Phosphate transfer reactions play a central role in metabolism, regulation and signaling. As phosphate esters are kinetically extremely stable, efficient catalysis is crucial for the control of these cellular processes. A snapshot of an enzyme in a high-energy state would be immensely useful, as it would allow the very interactions that bring about catalysis to be observed. However, high-energy intermediates and transition states are extremely elusive species, and hence, transition state analogues that bind tightly in an enzyme active site have been of paramount importance in defining the structural and energetic framework for catalysis. MgF3 is particularly interesting to characterize as a potentially highly accurate transition state analog (TSA). The kinetic analysis of an MgF3 adduct is difficult as compared to other metal fluoride TSAs, because it does not exist in solution because of its low formation constant and must be assembled and stabilized in the active site. In this article, we provided a kinetic analysis of fluoride inhibition of beta-phosphoglukomutase that allowed us to measure the stability constant and characteristics of the formation of the TSA.
COBISS.SI-ID: 26007001
17beta-Hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17beta-HSDcl) is currently the only fungal HSD member that has been described and represents one of the model enzymes of the cP1 classical subfamily of NADPH dependent SDR enzymes. A thorough crystallographic analysis has been performed to better understand the structural aspects of this subfamily and provided insights into the evolution of the HSD enzymes. The crystal structures of the 17beta-HSDcl apo, holo and coumestrol-inhibited ternary complex, and the active site Y167F mutant reveal subtle conformational differences in the substrate-binding loop that likely modulate the catalytic activity of 17beta-HSDcl. Coumestrol, a plant-derived non-steroidal compound with estrogenic activity, inhibits 17beta-HSDcl (IC50, 2.8 mM; at 100 muM substrate š4-estrene-3,17-dioneđ) by occupying the putative steroid-binding site. In addition to an extensive hydrogen-bonding network, coumestrol binding is further stabilized by pi-pi stacking interactions with Tyr-212. A stopped-flow kinetic experiment clearly showed the coenzyme dissociation as the slowest step of the reaction and in addition to the low steroid solubility it prevents the accumulation of enzyme-coenzyme-steroid ternary complex formation.
COBISS.SI-ID: 28926169
An efficient highthroughput method for the identification of biotechnologically interesting genes of extremotolerant fungi was developed by constructing a cDNA expression library in Saccharomyces cerevisiae and screening for gainoffunction transformants under stress conditions. The method was demonstrated with the identification genes from the halotolerant yeast Rhodotorula mucilaginosa that increased the halotolerance of S. cerevisiae and might also be useful for the improvement of halotolerance in other industrial microorganisms or genetically modified crops.
COBISS.SI-ID: 2514255
Human aldo-keto reductases 1C1-1C4 (AKR1C1-AKR1C4) function in vivo as 3-keto-, 17-keto- and 20-ketosteroid reductases, and regulate the activity of androgens, estrogens and progesterone and the occupancy and transactivation oftheir corresponding receptors. Aberrant expression and action of AKR1C enzymes can lead to different pathophysiological conditions. AKR1C enzymes thus represent important targets for development of new drugs. We performed a virtual high-throughput screen of a fragment library that was followed by biochemical evaluation on AKR1C1-AKR1C4 enzymes. Twenty-four structurally diverse compounds were discovered with low muM Ki values for AKR1C1, AKR1C3, or both. Two structural series included the salicylates and the N-phenylanthranilic acids and additionally a series of inhibitors with completely novel scaffolds was discovered. Two of the best selective AKR1C3 inhibitors had Ki values of 0.1 muM and 2.7 muM, exceeding expected activity for fragments. The compounds identified represent an excellent starting pointfor further hit-to-lead development.
COBISS.SI-ID: 30078937
The occurrence of adverse events and resistance to treatment is one of the major clinical problems in cancer treatment. It has been well established that genetic variability in drug pathways or DNA repair mechanisms might influence treatment outcome. In addition, genetic polymorphisms in DNA repair mechanisms may influence the risk of malignant disease. The aim of our study was to evaluate theinfluence of platinum pathway polymorphisms on treatment outcome in patients with MM. GST polymorphisms were not associated with treatment outcome in patients with MM. In the group of platinum-treated patients with MM, ERCC1 8092C/C genotype significantly influenced progression-free survival (PFS). XPD 312Asp/Asp and ERCC1 8092C/C wild-type genotypes also increased the odds of treatment-related toxic effects and of better PFS. Our results suggest that polymorphisms in RNA repair pathway influence platinum-treatment efficacy and toxicity. If confirmed as markers of clinical outcome in further pharmacogenetics studies, the analysis of these polymorphisms may contribute to the individualisation of cancer treatment.
COBISS.SI-ID: 28755417