Abstracts

16th Smyte (Small Meeting on Yeast Transport and Energetics) held in Častá-Papiernička, Slovakia September 23–27, 1998

Abstracts     

Dihydropyrimidine amidohydrolases and dihydroorotases share the same origin and several enzymatic properties

Slime mold, plant and insect dihydropyrimidine amidohydrolases (DHPases, EC 3.5.2.2), which catalyze the second step of pyrimidine and several anti-cancer drug degradations, were cloned and shown to functionally replace a defective DHPase enzyme in the yeast Saccharomyces kluyveri. The yeast and slime mold DHPases were over-expressed, shown to contain two zinc ions, characterized for their properties and compared to those of the calf liver enzyme. In general, the kinetic parameters varied widely among the enzymes, the mammalian DHPase having the highest catalytic efficiency. The ring opening was catalyzed most efficiently at pH 8.0 and competitively inhibited by the reaction product, N-carbamyl-β-alanine. At lower pH values DHPases catalyzed the reverse reaction, the closing of the ring. Apparently, eukaryote DHPases are enzymatically as well as phylogenetically related to the de novo biosynthetic dihydroorotase (DHOase) enzymes. Modeling studies showed that the position of the catalytically critical amino acid residues of bacterial DHOases and eukaryote DHPases overlap. Therefore, only a few modifications might have been necessary during evolution to convert the unspecialized enzyme into anabolic and catabolic ones.     

Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is unable to incorporate exogenous nucleosides into DNA. We have made a number of improvements to existing strategies to reconstitute an efficient thymidine salvage pathway in yeast. We have constructed strains that express both a nucleoside kinase as well as an equilibrative nucleoside transporter. By also deleting the gene encoding thymidylate synthase (CDC21) we have constructed strains that are entirely dependent upon exogenous thymidine for viability and that can grow with normal kinetics at low thymidine concentrations. Using this novel approach, we show that depletion of a single deoxyribonucleoside causes reversible arrest of cells in S phase with concomitant phosphorylation and activation of the S phase checkpoint kinase, Rad53. We show that this strain also efficiently incorporates the thymidine analogue, BrdU, into DNA and can be used for pulse–chase labelling.     

Inheritance and organisation of the mitochondrial genome differ between two Saccharomyces yeasts

Petite-positive Saccharomyces yeasts can be roughly divided into the sensu stricto, including Saccharomyces cerevisiae, and sensu lato group, including Saccharomyces castellii; the latter was recently studied for transmission and the organisation of its mitochondrial genome. S. castellii mitochondrial molecules (mtDNA) carrying point mutations, which confer antibiotic resistance, behaved in genetic crosses as the corresponding point mutants of S. cerevisiae. While S. castellii generated spontaneous petite mutants in a similar way as S. cerevisiae, the petites exhibited a different inheritance pattern. In crosses with the wild type strains a majority of S. castellii petites was neutral, and the suppressivity in suppressive petites was never over 50%. The two yeasts also differ in organisation of their mtDNA molecules. The 25,753 bp sequence of S. castellii mtDNA was determined and the coding potential of both yeasts is similar. However, the S. castellii intergenic sequences are much shorter and do not contain sequences homologous to the S. cerevisiae biologically active intergenic sequences, as ori/rep/tra, which are responsible for the hyper-suppressive petite phenotype found in S. cerevisiae. The structure of one suppressive S. castellii mutant, CA38, was also determined. Apparently, a short direct intergenic repeat was involved in the generation of this petite mtDNA molecule.     

Acceleration of Drosophila melanogaster acetylcholinesterase methanesulfonylation: peripheral ligand D-tubocurarine enhances the affinity for small methanesulfonylfluoride

D-Tubocurarine, a reversible peripheral inhibitor of cholinesterases accelerates methanesulfonylation of Drosophila melanogaster wild type and W359L mutant. The kinetic evaluation of the process was performed in a step-by-step analysis. The second order overall sulfonylation rate constants, determined from classical residual activity measurements, were used in the subsequent analysis of progress curves. The latter were obtained by measuring the hydrolysis of acetylthiocholine in a complex reaction system of enzyme, substrate, irreversible and reversible inhibitor. The underlying kinetic mechanisms, from such a complex data, could only be untangled by targeted inspection and successive incorporation of reaction steps for which experimental evidence existed. The study showed that the peripheral ligand D-tubocurarine, by binding at the entrance into the active site of the two investigated enzymes (Golicnik et al., Biochemistry 40 (2001) 1214), enhances the affinity for small methanesulfonylfluoride, rather to speeding up the formation of a stable covalent enzyme-inhibitor complex. The specific arrangements at the rim of the active site of each individual enzyme dictate the actual events which can be detected by kinetic means.     

Optimization of cultivation conditions in spin tubes for Chinese hamster ovary cells producing erythropoietin and the comparison of glycosylation patterns in different cultivation vessels

This article describes the optimization of cultivation factor settings, that is the shaking rate and working volume in 50 mL spin tubes for a Chinese hamster ovary cell line expressing recombinant human α‐erythropoietin, using a response D‐optimal surface method. The main objectives of the research were, firstly, to determine a setting in which the product titer and product quality attributes in spin tubes are equivalent to those in 250 mL shake flasks in a seven day batch and, secondly, to find a setting in which the product titer is maximal. The model for product titer prediction as a function of shaking rate and working volume in the defined design space was successfully applied to the optimization of cultivation conditions in spin tubes for the tested cell line. Subsequently, validation experiments were carried out simultaneously in spin tubes, shake flasks and bench scale bioreactors to compare cell culture performance parameters such as growth, productivity and product quality attributes in the form of isoform profiles and glycan antennarity structures. The results of the experiments showed that similar cell culture performance and product quality could be achieved in spin tubes when compared to shake flasks. Additionally, bioreactor titers could be reproduced in spin tubes at high shaking rates and low working volumes, but with differing product quality. Cultivation at lower shaking rates in spin tubes and shake flasks produced a glycoprotein with a product quality slightly comparable to that from bioreactors, but with titers being only two thirds. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The association between estrogen receptor alpha polymorphisms and the risk of prostate cancer in Slovak population

The aim of our study was to evaluate the effect of two polymorphisms in the estrogen receptor alpha, PvuII and XbaI, on the development of prostate cancer within Slovak population, as well as their correlation with selected clinical characteristics. The study was performed using 311 prostate cancer patients and 256 healthy male controls. Both polymorphisms were significantly associated with higher risk of prostate cancer development. At the same time, the CC genotype of PvuII polymorphism (OR = 1.98; 95 % CI 0.94–4.21; p = 0.05) and the AG genotype of XbaI polymorphism (OR = 1.74; 95 % CI 1.0–3.02; p = 0.04) significantly contributed to the development of low-grade carcinoma, while the AG and GG genotypes of the XbaI polymorphism contributed mainly to the development of high-grade prostate cancer (OR = 1.83; 95 % CI 1.12–3.01; p = 0.01 and OR = 2.13; 95 % CI 1.06–4.19; p = 0.03, respectively). Similarly, the AG and GG genotypes of XbaI polymorphism showed significant association with prostate cancer in patients with serum PSA level ≥10 ng/ml. Both polymorphisms were found at the same time to be more frequent in patients diagnosed before the age of 60. We conclude on the basis of these results that PvuII and XbaI polymorphisms of estrogen receptor alpha might be associated with prostate cancer risk within Slovak population. Although this is a pilot study and, as such, more detailed investigations are needed to confirm the role of these polymorphisms in prostate cancer development and progression within said Slovak population, our results might still provide a valuable basis for further research with larger patient groups.     

The crystal structure of Streptococcus pyogenes uridine phosphorylase reveals a distinct subfamily of nucleoside phosphorylases

Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 ? resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an α/β monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is ～7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.