Molecular rules for selectivity in lipase-catalysed acylation of lysine

International audienceThe selectivity of L-lysine acylation by lauric acid catalysed by Candida antarctica lipase B (CALB) was investigated combining experimental and theoretical methodologies. Experiments showed the near-exclusive acylation of lysine ε-amino group; only traces of product resulting from the acylation of lysine α-amino group were observed fleetingly. Molecular modelling simulations were performed aiming to understand the molecular rules for selectivity. Flexible docking simulations combined with structural investigations into lysine/CALB binding modes also suggested the preferential acylation of lysine ε-amino group without, however, excluding the acylation of the lysine α-amino group. Electrostatic interaction energy between lysine and the residues covering the catalytic cavity was calculated in order to understand the discrimination between the two lysine amino groups. The results suggests that the proximity of the carboxylate group hinders the binding of the substrate in configurations enabling the N-acylation. Key interactions with the polar region covering the catalytic triad were identified and a plausible explanation for selectivity was proposed

Uloga enzima u metabolizmu ugljikohidrata pri diferencijaciji staničnih linija šećerne repe

Plant development is influenced by changes in the levels and types of sugars produced metabolically. The normal (N), habituated organogenic (HO) and habituated nonorganogenic (HNO) sugar beet cell lines originate from the same mother plant but exhibit distinct levels of morphogenesis and differentiation, and contain different levels of simple carbohydrates. We aim to elucidate whether differences in the abundance and activity of enzymes involved in carbohydrate metabolism and sugar sensing/signalling help explain the different carbohydrate profiles and differentiation states of the cell lines. Using 13C NMR spectroscopy to analyze cultures of the cell lines over 28 days, we found that N cells accumulated sucrose; HO cells sucrose, glucose and fructose; and HNO cells glucose and fructose. Of three invertase isoforms, the activity of cell wall invertase (CWI) was highest in all the cell lines, and CWI activity was greatest in HNO line. The specific accumulation of intracellular carbohydrates during subculture correlated strongly with CWI activity but less so with the vacuolar and cytoplasmic invertase isoforms, or with sucrose synthase activity. Cell lines showed differences in how sugars regulated invertase and sucrose synthase activity. The role of sugar sensing in the regulation of CWI activity was investigated in the cell lines using glucose and sucrose, as well as carbohydrate analogues such as mannitol, 2-O-deoxyglucose and 3-O-methylglucose. Differences in the regulation of CWI activity by carbohydrates across the three cell lines suggest that CWI can be fine-tuned according to the specific carbohydrate requirements of each line during growth. Differences in sugar signalling pathways across the cell lines were explored using glucose and sucrose in the presence of inhibitors of protein kinases or phosphatases. Taken together, our findings suggest that specific regulation of CWI activity plays an important role in determining the intracellular carbohydrate levels of sugar beet cell lines, and possibly their differentiation state as well.Na razvoj biljke utječu promjene razine i tipa proizvedenog šećera. Iako sve stanične linije šećerne repe, i to normalna, prilagođena organogena i prilagođena neorganogena potječu od iste biljke, na različitim su stupnjevima diferencijacije i morfogeneze te sadrže različitu količinu jednostavnih ugljikohidrata. Svrha je rada bila utvrditi može li se razlikama u količini i aktivnosti enzima, koji sudjeluju u metabolizmu šećera te onih u detekciji i prijenosu signala šećera, objasniti razlika u stupnju diferencijacije staničnih linija te u udjelu ugljikohidrata u njima. Primjenom 13C NMR spektroskopije analizirane su stanične linije tijekom 28 dana kultivacije i ustanovljeno je da normalne stanične linije akumuliraju saharozu, prilagođena organogena stanična linija saharozu, glukozu i fruktozu, a prilagođena neorganogena stanična linija šećerne repe glukozu i fruktozu. Od 3 izoformna oblika invertaze najveća je aktivnost invertaze stanične stijenke u sve 3 stanične linije, osobito u prilagođenih neorganogenih stanica. Specifičnost akumulacije ugljikohidrata u stanici tijekom kultivacije uvelike je ovisila o aktivnosti invertaze stanične stijenke, a manje o onoj vakuolarne i citoplazmatske invertaze te saharoza sintetaze. Šećeri su različito regulirali aktivnost invertaze stanične stijenke i saharoza sintetaze, ovisno o tipu stanične linije. Njihova je uloga istražena pomoću glukoze i saharoze te njihovih analoga, kao što su manitol, 2-O-deoksiglukoza i 3-O-metilglukoza. Utvrđeno je da se aktivnost invertaze stanične stijenke može fino regulirati pomoću ugljikohidrata, ovisno o potrebi staničnih linija za izvorom šećera tijekom rasta. Razlike u putovima prijenosa signala šećera u staničnih linija istražene su pomoću glukoze i saharoze u prisutnosti inhibitora protein kinaza i fosfataza. Na kraju, rezultati autora potvrđuju da specifična regulacija aktivnosti invertaze stanične stijenke ima važnu ulogu u određivanju razine ugljikohidrata u staničnim linijama šećerne repe, a možda i stupnju diferencijacije stanica

Improvement in the amino-acid microbial production

Amino-acid microbial production can be improved by a process modification or by inducing changes in the metabolism of the biocatalyst. These two approaches will be illustrated and discussed using the examples of glutamic acid and valine productions with Corynebacterium glutamicum.La production d'acide aminé par voie microbienne peut être améliorée soit en jouant sur le procédé de culture soit en modifiant le métabolisme du micro-organisme producteur. Ces 2 approches seront illustrées et discutées grâce à deux exemples d'acides aminés produits par Coryne-bacterium glutamicum: l'acide glutamique et la valine

Ex vivo activity of the ACT new components pyronaridine and piperaquine in comparison with conventional ACT drugs against isolates of Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>The aim of the present work was to assess i) <it>ex vivo </it>activity of pyronaridine (PND) and piperaquine (PPQ), as new components of artemisinin-based combination therapy (ACT), to define susceptibility baseline, ii) their activities compared to other partner drugs, namely monodesethylamodiaquine (MDAQ), lumefantrine (LMF), mefloquine (MQ), artesunate (AS) and dihydroartemisinin (DHA) against 181 <it>Plasmodium falciparum </it>isolates from African countries, India and Thailand, and iii) <it>in vitro </it>cross-resistance with other quinoline drugs, chloroquine (CQ) or quinine (QN).</p> <p>Methods</p> <p>The susceptibility of the 181 <it>P. falciparum </it>isolates to the nine anti-malarial drugs was assessed using the standard 42-hours ³H-hypoxanthine uptake inhibition method.</p> <p>Results</p> <p>The IC₅₀values for PND ranged from 0.55 to 80.0 nM (geometric mean = 19.9 nM) and from 11.8 to 217.3 nM for PPQ (geometric mean = 66.8 nM). A significant positive correlation was shown between responses to PPQ and PND responses (<it>rho </it>= 0.46) and between PPQ and MDAQ (<it>rho </it>= 0.30). No significant correlation was shown between PPQ IC₅₀and responses to other anti-malarial drugs. A significant positive correlation was shown between responses to PND and MDAQ (<it>rho </it>= 0.37), PND and LMF (<it>rho </it>= 0.28), PND and QN (<it>rho </it>= 0.24), PND and AS (<it>rho </it>= 0.19), PND and DHA (<it>rho </it>= 0.18) and PND and CQ (<it>rho </it>= 0.16). All these coefficients of correlation are too low to suggest cross-resistance between PPQ or PND and the other drugs.</p> <p>Conclusions</p> <p>In this study, the excellent anti-malarial activity of PPQ and PND was confirmed. The absence of cross-resistance with quinolines and artemisinin derivatives is consistent with the efficacy of the combinations of PPQ and DHA or PND and AS in areas where parasites are resistant to conventional anti-malarial drugs.</p

Laser-induced ultrafast demagnetization in the presence of a nanoscale magnetic domain network

International audienceFemtosecond magnetization phenomena have been challenging our understanding for over a decade. Most experiments have relied on infrared femtosecond lasers, limiting the spatial resolution to a few micrometres. With the advent of femtosecond X-ray sources, nanometric resolution can now be reached, which matches key length scales in femtomagnetism such as the travelling length of excited 'hot' electrons on a femtosecond timescale. Here we study laser-induced ultrafast demagnetization in [Co/Pd]30 multilayer films, which, for the first time, achieves a spatial resolution better than 100 nm by using femtosecond soft X-ray pulses. This allows us to follow the femtosecond demagnetization process in a magnetic system consisting of alternating nanometric domains of opposite magnetization. No modification of the magnetic structure is observed, but, in comparison with uniformly magnetized systems of similar composition, we find a significantly faster demagnetization time. We argue that this may be caused by direct transfer of spin angular momentum between neighbouring domains

Combining metabolic flux analysis with proteomics to shed light on the metabolic flexibility: the case of Desulfovibrio vulgaris Hildenborough

IntroductionDesulfovibrio vulgaris Hildenborough is a gram-negative anaerobic bacterium belonging to the sulfate-reducing bacteria that exhibits highly versatile metabolism. By switching from one energy mode to another depending on nutrients availability in the environments„ it plays a central role in shaping ecosystems. Despite intensive efforts to study D. vulgaris energy metabolism at the genomic, biochemical and ecological level, bioenergetics in this microorganism remain far from being fully understood. Alternatively, metabolic modeling is a powerful tool to understand bioenergetics. However, all the current models for D. vulgaris appeared to be not easily adaptable to various environmental conditions.MethodsTo lift off these limitations, here we constructed a novel transparent and robust metabolic model to explain D. vulgaris bioenergetics by combining whole-cell proteomic analysis with modeling approaches (Flux Balance Analysis).ResultsThe iDvu71 model showed over 0.95 correlation with experimental data. Further simulations allowed a detailed description of D. vulgaris metabolism in various conditions of growth. Altogether, the simulations run in this study highlighted the sulfate-to-lactate consumption ratio as a pivotal factor in D. vulgaris energy metabolism.DiscussionIn particular, the impact on the hydrogen/formate balance and biomass synthesis is discussed. Overall, this study provides a novel insight into D. vulgaris metabolic flexibility

Zebularine, a DNA Methylation Inhibitor, Activates Anthocyanin Accumulation in Grapevine Cells

Through its role in the regulation of gene expression, DNA methylation can participate in the control of specialized metabolite production. We have investigated the link between DNA methylation and anthocyanin accumulation in grapevine using the hypomethylating drug, zebularine and Gamay Teinturier cell suspensions. In this model, zebularine increased anthocyanin accumulation in the light, and induced its production in the dark. To unravel the underlying mechanisms, cell transcriptome, metabolic content, and DNA methylation were analyzed. The up-regulation of stress-related genes, as well as a decrease in cell viability, revealed that zebularine affected cell integrity. Concomitantly, the global DNA methylation level was only slightly decreased in the light and not modified in the dark. However, locus-specific analyses demonstrated a decrease in DNA methylation at a few selected loci, including a CACTA DNA transposon and a small region upstream from the UFGT gene, coding for the UDP glucose:flavonoid-3-O-glucosyltransferase, known to be critical for anthocyanin biosynthesis. Moreover, this decrease was correlated with an increase in UFGT expression and in anthocyanin content. In conclusion, our data suggest that UFGT expression could be regulated through DNA methylation in Gamay Teinturier, although the functional link between changes in DNA methylation and UFGT transcription still needs to be demonstrated

Enquête auprès des médecins généralistes sur leurs connaissances en matière de dopage

RENNES1-BU Santé (352382103) / SudocSudocFranceF