Ergot alkaloid biosynthesis in Aspergillus fumigatus

Ergot alkaloid biosynthesis in Aspergillus fumigatus - Overproduction and biochemical characterization of a 4-dimethylallyltryptophan N-methyltransferase

Author(s): Rigbers O (Rigbers, Ole), Li SM (Li, Shu-Ming)1
Source: JOURNAL OF BIOLOGICAL CHEMISTRY
Volume: 283 Issue: 40 Pages: 26859-26868 Published: OCT 3 2008

Abstract: The putative gene fgaMT was identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus. The coding region of fgaMT was amplified by PCR from a cDNA library, cloned into pQE60, and overexpressed in Escherichia coli. FgaMT comprises 339 amino acids with a molecular mass of about 38.1 kDa. The soluble dimeric His(6)-FgaMT was purified to near homogeneity and characterized biochemically. FgaMT was found to catalyze the N-methylation of 4-dimethylallyltryptophan in the presence of S-adenosylmethionine, resulting in the formation of 4-dimethylallyl-L-abrine, which was identified by NMR and mass spectrometry analysis. Therefore, FgaMT represents the second pathway-specific enzyme in the biosynthesis of ergot alkaloids. The enzyme did not require metal ions for its enzymatic reaction and showed a relatively high specificity toward the prenyl moiety at position C-4 of the indole ring. 4-Dimethylallyltryptophan derivatives with modification at the indole ring were also accepted by FgaMT as substrates. Km values for 4-dimethylallyltryptophan and S-adenosylmethionine were determined at 0.12 and 2.4 mM, respectively. The turnover number was 2.0 s(-1).

Determination of Ergot Alkaloids

Determination of Ergot Alkaloids: Purity and Stability Assessment of Standards and Optimization of Extraction Conditions for Cereal Samples


Author(s): Krska R (Krska, Rudolf)1,2, Berthiller F (Berthiller, Franz)1, Schuhmacher R (Schuhmacher, Rainer)1, Nielsen KF (Nielsen, Kristian F.)3, Crews C (Crews, Colin)2

Source: JOURNAL OF AOAC INTERNATIONAL Volume: 91 Issue: 6 Pages: 1363-1371 Published: NOV-DEC 2008

Abstract: Results obtained from a purity study on standards of the 6 major ergot alkaloids ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, and ergocornine and their corresponding epimers are discussed. The 6 ergot alkaloids studied have been defined by the European Food Safety Authority as those that are the most common and physiologically active. The purity of the standards was investigated by means of liquid chromatography with diode array detection, electrospray ionization, and time-of-flight mass spectrometry (LC-DAD-ESI-TOF-MS). All of the standards assessed showed purity levels considerably above 98% apart from ergocristinine (94%), ergosine (96%), and ergosinine (95%). Also discussed is the optimization of extraction conditions presented in a recently published method for the quantitation of ergot alkaloids in food samples using solid-phase extraction with primary secondary amine (PSA) before LC/MS/MS. Based on the results obtained from these optimization studies, a mixture of acetonitrile with ammonium carbonate buffer was used as extraction solvent, as recoveries for all analyzed ergot alkaloids were significantly higher than those with the other solvents. Different sample-solvent ratios and extraction times showed just minor influences in extraction efficacy. Finally, the stability of the ergot alkaloids in both raw cereals and cereal-based processed food extracts was studied. According to these studies, extracts should be prepared and analyzed the same day or stored below ambient temperatures. Barley and rye extracts, which were stored at 4 and 15 degrees C after PSA cleanup, proved to be stable overnight. However, storage over a period of 14 days at 4 degrees C resulted in significant epimerization, which was most pronounced in rye and particularly for ergocornine, ergocryptine, and ergocristine.

The Venus of Hohle Fels.

The Venus of Hohle Fels. (Credit: Photo by H. Jensen; Copyright: Universität Tübingen)

Good News for Night Owls

Good News for Night Owls
By Elsa Youngsteadt
ScienceNOW Daily News
23 April 2009

Night owls seem to have a cognitive edge over early risers--at least when they're on their natural sleep schedule. That's one upshot of a new brain-imaging study that also gives surprising new insights into how the brain manages the urge to sleep and wake. The results, sleep researchers say, may improve predictions of when people are most at risk for drowsy accidents.
Two factors control our bedtime. The first is hardwired: A master clock in the brain regulates a so-called circadian rhythm, which synchronizes activity patterns to the 24-hour day. Some people's clocks tell them to go to bed at 9 p.m., others' at 3 a.m., (ScienceNOW, 24 June 2003). The second factor--called sleep pressure--depends not on time of day but simply on how long someone has been awake already.

Because sleep pressure accumulates during waking hours, logic suggests that we should be most alert--and hence sharpest--shortly after we get up versus right before we go to bed, regardless of whether we're night owls or larks.

But that's not what Christina Schmidt found. The doctoral student at the University of Liège in Belgium and her collaborators, led by sleep researcher Philippe Peigneux, recruited 16 morning people and 15 night people to take alertness tests in a brain scanner. Subjects had to pay attention to numbers on a computer screen and hit a button whenever the numbers began to change. To control for the effect of the circadian clock, the subjects were allowed to sleep on their own natural schedules and take the test 1.5 hours and 10.5 hours after waking, regardless of the actual time of day.

Both groups performed equally well on the test when they took it 1.5 hours after waking. But after 10.5 hours without sleep, the night owls pulled ahead. Their reaction times improved by about 6% relative to the morning people and to their own earlier performance, the researchers report in tomorrow's issue of Science. This suggests that once they wake up, sleep pressure builds up faster in early birds, says Peigneux, and that this hurts their cognition over time.

It's a result with "real-world consequences," says sleep researcher David Dinges of the University of Pennsylvania School of Medicine in Philadelphia. Current risk analyses use the time of day and hours worked to predict when people are in greatest danger of accidents--such as aviation errors. But now, Dinges says, they may need to take into account that morning people tend to lose their concentration faster. At the very least, according to sleep researcher Amita Sehgal, also at the University of Pennsylvania School of Medicine, this is a new and "intriguing" explanation for larks' and owls' different habits.

But the really provocative result, adds Dinges, came from the brain imaging. The night owls showed greater activity in the master-clock region of their brains--a cluster of cells known as the suprachiasmatic nucleus--than the larks when taking the later test. That suggests that sleep pressure and the circadian clock can influence each other directly--bringing together two systems that, for decades, had been thought to operate separately.

Homeostatic Sleep Pressure and Responses to Sustained Attention in the Suprachiasmatic Area

Science 24 April 2009:
Vol. 324. no. 5926, pp. 516 - 519
DOI: 10.1126/science.1167337
Prev | Table of Contents | Next
Reports
Homeostatic Sleep Pressure and Responses to Sustained Attention in the Suprachiasmatic Area
Christina Schmidt,1,2,* Fabienne Collette,1,2 Yves Leclercq,1 Virginie Sterpenich,1 Gilles Vandewalle,1 Pierre Berthomier,3 Christian Berthomier,3 Christophe Phillips,1 Gilberte Tinguely,1 Annabelle Darsaud,1 Steffen Gais,1 Manuel Schabus,1 Martin Desseilles,1 Thien Thanh Dang-Vu,1 Eric Salmon,1 Evelyne Balteau,1 Christian Degueldre,1 André Luxen,1 Pierre Maquet,1 Christian Cajochen,4 Philippe Peigneux1,5,*

Throughout the day, cognitive performance is under the combined influence of circadian processes and homeostatic sleep pressure. Some people perform best in the morning, whereas others are more alert in the evening. These chronotypes provide a unique way to study the effects of sleep-wake regulation on the cerebral mechanisms supporting cognition. Using functional magnetic resonance imaging in extreme chronotypes, we found that maintaining attention in the evening was associated with higher activity in evening than morning chronotypes in a region of the locus coeruleus and in a suprachiasmatic area (SCA) including the circadian master clock. Activity in the SCA decreased with increasing homeostatic sleep pressure. This result shows the direct influence of the homeostatic and circadian interaction on the neural activity underpinning human behavior.

2 micron plasmid hitchhikes on mitotic mechanism of Saccharomyces cerevisiae to maintain equal distribution in host

Published online April 13, 2009
doi:10.1083/jcb.200810130
The Journal of Cell Biology, Vol. 185, No. 2, 251-264
The Rockefeller University Press, 0021-9525 $30.00
© 2009 Cui et al.

The selfish yeast plasmid uses the nuclear motor Kip1p but not Cin8p for its localization and equal segregation

Hong Cui, Santanu K. Ghosh, and Makkuni Jayaram
Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712

Correspondence to Makkuni Jayaram: jayaram@icmb.utexas.edu


The 2 micron plasmid of Saccharomyces cerevisiae uses the Kip1 motor, but not the functionally redundant Cin8 motor, for its precise nuclear localization and equal segregation. The timing and lifetime of Kip1p association with the plasmid partitioning locus STB are consistent with Kip1p being an authentic component of the plasmid partitioning complex. Kip1–STB association is not blocked by disassembling the mitotic spindle. Lack of Kip1p disrupts recruitment of the cohesin complex at STB and cohesion of replicated plasmid molecules. Colocalization of a 2 micron reporter plasmid with Kip1p in close proximity to the spindle pole body is reminiscent of that of a CEN reporter plasmid. Absence of Kip1p displaces the plasmid from this nuclear address, where it has the potential to tether to a chromosome or poach chromosome segregation factors. Exploiting Kip1p, which is subsidiary to Cin8p for chromosome segregation, to direct itself to a "partitioning center" represents yet another facet of the benign parasitism of the yeast plasmid.

Biogeochemistry: Less nickel for more oxygen

News and Views
Nature 458, 714-715 (9 April 2009) | doi:10.1038/458714a; Published online 8 April 2009


Biogeochemistry: Less nickel for more oxygen
Mak A. Saito1

Top of pageAbstractThe availability (or lack) of oceanic trace elements is providing fresh lines of explanation for turning points in Earth's history — the Great Oxidation Event being one such momentous occasion.

About 2.4 billion years ago, the oxygen content of Earth's atmosphere increased in what is called the Great Oxidation Event (GOE). This marked the beginning of the most significant series of chemical changes Earth has ever experienced, setting the stage for oxidative weathering of the continents, successive changes in ocean chemistry, and the eventual rise of multicellular life.

Yet the sequence of events leading up to the GOE is not well understood. Most researchers agree that the evolution of oxygenic photosynthesis within a group called the cyanobacteria was the source of the molecular oxygen that caused the GOE1. But the timing of the rise of these bacteria is uncertain2, 3, and there may have been a period of inertia — due, for example, to chemical reactions with methane that consumed oxygen4 — that prevented a swift increase in atmospheric oxygen. It remains a matter of debate how these two phenomena might have induced the GOE: an early rise of cyanobacteria and slow crumbling of chemical resistance3, 4; or a late rise of cyanobacteria leading to rapid initiation of the GOE5.

On page 750 of this issue6, Konhauser et al. report evidence for an alternative driving mechanism of the GOE, one that would have decreased microbial methane production in the oceans and paved the way for increased oxygen abundances. The authors find significant decreases in the nickel-to-iron ratios in ancient rocks, known as banded iron formations, that provide records of element concentrations in the oceans (Fig. 1). They estimate that a major decrease in the oceanic inventory of nickel must have occurred around 2.7 billion years ago. This, they conclude, led to a cascade of events in which methanogens, with their gluttonous appetite for nickel to feed three nickel-containing metalloenzymes, would have become starved of the element and so have produced much less methane. With the decrease in chemical inertia associated with methane4, the stage was set for cyanobacterial oxygen to accumulate, leading to the GOE. Moreover, although Konhauser et al. don't go into detail, the decline in atmospheric methane, a powerful greenhouse gas, is believed to help account for the initiation of a planetary-scale glaciation known as Snowball Earth that is thought to have begun between 2.3 billion and 2.2 billion years ago4, 5.

Figure 1: Record site.
This is a view of Dales Gorge, northwest Australia, one of the banded iron formations sampled by Konhauser et al.6.

High resolution image and legend (151K)



The idea that significant changes in seawater trace-metal abundance have occurred during Earth's history is becoming popular7, 8. For example, it is thought that iron and cobalt were abundant in ancient oceans, whereas zinc and copper were probably extremely scarce owing to precipitation with sulphides8. When the oceans became oxygenated, it is likely that this scheme was reversed, with iron and cobalt becoming scarce through oxidation and precipitation as oxyhydroxides, and zinc and copper becoming much more abundant upon the oxidation of sulphide to sulphate in sea water. These predictions of broad changes in ocean chemistry are mirrored by the physiological and genomic traits of archaea and bacteria, relative to those of the later-evolving eukaryotes8, 9.

Nickel has largely been left out of this intriguing story. On the evidence of chemical modelling8, it seems that nickel was not as strongly affected by the variations in sulphide and oxygen during Earth's history. But such a conclusion does not take into account the possible involvement of external factors. Konhauser et al. show how such a factor might have come into play, with the cooling of Earth's mantle resulting in decreased eruption of nickel-rich rocks and causing an estimated 50% fall in the oceanic nickel inventory.

Konhauser and colleagues' thinking6 may come as a surprise to those familiar with the chemistry of the modern oceans. Trace metals — as their name suggests — are extraordinarily scarce in sea water. In vast regions of the modern oceans, photosynthesis is limited by low iron availability, with iron concentrations often being less than 0.05 nanomoles per litre10. Yet, of the trace metals required by life, nickel is one of the more abundant in sea water, with surface water concentrations of 1–2 nanomoles per litre11. In this modern context, the idea of a nickel famine seems odd. But the nickel requirements of methanogens are reported6 to be in the hundreds of nanomoles per litre, suggesting that methanogens cannot live in the modern oceans and are perhaps relegated to sedimentary, coastal and freshwater environments, where nickel is more abundant.

By connecting changes in mantle temperature to nickel fluxes and methanogens, Konhauser and colleagues' study is particularly satisfying. Instead of relying on the uncertain timing of the rise of cyanobacteria to explain the GOE, that event can instead be tied to a specific mechanism recorded in the banded iron formations. In addition, this 'nickel famine' mechanism is consistent with evidence12 of 'whiffs of oxygen' that occurred more than 50 million years before the GOE. But I cannot help but wonder whether there is a reason — such as the slow chemical kinetics of nickel ions — why methanogens could not evolve a high-affinity nickel-uptake mechanism similar to those that exist for the uptake of iron, zinc and cobalt13, 14, 15.

Finally, there is another context in which the research of Konhauser et al. is set — the exciting endeavour of trying to understand how the elemental cycles (of nickel, carbon, iron, nitrogen and so on) have 'co-evolved' with microbial life. Many of the changes in element cycling were probably caused by the rise and fall of specific microbial metabolisms, while also strongly affecting the trajectory and composition of life on Earth. Life and the cycling of elements have both been changing throughout Earth's history, often influencing each other profoundly along the way. One of the sobering realizations of studies such as this is that, although natural selection provides a clear, single positive-feedback mechanism for the continuation of life, elemental cycles are instead influenced by an aggregate of mechanisms, including biological evolution, chemical reactions, changes in ocean circulation and geological events. If, as Konhauser et al. suggest, a single geological change can starve a major oceanic microbial community, and thereby change the trajectory of life on Earth, it suggests that there is a fragility to Earth's elemental cycles that we are only beginning to uncover.