catalyze dopamine oxidation. These results provide a reference point for further structural studies of TyrH and the other aromatic amino acid hydroxylases, and for similar studies of other enzymes containing this ironbinding motif.

O ácido fólico, a forma mais estável entre os folatos, é indicado como forma de prevenção sendo encontrado em forma de suplementação medicamentosa e alimentos fortificados, como leite e derivados. O folato pode reagir com a riboflavina singlete excitada, 1kq = 4,8·1010 L·mol-1·s-1, como determinado por desativação da fluorescência do estado estacionário, e com a riboflavina triplete excitada, com uma reação um pouco mais lenta, 3kq= 4,8·108 L·mol-1·s-1, como determinado por fotólise de pulso de laser e espectroscopia de absorção de transientes verificando-se que ambos os processos são competitivos e próximos ao limite de difusão. A preferência cinética de um a outro depende da matriz alimentar. O rendimento quântico para solução de riboflavina e de folato preparado em solvente aquoso e deuterado e em meio anaeróbico e aeróbico mostrou a prevalência do mecanismo fotoreacional do Tipo I. A voltametria cíclica apresentou um processo irreversível anódico de dois elétrons para o ácido fólico (E= 1,14 V vs. NHE). Os principais produtos da fotodegradação do folato sensibilizado pela riboflavina foram identificados por LC-IT-MS/MS como: 6-carboxipterina,p-aminobenzoil-L-ácido glutâmico e oxaziridina derivada do ácido fólico, como confirmando a desativação química do estado triplete excitado da riboflavina por transferência de elétrons com subseqüente clivagem oxidativa entre N(10) e C(9) no ácido fólico. / Folic acid, the most stable among folate, is recommended as prevention and it is found in supplementation and fortified foods such as milk and dairy products. The folate can react with singlet-excited state of riboflavin, 1kq= 4.8·1010 L·mol-1·s-1, as determined by steady-state fluorescence quenching, and with triplet-excited state of riboflavin in a slower reaction with 3kq= 4.8·108 L·mol-1·s-1, as determined by laser flash photolysis and transient absorption spectroscopy, verifying that both the processes are competitive and they are near limited diffusion. The kinetic preference depends on the matrix food. The quantum yield for the solution of riboflavin and folate prepared in aqueous and deuterated solvents and in anaerobic and aerobic medium showed the prevalence of the mechanism Type I. The cyclic voltammetry showed an irreversible two-electron anodic process for folate (E = 1.14 V vs. NHE). The main products of folate photodegradation sensitized by riboflavin were identified by LC-IT-MS/MS as: pterin-6-carboxylic acid, p-aminobenzoyl-L-glutamic acid and oxaziridine derivative of folic acid, as confirming chemical quenching of the triplet-excited state of riboflavin by electron transfer with subsequent oxidative cleavage between N(10) and C(9) in folic acid.

The Fdc1 and Pad1 decarboxylase system from Saccharomyces cerevisiae has been identified as a potential candidate to feature in novel biofuel production pathways based on its ability to catalyze the transformation of sorbic acid, an antimicrobial compound, to 1, 3-pentadiene, a volatile hydrocarbon. Although information on the system is currently insufficient to permit a full assessment of its potential for future commercialization, it is hoped that (rational) engineering approaches can be used to evolve the enzymes to produce more desirable hydrocarbons. This requires biochemical characterization of the proteins. Genetic manipulation experiments have indicated that both enzymes are required for activity. However, no in vitro studies were conducted to verify the function, determine the relationship or establish the cofactor requirements of Fdc1 and Pad1. Results reported here establish that Fdc1 is the enzyme responsible for catalyzing decarboxylation, requiring a novel cofactor synthesized by Pad1 (or the bacterial homologue UbiX) for activity. High resolution crystal structures and mass spectrometry data from Fdc1 co-expressed with UbiX have indicated that the cofactor corresponds to a modified flavin mononucleotide (FMN) that has been extended with a C5-unit through linkages at the N5 and C6 atoms, creating a fourth, non-aromatic ring on the isoalloxazine group. Subsequent solution studies have established that this modification is achieved through isoprene chemistry, with UbiX facilitating prenyl transfer from dimethylallyl monophosphate (DMAP) to FMN. Analysis of wild type and mutant UbiX constructs by kinetic X-ray crystallography has allowed several distinct stages of the prenyl transfer reaction to be trapped, establishing that the protein uses a number of chemical strategies similar to terpene synthases to generate its product. The active site is dominated by pi systems, which aid heterolytic cleavage of the isoprene precursors phosphate-C1’ bond following FMN reduction, leading to the formation of an N5-C1’ intermediate. UbiX then acts as a chaperone for adduct reorientation, potentially via a transient tertiary carbocation, ultimately resulting in ring closure between the C6 and C3’. This work has established the biochemical principles underpinning the Fdc1 and Pad/UbiX decarboxylase system, providing a platform from which rational evolution approaches can be applied to the enzymes, specifically Fdc1, to improve their validity in the biofuels industry. It has also identified a novel cofactor that extends the previously well-documented flavin and isoprenoid repertoire.

No description available.

Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus during infection. This siderophore includes N5-hydroxylated L-ornithine in the peptide backbone that serve as iron chelators. Af SidA is the L-ornithine N5-hydroxylase, which performs the first enzymatic step in the biosynthesis of ferrichrome. In this study, Af SidA was recombinantly expressed and purified as a soluble tetramer with a bound FAD cofactor. The enzyme demonstrated typical Michaelis-Menten kinetics in a product formation assay with respect to L-ornithine, but similar experiments as a function NADH and NADPH indicated inhibition at high coenzyme concentrations. Af SidA is highly specific for its substrate; however, it is promiscuous with respect to its coenzyme requirement. A multi-functional role of NADPH is observed since NADP+ is a competitive inhibitor with respect to NADPH and steady-state kinetic experiments indicate that Af SidA forms a ternary complex with NADP+ and L-ornithine for catalysis. Furthermore, in the absence of substrate, Af SidA forms a stable C4a-(hydro)peroxyflavin intermediate that is stable on the second time scale. Af SidA is also inhibited by several halides and the arginine-reactive reagent, phenylglyoxal. Biochemical comparison of Af SidA to other flavin-containing monooxygenases reveal that Af SidA likely proceeds by a sequential-ordered mechanism. / Master of Science in Life Sciences