Computational study of conformational changes in human 3-hydroxy-3-methylglutaryl coenzyme reductase induced by substrate binding

Abstract

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is mainly involved in the regulation of cholesterol biosynthesis. HMGR catalyses the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate at the expense of two NADPH molecules in a two-step reversible reaction. In the present study, we constructed a model of human HMGR (hHMGR) to explore the conformational changes of HMGR in complex with HMG-CoA and NADPH. In addition, we analysed the complete sequence of the Flap domain using molecular dynamics (MD) simulations and principal component analysis (PCA). The simulations revealed that the Flap domain plays an important role in catalytic site activation and substrate binding. The apo form of hHMGR remained in an open state, while a substrate-induced closure of the Flap domain was observed for holo hHMGR. Our study also demonstrated that the phosphorylation of Ser872 induces significant conformational changes in the Flap domain that lead to a complete closure of the active site, suggesting three principal conformations for the first stage of hHMGR catalysis. Our results were consistent with previous proposed models for the catalytic mechanism of hHMGR.

Communicated by Ramaswamy H. Sarma     

Effect of seven species of the family Asteraceae on longevity and nutrient levels of Episyrphus balteatus

Adult hoverflies feed on pollen, nectar and honeydews. Thus, the implementation of floristically diverse resources in agroecosystems can enhance biological control of pests by increasing populations of these natural enemies. The objective of this work was to study the effect of seven species of Asteraceae (Andryala integrifolia L., Anthemis arvensis L., Calendula arvensis L., Carduus tenuiflorus Curtis, Chamaemelum nobile (L.) All., Coleostephus myconis (L.) Rchb.f. and Crepis vesicaria L.) on adult longevity and nutrient levels of Episyrphus balteatus (De Geer) (Diptera: Syrphidae). Crepis vesicaria and C. nobile gave the highest mean longevities while, for the other plant species, longevities were not significantly different from those in the water only treatment. In addition, all species tested resulted in significantly lower longevities than the positive control (glucose). In terms of nutrient levels, C. nobile generated high levels of total sugars and C. vesicaria high levels of lipids which suggests the consumption of flower resources. However, the majority of the plants tested would not allow hoverflies to complete their life cycle. Thus, further experimentation needs to be done before considering these plants for implementation in agroecosystems.     

Systemic and regional hemodynamic effects of angiotensin-(1-7) in rats

The systemic and regional hemodynamics effects of ANG-(1-7) were examined in urethane-anesthetized rats. The blood flow distribution (kidneys, skin, mesentery, lungs, spleen, brain, muscle, and adrenals), cardiac output, and total peripheral resistance were investigated by using fluorescent microspheres. Blood pressure and heart rate were recorded from the brachial artery. ANG-(1-7) infusion (110 fmol x min(-1) x 10 min(-1) iv) significantly increased blood flow to the kidney (5.10 +/- 1.07 to 8.30 +/- 0.97 ml x min(-1) x g(-1)), mesentery (0.73 +/- 0.16 to 1.17 +/- 0.49 ml x min(-1) x g(-1)), brain (1.32 +/- 0.44 to 2.18 +/- 0.85 ml x min(-1) x g(-1)), and skin (0.07 +/- 0.02 to 0.18 +/- 0.07 ml x min(-1) x g(-1)) and the vascular conductance in these organs. ANG-(1-7) also produced a significant increase in cardiac index (30%) and a decrease in total peripheral resistance (2.90 +/- 0.55 to 2.15 +/- 0.28 mmHg x ml(-1) x min x 100 g). Blood flow to the spleen, muscle, lungs, and adrenals, as well as the blood pressure and heart rate, were not altered by the ANG-(1-7) infusion. The selective ANG-(1-7) antagonist A-779 reduced the blood flow in renal, cerebral, mesenteric, and cutaneous beds and blocked the ANG-(1-7)-induced vasodilatation in the kidney, mesentery, and skin, suggesting a significant role of endogenous ANG-(1-7) in these territories. The effects of ANG-(1-7) on the cerebral blood flow, cardiac index, systolic volume, and total peripheral resistance were partially attenuated by A-779. A high dose of ANG-(1-7) (11 pmol x min(-1) x 10 min(-1)) caused an opposite effect of that produced by the low dose. Our results show for the first time that ANG-(1-7) has a previously unsuspected potent effect in the blood flow distribution and systemic hemodynamics.     

Larvicidal isoxazoles: Synthesis and their effective susceptibility towards Aedes aegypti larvae

Twenty 3,5-disubstituted isoxazoles have been synthesized and tested against fourth instar Aedes aegypti larvae. In the synthesis of title compounds, modifications have been made in the C-5 side-chain with a view to test their larvicidal activity. These isoxazoles have been obtained by 1,3-dipolar cycloaddition of arylnitrile oxides to terminal alkynes which furnished the desired products in 20% to 79% yields. A comparative study of the larvicidal activity between 3-(3-aryl-isoxazol-5-yl)-propan-1-ols and 3-(3-aryl-isoxazol-5-yl)-propionic acids clearly demonstrated that the latter compounds possess much better larvicidal activity than the former. We also tested two esters, viz., methyl 3-[3-(phenyl)-isoxazole-5-yl] propionate and methyl 3-[3-(4-chlorophenyl)-isoxazole-5-yl] propionate, where the latter presented an excellent larvicidal profile.     

A practical approach to the choice of a suitable detergent and optimal conditions for solubilizing a membrane protein

The main objective of this laboratory practical class was to teach students how a detergent and the best experimental conditions are chosen to solubilize a given membrane protein. Kidney Na,K-ATPase was chosen as the protein of interest and anionic, neutral and zwitterionic detergents were tested. Simple laboratory experiments were designed to study the effect of the detergent on the activity of the enzyme, the effect of detergent concentration on solubilization, the effect of protein concentration on enzyme solubilization, and the effect of time and temperature of incubation during enzyme solubilization. This resulted in the selection of an appropriate detergent for the solubilization of the protein taking into account smaller inactivation factors, more effective solubilization (more effective solubilization with a better detergent-protein relationship), lower inactivation temperature and time of incubation of the membrane protein with the detergent. The results obtained showed that instantaneous incubation of Na,K-ATPase with C(12)E(8) (1:1 w/w) at 4 degrees C resulted in a more efficient solubilization and had a smaller denaturing effect on the solubilized enzyme.     

Transfer RNA structural change is a key element in the reassignment of the CUG codon in Candida albicans.

The human pathogenic yeast Candida albicans and a number of other Candida species translate the standard leucine CUG codon as serine. This is the latest addition to an increasing number of alterations to the standard genetic code which invalidate the theory that the code is frozen and universal. The unexpected finding that some organisms evolved alternative genetic codes raises two important questions: how have these alternative codes evolved and what evolutionary advantages could they create to allow for their selection? To address these questions in the context of serine CUG translation in C.albicans, we have searched for unique structural features in seryl-tRNA(CAG), which translates the leucine CUG codon as serine, and attempted to reconstruct the early stages of this genetic code switch in the closely related yeast species Saccharomyces cerevisiae. We show that a purine at position 33 (G33) in the C.albicans Ser-tRNA(CAG) anticodon loop, which replaces a conserved pyrimidine found in all other tRNAs, is a key structural element in the reassignment of the CUG codon from leucine to serine in that it decreases the decoding efficiency of the tRNA, thereby allowing cells to survive low level serine CUG translation. Expression of this tRNA in S.cerevisiae induces the stress response which allows cells to acquire thermotolerance. We argue that acquisition of thermotolerance may represent a positive selection for this genetic code change by allowing yeasts to adapt to sudden changes in environmental conditions and therefore colonize new ecological niches.     

Glucose Metabolism and Kinetics of Phosphorus Removal by the Fermentative Bacterium Microlunatus phosphovorus

Phosphorus and carbon metabolism in Microlunatus phosphovorus was investigated by using a batch reactor to study the kinetics of uptake and release of extracellular compounds, in combination with ³¹P and ¹³C nuclear magnetic resonance (NMR) to characterize intracellular pools and to trace the fate of carbon substrates through the anaerobic and aerobic cycles. The organism was subjected to repetitive anaerobic and aerobic cycles to induce phosphorus release and uptake in a sequencial batch reactor; an ultrafiltration membrane module was required since cell suspensions did not sediment. M. phosphovorus fermented glucose to acetate via an Embden-Meyerhof pathway but was unable to grow under anaerobic conditions. A remarkable time shift was observed between the uptake of glucose and excretion of acetate, resulting in an intracellular accumulation of acetate. The acetate produced was oxidized in the subsequent aerobic stage. Very high phosphorus release and uptake rates were measured, 3.34 mmol g of cell⁻¹ h⁻¹ and 1.56 mmol g of cell⁻¹ h⁻¹, respectively, values only comparable with those determined in activated sludge. In the aerobic period, growth was strictly dependent on the availability of external phosphate. Natural abundance ¹³C NMR showed the presence of reserves of glutamate and trehalose in cell suspensions. Unexpectedly, [1-¹³C]glucose was not significantly channeled to the synthesis of internal reserves in the anaerobic phase, and acetate was not during the aerobic stage, although the glutamate pool became labeled via the exchange with intermediates of the tricarboxylic acid cycle at the level of glutamate dehydrogenase. The intracellular pool of glutamate increased under anaerobic conditions and decreased during the aerobic period. The contribution of M. phosphovorus for phosphorus removal in wastewater treatment plants is discussed on the basis of the metabolic features disclosed by this study.     

The antimicrobial activity of lapachol and its thiosemicarbazone and semicarbazone derivatives

Lapachol was chemically modified to obtain its thiosemicarbazone and semicarbazone derivatives. These compounds were tested for antimicrobial activity against several bacteria and fungi by the broth microdilution method. The thiosemicarbazone and semicarbazone derivatives of lapachol exhibited antimicrobial activity against the bacteria Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentrations (MICs) of 0.05 and 0.10 µmol/mL, respectively. The thiosemicarbazone and semicarbazone derivatives were also active against the pathogenic yeast Cryptococcus gattii (MICs of 0.10 and 0.20 µmol/mL, respectively). In addition, the lapachol thiosemicarbazone derivative was active against 11 clinical isolates of Paracoccidioides brasiliensis, with MICs ranging from 0.01-0.10 µmol/mL. The lapachol-derived thiosemicarbazone was not cytotoxic to normal cells at the concentrations that were active against fungi and bacteria. We synthesised, for the first time, thiosemicarbazone and semicarbazone derivatives of lapachol. The MICs for the lapachol-derived thiosemicarbazone against S. aureus, E. faecalis, C. gattii and several isolates of P. brasiliensis indicated that this compound has the potential to be developed into novel drugs to treat infections caused these microbes.