Impact of ancestral sequence reconstruction on mechanistic and structural enzymology

Ancestral sequence reconstruction (ASR) provides insight into the changes within a protein sequence across evolution. More specifically, it can illustrate how specific amino acid changes give rise to different phenotypes within a protein family. Over the last few decades it has established itself as a powerful technique for revealing molecular common denominators that govern enzyme function. Here, we describe the strength of ASR in unveiling catalytic mechanisms and emerging phenotypes for a range of different proteins, also highlighting biotechnological applications the methodology can provide.</p

Identification and characterization of archaeal and bacterial F420-dependent thioredoxin reductases

The thioredoxin pathway is an antioxidant system present in most organisms. Electrons flow from a thioredoxin reductase to thioredoxin at the expense of a specific electron donor. Most known thioredoxin reductases rely on NADPH as reducing cofactor. Yet, in 2016 a new type of thioredoxin reductase was discovered in archaea which utilizes instead a reduced deazaflavin cofactor (F 420 H 2 ). For this reason, the respective enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). To have a broader understanding of the biochemistry of DFTRs, we identified and characterized two other archaeal representatives. A detailed kinetic study, which included pre-steady state kinetic analyses, revealed these two DFTRs are highly specific for F 420 H 2 while displaying marginal activity with NADPH. Nevertheless, they share mechanistic features with the canonical thioredoxin reductases that dependent on NADPH (NTRs). A detailed structural analysis led the identification of two key residues that tune cofactor specificity of DFTRs. This allowed us to propose a DFTR-specific sequence motif that enabled for the first time the identification and experimental characterization of a bacterial DFTR. </p

Vanillyl alcohol oxidase from Diplodia corticola:Residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives

Vanillyl alcohol oxidases belong to the 4-phenol oxidases family and are found predominantly in lignin-degrading ascomycetes. Systematical investigation of the enzyme family at the sequence level resulted in discovery and characterization of the second recombinantly produced VAO member, DcVAO, from Diplodia corticola. Remarkably high activities for 2,6-substituted substrates like 4-allyl-2,6-dimethoxy-phenol (3.5 ± 0.02 U mg -1) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3 ± 0.5 U mg -1) were observed which could be attributed to a Phe to Ala exchange in the catalytic center. In order to rationalize this rare substrate preference among VAOs, we resurrected and characterized three ancestral enzymes and performed mutagenesis analyses. The results indicate that a Cys/Glu exchange was required to retain activity for ɣ-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0 ± 0.1 U mg -1). Our findings contribute to the understanding of the functionality of VAO enzyme group, and with DcVAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts. </p

Mutational and structural analysis of an ancestral fungal dye decolorizing peroxidase

Dye-decolorizing peroxidases (DyPs) constitute a superfamily of heme-containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A-C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D-type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D-type DyP ancestor, AncDyPD-b1. Expression of AncDyPD-b1 in Escherichia coli results in large amounts of a heme-containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV-Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady-state kinetics and the crystal structure, reveal fine pH-dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long-range electron transfer process. Database: Structural data are available in the PDB database under the accession number 7ANV.</p

A typology for unpacking the diversity of social innovation in energy transitions

Academic research and policy have focused on sustainable energy transitions for addressing the societal challenge of climate change for a long time, but the concept of ‘social innovation’ has only been recently taken up. This concept refers to different social phenomena relating to changes in socio-technical systems. Moving beyond narrow perspectives on social innovation, this article asks how we can capture the diversity of social innovation, taking the example of the energy sector. It proposes a comprehensive typology of social innovation that allows to capture the phenomenon in its empirical diversity, and to more systematically investigate processes of social innovation and their contributions to making socio-technical systems more sustainable. The typology is based on a conceptual understanding of social innovation in energy (SIE) as comprising changing social relations involving new ways of doing, thinking and/or organising energy. It is empirically grounded in mapping and analysing 500 SIE-initiatives across eight European countries. Such a conceptually-informed and empirically-grounded understanding of SIE sheds light on the ‘social’ as an object of innovation, the agency of a multitude of actors, and the different types of social relations and activities pursued by energy system actors. It also opens the possibility to publicly discuss the diverse social innovations and their interdependencies, (un)desirability, as well as transformative potentials in energy transitions

Power to, over and with: Exploring power dynamics in social innovations in energy transitions across Europe

This paper explores how power relations are manifested, altered and/or reproduced in processes of social innovations in energy transitions (SIE). We explore this research question by developing an interdisciplinary and transdisciplinary power heuristic building on different dimensions of power: power to, power over and power with. This conceptual framework helps us analyse the power dynamics in multiple types of SIEs that aim to contribute to sustainable energy transitions across three different national contexts: Germany, Poland, and the United Kingdom (UK). Our findings show how social innovation involves different dimensions of power to/over/with, and how power relations are both altered and reproduced. The cases under study also lead us to argue that understanding how power dynamics develop requires the analysis of the interplay between different power dimensions across the multiplicity of actors within different SIE-fields and their initiatives

Power to, over and with:Exploring power dynamics in social innovations in energy transitions across Europe

This paper explores how power relations are manifested, altered and/or reproduced in processes of social innovations in energy transitions (SIE). We explore this research question by developing an interdisciplinary and transdisciplinary power heuristic building on different dimensions of power: power to, power over and power with. This conceptual framework helps us analyse the power dynamics in multiple types of SIEs that aim to contribute to sustainable energy transitions across three different national contexts: Germany, Poland, and the United Kingdom (UK). Our findings show how social innovation involves different dimensions of power to/over/with, and how power relations are both altered and reproduced. The cases under study also lead us to argue that understanding how power dynamics develop requires the analysis of the interplay between different power dimensions across the multiplicity of actors within different SIE-fields and their initiatives.</p

Who finances renewable energy in Europe? Examining temporality, authority and contestation in solar and wind subsidies in Poland, the Netherlands and the United Kingdom

In this paper, we explore the development of financing and subsidies for renewable energy in three fossil-fuelled European countries: Poland, the Netherlands, and the United Kingdom. Financing for renewable energy is an existing arena involving multi-actor activities and practices that develop and implement (innovative) financial instruments to facilitate investments in renewable energy. This means that the paper focuses on different financial mechanisms – such as grants, awards, subsidies, crowdfunding, community bonds, ventures, social investment, as long as these funding instruments finance sustainable energy infrastructure and activities. The extent to which this is changing social relations and comes with new ways of doing, thinking and/or organizing is an empirical topic explicitly examined in the study. We first briefly define and conceptualize financial mechanisms and subsidies before explicating our mixed methods research design consisting of scoping, document analysis, 22 original expert interviews, and observational data from eight meetings and events. We then compare the recent history of solar and wind energy financing and subsidies in our three countries. These comparative cases reveal the temporality of subsidization, indicating fundamental changes in the patterns and logics of financing over the past two decades. They reveal shifts in authority and an expansion of actors involved in financing. They lastly reveal tensions and contestations in financing, including gaps in coverage and conflicts among stakeholder groups. We conclude with future insights for renewable energy diffusion, innovation, and policy

Evaluation of a paper by Guarnaccia et al. (2017) on the first report of Phyllosticta citricarpa in Europe

27The Plant Health Panel reviewed the paper by Guarnaccia et al. (2017) and compared theirfindingswith previous predictions on the establishment ofPhyllosticta citricarpa. Four species ofPhyllostictawere found by Guarnaccia et al. (2017) in Europe.P. citricarpaandP. capitalensisare well-definedspecies, withP. citricarparecorded for thefirst time in Europe, confirming predictions by Magareyet al. (2015) and EFSA (2008, 2014, 2016) thatP. citricarpacan establish in some European citrus-growing regions. Two new speciesP. paracitricarpaandP. paracapitalensiswere also described, withP. paracitricarpa(found only in Greece) shown to be pathogenic on sweet orange fruits.Genotyping oftheP. citricarpaisolates suggests at least two independent introductions, with the population inPortugal being different from that present in Malta and Italy.P. citricarpaandP. paracitricarpawereisolated only from leaf litter in backyards. However, sinceP. citricarpadoes not infect or colonise deadleaves, the pathogen must have infected the above living leaves in citrus trees nearby. Guarnacciaet al. (2017) considered introduction to be a consequence ofP. citricarpahaving long been present orof illegal movement of planting material. In the Panel’s view, the fruit pathway would be an equally ormore likely origin. The authors did not report how surveys for citrus black spot (CBS) disease werecarried out, therefore their claim that there was no CBS disease even where the pathogen was presentis not supported by the results presented. From previous simulations, the locations where Guarnacciaet al. (2017) foundP. citricarpaorP. paracitricarpawere conducive forP. citricarpaestablishment, withnumber of simulated infection events by pycnidiospores comparable to sites of CBS occurrence outsideEurope. Preliminary surveys by National Plant Protection Organisations (NPPOs) have not confirmed sofar thefindings by Guarnaccia et al. (2017) but monitoring is still ongoingopenopenJeger, Michael; Bragard, Claude; Caffier, David; Candresse, Thierry; Chatzivassiliou, Elisavet; Dehnen‐Schmutz, Katharina; Gilioli, Gianni; Grégoire, Jean‐Claude; Jaques Miret, Josep Anton; MacLeod, Alan; Navajas Navarro, Maria; Niere, Björn; Parnell, Stephen; Potting, Roel; Rafoss, Trond; Rossi, Vittorio; Urek, Gregor; Van Bruggen, Ariena; Van Der Werf, Wopke; West, Jonathan; Winter, Stephan; Baker, Richard; Fraaije, Bart; Vicent, Antonio; Behring, Carsten; Mosbach Schulz, Olaf; Stancanelli, GiuseppeJeger, Michael; Bragard, Claude; Caffier, David; Candresse, Thierry; Chatzivassiliou, Elisavet; Dehnen‐schmutz, Katharina; Gilioli, Gianni; Grégoire, Jean‐claude; Jaques Miret, Josep Anton; Macleod, Alan; Navajas Navarro, Maria; Niere, Björn; Parnell, Stephen; Potting, Roel; Rafoss, Trond; Rossi, Vittorio; Urek, Gregor; Van Bruggen, Ariena; Van Der Werf, Wopke; West, Jonathan; Winter, Stephan; Baker, Richard; Fraaije, Bart; Vicent, Antonio; Behring, Carsten; Mosbach Schulz, Olaf; Stancanelli, Giusepp