Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.</p

Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin auxotrophic Escherichia coli strain and maltose-binding protein as solubility tag, we produced the apoprotein of Thermus thermophilus ProDH (MBP-TtProDH). Remarkably, reconstitution with FAD or FMN revealed that MBP-TtProDH has no preference for either of the two prosthetic groups. Kinetic parameters of both holo forms are similar, as are the dissociation constants for FAD and FMN release. Furthermore, we show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In line with this flavin content, the crystal structure of TtProDH variant ¿ABC, which lacks helices aA, aB and aC, shows no electron density for an AMP moiety of the cofactor. To the best of our knowledge, this is the first example of a flavoenzyme that does not discriminate between FAD and FMN as cofactor. Therefore, classification of TtProDH as an FAD-binding enzyme should be reconsidered

Editorial: Actinobacteria, a Source of Biocatalytic Tools

Actinobacteria (Actinomycetes) represent one of the largest and most diverse phyla among the Bacteria. The characteristics and phylogeny of actinobacteria have been well-described throughout the years (Anteneh and Franco; Embley et al., 1994; Stackebrandt et al., 1997a,b; Stach and Bull, 2005; Stackebrandt and Schumann, 2006; Ventura et al., 2007; Gao and Gupta, 2012; Goodfellow, 2012a,b; Schrempf, 2013; Lawson, 2018; Lewin et al., 2016). Still actinobacteria are hotspots for discovery of new biomolecules and enzyme activities, fueling an active field of research. The remarkable diversity is displayed by various lifestyles, distinct morphologies, a wide spectrum of physiological and metabolic activities, as well as genetics

Mixed-Integer MPC Strategies for Fueling and Density Control in Fusion Tokamaks

Model predictive control (MPC) is promising for fueling and core density feedback control in nuclear fusion tokamaks, where the primary actuators, frozen hydrogen fuel pellets fired into the plasma, are discrete. Previous density feedback control approaches have only approximated pellet injection as a continuous input due to the complexity that it introduces. In this letter, we model plasma density and pellet injection as a hybrid system and propose two MPC strategies for density control: mixed-integer (MI) MPC using a conventional mixed-integer programming (MIP) solver and MPC utilizing our novel modification of the penalty term homotopy (PTH) algorithm. By relaxing the integer requirements, the PTH algorithm transforms the MIP problem into a series of continuous optimization problems, reducing computational complexity. Our novel modification to the PTH algorithm ensures that it can handle path constraints, making it viable for constrained hybrid MPC in general. Both strategies perform well with regards to reference tracking without violating path constraints and satisfy the computation time limit for real-time control of the pellet injection system. However, the computation time of the PTH-based MPC strategy consistently outpaces the conventional MI-MPC strategy

A massive urban symbiosis:A preliminary review of the Urban Mining Pilot Bases Programme in China

Waste recycling helps to establish a circular loop of resource flow between production and consumption, achieving a certain symbiosis between the industrial and urban sector. Since more and more resources are accumulated in the urban sector, urban mining as form of waste recycling in a massive way becomes an outstanding way to achieve industrial and urban symbiosis. In 2010 China initiated a national urban mining pilot base (UMPB) programme with the objective of developing the recycling industry and relieving environmental and resource constrains. This study aims to provide policy review of the programme. We find that the UMPB programme was developed from past circular economy policies and attains legacy assurance from current laws and national plans. But this did not formulate a perfect governance context for its implementation. A multi-ministerial cross-management network led to policy conflicts, and recycling-oriented legislation remained absent. These became the main barriers for the good implementation of those urban mining pilots. Comparing with the eco-town programme in Japan, it shows that both programmes share some similarities of partial policy objectives but also show variety in the scope of urban symbiosis due to the different problems they focus on and the slightly different policy objectives under the different economic and social development phases

Targeting a Versatile Actuator for EU-DEMO: Real Time Monitoring of Pellet Delivery to Facilitate Burn Control

Core particle fueling, an essential task in the European demonstration fusion power plant EU-DEMO, relies on adequate pellet injection. However, pellets are fragile objects, and their delivery efficiency can hardly be assumed to be unity. Exploring kinetic control of the EU-DEMO1 scenario indicates that such missed-out pellets do cause a considerable problem for keeping a burning plasma. Missed-out pellets can cause a severe drop of plasma density that in turn results in a potential drastic loss of burn power. Efforts are under way at the ASDEX Upgrade (AUG) tokamak aiming to provide real-time monitoring of pellet arrival and announcement of missed-out cases to the control systems. To further optimize the controllers, system identification experiments have been performed to identify the dynamic response of the system to the actuator

The secretome of Agaricus bisporus: Temporal dynamics of plant polysaccharides and lignin degradation

Despite substantial lignocellulose conversion during mycelial growth, previous transcriptome and proteome studies have not yet revealed how secretomes from the edible mushroom Agaricus bisporus develop and whether they modify lignin models in vitro. To clarify these aspects, A. bisporus secretomes collected throughout a 15-day industrial substrate production and from axenic lab-cultures were subjected to proteomics, and tested on polysaccharides and lignin models. Secretomes (day 6-15) comprised A. bisporus endo-acting and substituent-removing glycoside hydrolases, whereas β-xylosidase and glucosidase activities gradually decreased. Laccases appeared from day 6 onwards. From day 10 onwards, many oxidoreductases were found, with numerous multicopper oxidases (MCO), aryl alcohol oxidases (AAO), glyoxal oxidases (GLOX), a manganese peroxidase (MnP), and unspecific peroxygenases (UPO). Secretomes modified dimeric lignin models, thereby catalyzing syringylglycerol-β-guaiacyl ether (SBG) cleavage, guaiacylglycerol-β-guaiacyl ether (GBG) polymerization, and non-phenolic veratrylglycerol-β-guaiacyl ether (VBG) oxidation. We explored A. bisporus secretomes and insights obtained can help to better understand biomass valorization