The First Step of Neurospora crassa Molybdenum Cofactor Biosynthesis: Regulatory Aspects under N-Derepressing and Nitrate-Inducing Conditions

Molybdenum cofactor (Moco) is the active site prosthetic group found in all Moco dependent enzymes, except for nitrogenase. Mo-enzymes are crucial for viability throughout all kingdoms of life as they catalyze a diverse set of two electron transfer reactions. The highly conserved Moco biosynthesis pathway consists of four different steps in which guanosine triphosphate is converted into cyclic pyranopterin monophosphate, molybdopterin (MPT), and subsequently adenylated MPT and Moco. Although the enzymes and mechanisms involved in these steps are well characterized, the regulation of eukaryotic Moco biosynthesis is not. Within this work, we described the regulation of Moco biosynthesis in the filamentous fungus Neurospora crassa, which revealed the first step of the multi-step pathway to be under transcriptional control. We found, that upon the induction of high cellular Moco demand a single transcript variant of the nit-7 gene is increasingly formed pointing towards, that essentially the encoded enzyme NIT7-A is the key player for Moco biosynthesis activity in Neurospora

The Molybdenum Cofactor Biosynthesis Network: In vivo Protein-Protein Interactions of an Actin Associated Multi-Protein Complex

Survival of plants and nearly all organisms depends on the pterin based molybdenum cofactor (Moco) as well as its effective biosynthesis and insertion into apo-enzymes. To this end, both the central Moco biosynthesis enzymes are characterized and the conserved four-step reaction pathway for Moco biosynthesis is well-understood. However, protection mechanisms to prevent degradation during biosynthesis as well as transfer of the highly oxygen sensitive Moco and its intermediates are not fully enlightened. The formation of protein complexes involving transient protein-protein interactions is an efficient strategy for protected metabolic channelling of sensitive molecules. In this review, Moco biosynthesis and allocation network is presented and discussed. This network was intensively studied based on two in vivo interaction methods: bimolecular fluorescence complementation (BiFC) and split-luciferase. Whereas BiFC allows localisation of interacting partners, split-luciferase assay determines interaction strengths in vivo. Results demonstrate (i) interaction of Cnx2 and Cnx3 within the mitochondria and (ii) assembly of a biosynthesis complex including the cytosolic enzymes Cnx5, Cnx6, Cnx7, and Cnx1, which enables a protected transfer of intermediates. The whole complex is associated with actin filaments via Cnx1 as anchor protein. After biosynthesis, Moco needs to be handed over to the specific apo-enzymes. A potential pathway was discovered. Molybdenum-containing enzymes of the sulphite oxidase family interact directly with Cnx1. In contrast, the xanthine oxidoreductase family acquires Moco indirectly via a Moco binding protein (MoBP2) and Moco sulphurase ABA3. In summary, the uncovered interaction matrix enables an efficient transfer for intermediate and product protection via micro-compartmentation

The Kinematics of Massive Quiescent Galaxies at 1.4 < z < 2.1: Dark Matter Fractions, IMF Variation, and the Relation to Local Early-type Galaxies

We study the dynamical properties of massive quiescent galaxies at 1.4 <z <2.1 using deep Hubble Space Telescope WFC3/F160W imaging and a combination of literature stellar velocity dispersion measurements and new near-infrared spectra obtained using the K-band Multi Object Spectrograph(KMOS) on the ESO Very Large Telescope. We use these data to show that the typical dynamical-to-stellar mass ratio has increased by∼0.2 dex from z = 2 to the present day, and we investigate this evolution in the context of possible changes in the stellar initial mass function(IMF) and/or fraction of dark matter contained within the galaxy effective radius,fDM[<re]. Comparing our high-redshift sample to their likely descendants at low redshift, we find that fDM[<re] has increased by a factor of more than 4 since z ≈ 1.8, from fDM[<re] = 6.6% +-1.0% to∼24%. The observed increase appears robust to changes in the methods used to estimate dynamical masses or match progenitors and descendants. We quantify possible variation of the stellar IMF through the offset parameter α, defined as the ratio of dynamical mass in stars to the stellar mass estimated using a Chabrier IMF. We demonstrate that the correlation between stellar velocity dispersion and α reported among quiescent galaxies at low redshift is already in place atz = 2, and we argue that subsequent evolution through (mostly minor) merging should act to preserve this relation while contributing significantly to galaxies'overall growth in size and stellar mass.J.T.M. acknowledges the support of the Australian Research Council Centre of Excellence for All Sky Astrophysics in 3 Dimensions (ASTRO 3D), through project No. CE170100013. D.J.W. and M.F. acknowledge the support of the Deutsche Forschungsgemeinschaft via Project IDs 3871/1-1 and 3871/1-2. M.F. has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 757535)

Sulphur flux through the sulphate assimilation pathway is differently controlled by adenosine 5′-phosphosulphate reductase under stress and in transgenic poplar plants overexpressing γ-ECS, SO, or APR

Sulphate assimilation provides reduced sulphur for the synthesis of cysteine, methionine, and numerous other essential metabolites and secondary compounds. The key step in the pathway is the reduction of activated sulphate, adenosine 5′-phosphosulphate (APS), to sulphite catalysed by APS reductase (APR). In the present study, [35S]sulphur flux from external sulphate into glutathione (GSH) and proteins was analysed to check whether APR controls the flux through the sulphate assimilation pathway in poplar roots under some stress conditions and in transgenic poplars. (i) O-Acetylserine (OAS) induced APR activity and the sulphur flux into GSH. (ii) The herbicide Acetochlor induced APR activity and results in a decline of GSH. Thereby the sulphur flux into GSH or protein remained unaffected. (iii) Cd treatment increased APR activity without any changes in sulphur flux but lowered sulphate uptake. Several transgenic poplar plants that were manipulated in sulphur metabolism were also analysed. (i) Transgenic poplar plants that overexpressed the γ-glutamylcysteine synthetase (γ-ECS) gene, the enzyme catalysing the key step in GSH formation, showed an increase in sulphur flux into GSH and sulphate uptake when γ-ECS was targeted to the cytosol, while no changes in sulphur flux were observed when γ-ECS was targeted to plastids. (ii) No effect on sulphur flux was observed when the sulphite oxidase (SO) gene from Arabidopsis thaliana, which catalyses the back reaction of APR, that is the reaction from sulphite to sulphate, was overexpressed. (iii) When Lemna minor APR was overexpressed in poplar, APR activity increased as expected, but no changes in sulphur flux were observed. For all of these experiments the flux control coefficient for APR was calculated. APR as a controlling step in sulphate assimilation seems obvious under OAS treatment, in γ-ECS and SO overexpressing poplars. A possible loss of control under certain conditions, that is Cd treatment, Acetochlor treatment, and in APR overexpressing poplar, is discussed

Impact of SO2 on Arabidopsis thaliana transcriptome in wildtype and sulfite oxidase knockout plants analyzed by RNA deep sequencing

Hamisch D, Randewig D, Schliesky S, et al. Impact of SO2 on Arabidopsis thaliana transcriptome in wildtype and sulfite oxidase knockout plants analyzed by RNA deep sequencing. New Phytologist. 2012;196(4):1074-1085.High concentrations of sulfur dioxide (SO2) as an air pollutant, and its derivative sulfite, cause abiotic stress that can lead to cell death. It is currently unknown to what extent plant fumigation triggers specific transcriptional responses. To address this question, and to test the hypothesis that sulfite oxidase (SO) is acting in SO2 detoxification, we compared Arabidopsis wildtype (WT) and SO knockout lines (SO-KO) facing the impact of 600 nl l (1) SO2, using RNAseq to quantify absolute transcript abundances. These transcriptome data were correlated to sulfur metabolism-related enzyme activities and metabolites obtained from identical samples in a previous study. SO-KO plants exhibited remarkable and broad regulative responses at the mRNA level, especially in transcripts related to sulfur metabolism enzymes, but also in those related to stress response and senescence. Focusing on SO regulation, no alterations were detectable in the WT, whereas in SO-KO plants we found up-regulation of two splice variants of the SO gene, although this gene is not functional in this line. Our data provide evidence for the highly specific coregulation between SO and sulfur-related enzymes like APS reductase, and suggest two novel candidates for involvement in SO2 detoxification: an apoplastic peroxidase, and defensins as putative cysteine mass storages

Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo

Meeting Abstracts: Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo Clearwater Beach, FL, USA. 9-11 June 201

The History of the Molybdenum Cofactor—A Personal View

The transition element molybdenum (Mo) is an essential micronutrient for plants, animals, and microorganisms, where it forms part of the active center of Mo enzymes. To gain biological activity in the cell, Mo has to be complexed by a pterin scaffold to form the molybdenum cofactor (Moco). Mo enzymes and Moco are found in all kingdoms of life, where they perform vital transformations in the metabolism of nitrogen, sulfur, and carbon compounds. In this review, I recall the history of Moco in a personal view, starting with the genetics of Moco in the 1960s and 1970s, followed by Moco biochemistry and the description of its chemical structure in the 1980s. When I review the elucidation of Moco biosynthesis in the 1990s and the early 2000s, I do it mainly for eukaryotes, as I worked with plants, human cells, and filamentous fungi. Finally, I briefly touch upon human Moco deficiency and whether there is life without Moco

Why do we die of a defect in molybdenum metabolism?

Das Spurenelement Molybdän (Mo) ist lebensnotwendig für den Menschen, bei dem es als katalytisch aktives Metall Bestandteil von vier Enzymen ist. Oxidiertes Mo wird als Molybdat aus der Nahrung aufgenommen und an ein chemisches Grundgerüst (Pterin) gebunden, wodurch es zum Molybdän-Cofaktor (Moco) wird. Nur in dieser Form kann Mo biologisch aktiv werden. Die Biosynthese des Moco ist ein Mehrschrittprozess, der in den Mitochondrien beginnt und im Cytoplasma abgeschlossen wird. Ein genetischer Defekt in der Moco-Biosynthese führt zum Ausfall der Aktivitäten aller vier Mo-Enzyme des Menschen, wobei der Ausfall der Sulfitoxidase dramatische Folgen hat. Das hochreaktive Sulfit akkumuliert und schädigt irreversibel Proteine und Metabolite, worauf neuronale Zellen in Neugeborenen am empfindlichsten reagieren. Die neurodegenerativen Symptome (Krämpfe, Störung der Gehirnentwicklung) führen in den meisten Fällen zum Tod der kleinen Patienten. Isolierter Moco ist zu instabil, um ihn den betroffenen Neugeborenen als Ersatz zu spritzen, aber für Patienten mit einer Mutation im ersten Schritt der Moco-Biosynthese gibt es eine Therapie. Ihnen wird das fehlende Biosynthese-Intermediat cPMP injiziert, was den genetischen Ausfall kompensiert und eine normale Kindesentwicklung ermöglicht. Das Medikament wurde 2021 in den USA und 2022 in Europa zugelassen.The trace element molybdenum (Mo) is vital for humans. As catalytically active metal it is part of four enzymes. Oxidized Mo is taken up from our food as molybdate and bound to a chemical scaffold (pterin) thus becoming the molybdenum cofactor (Moco). Only in this form Mo can become biologically active. The biosynthesis of Moco is a multi-step process starting in the mitochondria and completed in the cytoplasm. A genetic defect in Moco biosynthesis leads to the activity loss of all four Mo-enzymes in humans with most dramatic effects through the loss of sulfite oxidase. The highly reactive sulfite accumulates and damages proteins and metabolites irreversibly. In newborns, neuronal cells react most sensitively to sulfite, and in most cases neurodegenerative symptoms (spasms, impairment of brain development) lead to the death of the little patients. Moco in its isolated form is too unstable to be injected into the newborns as therapeutic medication, but there is a therapy for patients with a mutation in the first step of Moco-biosynthesis. Those patients receive the missing biosynthesis intermediate cPMP by injection thus compensating for the genetic loss and permitting a normal child development. The medication was approved in 2021 in the USA and in 2022 in Europe

Molybdenum cofactor biology, evolution and deficiency

The molybdenum cofactor (Moco) represents an ancient metal-sulfur cofactor, which participates as catalyst in carbon, nitrogen and sulfur cycles, both on individual and global scale. Given the diversity of biological processes dependent on Moco and their evolutionary age, Moco is traced back to the last universal common ancestor (LUCA), while Moco biosynthetic genes underwent significant changes through evolution and acquired additional functions. In this review, focused on eukaryotic Moco biology, we elucidate the benefits of gene fusions on Moco biosynthesis and beyond. While originally the gene fusions were driven by biosynthetic advantages such as coordinated expression of functionally related proteins and product/substrate channeling, they also served as origin for the development of novel functions. Today, Moco biosynthetic genes are involved in a multitude of cellular processes and loss of the according gene products result in severe disorders, both related to Moco biosynthesis and secondary enzyme functions