Klaus D. Schnackerz

Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anti-cancer drug 5-fluorouracil

Dihydropyrimidine dehydrogenase catalyzes the first step in pyrimidine degradation: the NADPH‐dependent reduction of uracil and thymine to the corresponding 5,6‐dihydropyrimidines. Its controlled inhibition has become an adjunct target for cancer therapy, since the enzyme is also responsible for the rapid breakdown of the chemotherapeutic drug 5‐fluorouracil. The crystal structure of the homodimeric pig liver enzyme (2× 111 kDa) determined at 1.9 Å resolution reveals a highly modular subunit organization, consisting of five domains with different folds. Dihydropyrimidine dehydrogenase contains two FAD, two FMN and eight [4Fe–4S] clusters, arranged in two electron transfer chains that pass the dimer interface twice. Two of the Fe–S clusters show a hitherto unobserved coordination involving a glutamine residue. The ternary complex of an inactive mutant of the enzyme with bound NADPH and 5‐fluorouracil reveals the architecture of the substrate‐binding sites and residues responsible for recognition and binding of the drug.

A gene duplication led to specialized gamma-aminobutyrate and beta-alanine aminotransferase in yeast.

In humans, β‐alanine (BAL) and the neurotransmitter γ‐aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, and SkPYD4 encodes an enzyme involved in both BAL and GABA transamination. SkPYD4 and SkUGA1 as well as S. cerevisiaeUGA1 and Schizosaccharomyces pombeUGA1 were subcloned, over‐expressed and purified. One discontinuous and two continuous coupled assays were used to characterize the substrate specificity and kinetic parameters of the four enzymes. It was found that the cofactor pyridoxal 5′‐phosphate is needed for enzymatic activity and α‐ketoglutarate, and not pyruvate, as the amino group acceptor. SkPyd4p preferentially uses BAL as the amino group donor (Vmax/Km = 0.78 U·mg−1·mm−1), but can also use GABA (Vmax/Km = 0.42 U·mg−1·mm−1), while SkUga1p only uses GABA (Vmax/Km = 4.01 U·mg−1·mm−1). SpUga1p and ScUga1p transaminate only GABA and not BAL. While mammals degrade BAL and GABA with only one enzyme, but in different tissues, S. kluyveri and related yeasts have two different genes/enzymes to apparently ‘distinguish’ between the two reactions in a single cell. It is likely that upon duplication ∼200 million years ago, a specialized Uga1p evolved into a ‘novel’ transaminase enzyme with broader substrate specificity.

NMR and circular dichroism studies of synthetic peptides derived from the third intracellular loop of the β-adrenoceptor

The C‐terminal part of the third intracellular loop of the β‐adrenoceptor is capable of stimulating adenylate cyclase in the presence of phospholipid vesicles via the stimulatory guanine nucleotide binding protein (Gs) [Palm et al. (1989) FEBS Lett. 254, 89–93]. We have investigated the structure of synthetic peptides corresponding to residues 284–295 of the turkey erythrocyte adrenoceptor in micelles, trifluoroethanol and aqueous solution, by using 2D 1H NMR and CD. In the presence of phospholipid micelles the peptides display a C‐terminal α‐helical region, whereas the N‐terminal part was found to be highly flexible.

Crystal structure of the productive ternary complex of dihydropyrimidine dehydrogenase with NADPH and 5-iodouracil : Implications for mechanism of inhibition and electron transfer

Dihydroprymidine dehydrogenase catalyzes the first and rate-limiting step in pyrimidine degradation by converting pyrimidines to the corresponding 5,6- dihydro compounds. The three-dimensional structures of a binary complex with the inhibitor 5-iodouracil and two ternary complexes with NADPH and the inhibitors 5-iodouracil and uracil-4-acetic acid were determined by x-ray crystallography. In the ternary complexes, NADPH is bound in a catalytically competent fashion, with the nicotinamide ring in a position suitable for hydride transfer to FAD. The structures provide a complete picture of the electron transfer chain from NADPH to the substrate, 5-iodouracil, spanning a distance of 56 Å and involving FAD, four [Fe-S] clusters, and FMN as cofactors. The crystallographic analysis further reveals that pyrimidine binding triggers a conformational change of a flexible active-site loop in the α/β-barrel domain, resulting in placement of a catalytically crucial cysteine close to the bound substrate. Loop closure requires physiological pH, which is also necessary for correct binding of NADPH. Binding of the voluminous competitive inhibitor uracil-4-acetic acid prevents loop closure due to steric hindrance. The three-dimensional structure of the ternary complex enzyme-NADPH-5-iodouracil supports the proposal that this compound acts as a mechanism-based inhibitor, covalently modifying the active-site residue Cys-671, resulting in S-(hexahydro-2,4-dioxo-5-pyrimidinyl)cysteine.

Preservation of left ventricular mechanical function and energy metabolism in rats after myocardial infarction by the angiotensin-converting enzyme inhibitor quinapril.

We tested whether angiotensin-converting enzyme (ACE) inhibitor therapy with quinapril prevents the deterioration of mechanical function and high-energy phosphate metabolism that occurs in chronically infarcted heart. Rats were subjected to ligation of the left anterior descending coronary artery (LAD) or sham operation. Four groups were studied: sham-operated rats (n = 10), rats with myocardial infarction (MI, n = 9), sham-operated quinapril-treated rats (n = 8), and infarcted quinapril-treated (n = 13) rats. Treated rats received 6 mg/kg/day of the ACE inhibitor quinapril orally, initiated 1 h after MI or sham operation. Eight weeks after LAD ligation or sham operation, hearts were isolated and buffer-perfused isovolumically. High-energy phosphate metabolism and intracellular pH were continuously recorded with 31P-nuclear magnetic resonance (NMR) spectroscopy. Hearts were subjected to 15-min control, 30-min hypoxia (95% N2/5% CO2, and 30-min reoxygenation. Left ventricular developed pressure (LVDP) was reduced in infarcted hearts (58 +/- 10 vs. 98 +/- 9 mm Hg in sham, p < 0.05), and this reduction was partially prevented by quinapril (78 +/- 8 mm Hg). ATP content of residual intact myocardium after sham operation or MI was unchanged. Creatine phosphate was reduced in infarcted hearts (107 +/- 10 vs. 138 +/- 5% of control ATP, p < 0.05), and quinapril prevented this decrease (131 +/- 8%). Therefore, quinapril preserved both function and high-energy phosphate metabolism in the chronically infarcted heart. However, when hearts were subjected to acute hypoxia, susceptibility to acute metabolic stress was substantially increased in both quinapril-treated groups: ATP content at end-hypoxia was reduced to 31 +/- 7 and 37 +/- 6% in sham and infarcted quinapril-treated groups, whereas ATP in untreated sham and infarcted hearts was 66 +/- 6 and 66 +/- 3% of baseline values (p < 0.05 untreated vs. quinapril treated). Likewise, recovery of LVDP during reoxygenation was impaired by quinapril treatment (15 +/- 7 and 15 +/- 4 mm Hg in quinapril-treated sham and MI vs. 73 +/- 9 and 46 +/- 9 mm Hg in untreated sham and MI groups, p < 0.05 untreated vs. quinapril treated). The most likely explanation for the unexpected finding of increased susceptibility to acute metabolic stress in the quinapril-treated groups is reduced wall thickness leading to increased wall stress. The preservation of high-energy phosphate content in residual intact hearts after MI may contribute to the beneficial effects of ACE inhibitors after MI.

Porcine recombinant dihydropyrimidine dehydrogenase: comparison of the spectroscopic and catalytic properties of the wild-type and C671A mutant enzymes

Dihydropyrimidine dehydrogenase catalyzes, in the rate-limiting step of the pyrimidine degradation pathway, the NADPH-dependent reduction of uracil and thymine to dihydrouracil and dihydrothymine, respectively. The porcine enzyme is a homodimeric iron-sulfur flavoprotein (2 x 111 kDa). C671, the residue postulated to be in the uracil binding site and to act as the catalytically essential acidic residue of the enzyme oxidative half-reaction, was replaced by an alanyl residue. The mutant enzyme was overproduced in Escherichia coli DH5alpha cells, purified to homogeneity, and characterized in comparison with the wild-type species. An extinction coefficient of 74 mM-1 cm-1 was determined at 450 nm for the wild-type and mutant enzymes. Chemical analyses of the flavin, iron, and acid-labile sulfur content of the enzyme subunits revealed similar stoichiometries for wild-type and C671A dihydropyrimidine dehydrogenases. One FAD and one FMN per enzyme subunit were found. Approximately 16 iron atoms and 16 acid-labile sulfur atoms were found per wild-type and mutant enzyme subunit. The C671A dihydropyrimidine dehydrogenase mutant exhibited approximately 1% of the activity of the wild-type enzyme, thus preventing its steady-state kinetic analysis. Therefore, the ability of the C671A mutant and, for comparison, of the wild-type enzyme species to interact with reaction substrates, products, or their analogues were studied by absorption spectroscopy. Both enzyme forms did not react with sulfite. The wild-type and mutant enzymes were very similar to each other with respect to the spectral changes induced by binding of the reaction product NADP+ or of its nonreducible analogue 3-aminopyridine dinucleotide phosphate. Uracil also induced qualitatively and quantitatively similar absorbance changes in the visible region of the absorbance spectrum of the two enzyme forms. However, the calculated Kd of the enzyme-uracil complex was significantly higher for the C671A mutant (9.1 +/- 0.7 microM) than for the wild-type dihydropyrimidine dehydrogenase (0.7 +/- 0.09 microM). In line with these observations, the two enzyme forms behaved in a similar way when titrated anaerobically with a NADPH solution. Addition of an up to 10-fold excess of NADPH to both dihydropyrimidine dehydrogenase forms led to absorbance changes consistent with reduction of approximately 0.5 flavin per subunit, with no indication of reduction of the enzyme iron-sulfur clusters. Absorbance changes consistent with reduction of both enzyme flavins were obtained by removing NADP+ with a NADPH-regenerating system. On the contrary, the two enzyme species differed significantly with respect to their reactivity with dihydrouracil. Addition of dihydrouracil to the wild-type enzyme species, under anaerobic conditions, led to absorbance changes that could be interpreted to result from both partial flavin reduction and the formation of a complex between the enzyme and (dihydro)uracil. In contrast, only spectral changes consistent with formation of a complex between the oxidized enzyme and dihydrouracil were observed when a C671A mutant enzyme solution was titrated with this compound. Furthermore, enzyme-monitored turnover experiments were carried out anaerobically in the presence of a limiting amount of NADPH and excess uracil with the two enzyme forms in a stopped-flow apparatus. These experiments directly demonstrated that the substitution of an alanyl residue for C671 in dihydropyrimidine dehydrogenase specifically prevents enzyme-catalyzed reduction of uracil. Finally, sequence analysis of dihydropyrimidine dehydrogenase revealed that it exhibits a modular structure; the N-terminal region, similar to the beta subunit of bacterial glutamate synthases, is proposed to be responsible for NADPH binding and oxidation with reduction of the FAD cofactor of dihydropyrimidine dehydrogenase. The central region, similar to the FMN subunit of dihydroorotate dehydrogenases, is likely to harbor the site o

Amidohydrolases of the reductive pyrimidine catabolic pathway: Purification, characterization, structure, reaction mechanisms and enzyme deficiency

In the reductive pyrimidine catabolic pathway uracil and thymine are converted to beta-alanine and beta-aminoisobutyrate. The amidohydrolases of this pathway are responsible for both the ring opening of dihydrouracil and dihydrothymine (dihydropyrimidine amidohydrolase) and the hydrolysis of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyrate (beta-alanine synthase). The review summarizes what is known about the properties, kinetic parameters, three-dimensional structures and reaction mechanisms of these proteins. The two amidohydrolases of the reductive pyrimidine catabolic pathway have unrelated folds, with dihydropyrimidine amidohydrolase belonging to the amidohydrolase superfamily while the beta-alanine synthase from higher eukaryotes belongs to the nitrilase superfamily. beta-Alanine synthase from Saccharomyces kluyveri is an exception to the rule and belongs to the Acyl/M20 family.

31P-NMR spectroscopy of human blood and serum: first results from volunteers and patients with congestive heart failure, diabetes mellitus and hyperlipidaemia

Abstract31P-containing metabolites in human blood, serum and erythrocytes were measured or calculated. Phosphodiesters were found in serum, but not in erythrocytes. 2,3-diphosphoglycerate and 2,3-diphosphoglycerate/ATP ratios were increased in patients with congestive heart failure (2,3-diphosphoglycerate by 13% in mild to moderate, 31% in severe congestive heart failure, 2,3-diphosphoglycerate/ATP ratio by 9% in mild to moderate, 38% in severe congestive heart failure); phosphodiesters were increased in diabetes mellitus (by 26%) and even more so in hyperlipidaemia (by 57%). Changes of blood31P compounds with disease states may have diagnostic potential and should be recognized for correction of organ spectra.

Phosphorus-31 nuclear magnetic resonance study of pyridoxal 5′-phosphate binding to Escherichiacoli tryptophan synthase

Abstract The pyridoxal phosphate dependent bienzyme complex tryptophan synthase from Escherichia coli has been investigated using 31P nuclear magnetic resonance (NMR) at 72.86 MHz. In both the isolated β2 subunit and the fully assembled α2 holo β2 complex, the pyridoxal phosphate 31P chemical shift is pH independent, indicating that the phosphate group of the cofactor is excluded from interaction with water and fixed in its dianionic form to the protein moiety. The line width of the phosphate signal of the coenzyme in the β2 subunit is unusually large pointing to different equilibrium conformations of the protein. In the native holo complex, however, the corresponding line width may be interpreted to result from moderate mobility of the cofactor leading to a higher rate of the transaldimination step of the catalytic mechanism.

31P-Nuclear Magnetic Resonance Spectroscopy of Blood: A Species Comparison

31P-nuclear magnetic resonance (NMR) spectroscopy isfrequently used as a tool in the study of organs from various animal species and humans. Because signals arising from the presence of blood are visible in in vivo 31P-NMR spectra of blood-filled organs, such as the heart, it is necessary to correct these spectra for the contribution of blood to the signal. It is unknown whether species differences in 31P signals of blood exist. 31P-containing metabolites of blood from various species were therefore quantified by means of 31P-NMR spectroscopy. Signals of 2,3-bisphosphoglycerate (2,3-DPG); phosphodiesters (PDE); and gamma-, alpha-, and beta-ATP were detected in all 31P-NMR spectra of blood. 2,3-DPG/ATP ratios were significantly higher in dogs, rats, and guinea pigs than in humans but lower in sheep. Pig and rabbit were the only animals with a 2,3-DPG/ATP ratio similar to that of humans. PDE levels varied among species but were significantly lower than in humans only in guinea pigs. The PDE/ATP ratio was relatively similar among all species compared with humans, except dog and guinea pig, where it was significantly higher and lower, respectively. We conclude that because of large species differences, species-specific 31P metabolite ratios should be applied for the correction of in vivo 31P-NMR spectra.