Laura Bowater

Bacillus subtilis YxaG is a novel Fe-containing quercetin 2,3-dioxygenase

The Bacillus subtilis genome contains genes for three hypothetical proteins belonging to the bicupin family, two of which we have previously shown to be Mn(II)‐dependent oxalate decarboxylases. We have now shown that the third, YxaG, exhibits quercetin 2,3‐dioxygenase activity and that it contains Fe ions. This contrasts with the eukaryotic enzyme which contains a Cu ion. YxaG is the first prokaryotic carbon monoxide‐forming enzyme that utilises a flavonol to be characterised and is only the second example of a prokaryotic dioxygenolytic carbon monoxide‐forming enzyme known to contain a cofactor. It is proposed to rename the B. subtilis gene qdoI.

The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations.

Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Delta162-163 or Delta162-164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Delta162-163 and S161A) strongly support the hypothesis that site 2 is purely structural.

pH-Dependent Structures of the Manganese Binding Sites in Oxalate Decarboxylase as Revealed by High-Field Electron Paramagnetic Resonance

A high-field electron paramagnetic resonance (HFEPR) study of oxalate decarboxylase (OxdC) is reported. OxdC breaks down oxalate to carbon dioxide and formate and possesses two distinct manganese(II) binding sites, referred to as site-1 and -2. The Mn(II) zero-field interaction was used to probe the electronic state of the metal ion and to examine chemical/mechanistic roles of each of the Mn(II) centers. High magnetic-fields were exploited not only to resolve the two sites, but also to measure accurately the Mn(II) zero-field parameters of each of the sites. The spectra exhibited surprisingly complex behavior as a function of pH. Six different species were identified based on their zero-field interactions, two corresponding to site-1 and four states to site-2. The assignments were verified using a mutant that only affected site-1. The speciation data determined from the HFEPR spectra for site -2 was consistent with a simple triprotic equilibrium model, while the pH dependence of site-1 could be described by a single pK(a). This pH dependence was independent of the presence of the His-tag and of whether the preparations contained 1.2 or 1.6 Mn per subunit. Possible structures of the six species are proposed based on spectroscopic data from model complexes and existing protein crystallographic structures obtained at pH 8 are discussed. Although site-1 has been identified as the active site and no role has been assigned to site-2, the pronounced changes in the electronic structure of the latter and its pH behavior, which also matches the pH-dependent activity of this enzyme, suggests that even if the conversion of oxalate to formate is carried out at site-1, site-2 likely plays a catalytically relevant role.

Detection of Transglucosidase-Catalyzed Polysaccharide Synthesis on a Surface in Real Time Using Surface Plasmon Resonance Spectroscopy

Biological systems that involve enzyme catalysis at surfaces, particularly strategically important ones that involve insoluble substrates/products such as the cell wall and the starch granule, require analyses beyond classical solution state enzymology. Using a model system, we have demonstrated the real-time measurement of transglucosidase activity on a surface using surface plasmon resonance (SPR) spectroscopy. We monitored the extension of a (partially carboxymethylated) dextran surface with alternansucrase and sucrose as a glycosyl donor. Conditions were used where surface polymer synthesis rates were a function of enzyme concentration and proportional to the extent of enzyme binding to the surface. A method to determine the turnover number of the enzyme on the surface was also developed. The presence of a new amorphous polysaccharide was observed optically, detected by lectin binding and imaged by atomic force microscopy. This surface method will have utility in a wide range of carbohydrate enzyme systems including screens.

Promoting microbiology education through the iGEM synthetic biology competition

Synthetic biology has developed rapidly in the 21st century. It covers a range of scientific disciplines that incorporate principles from engineering to take advantage of and improve biological systems, often applied to specific problems. Methods important in this subject area include the systematic design and testing of biological systems and, here, we describe how synthetic biology projects frequently develop microbiology skills and education. Synthetic biology research has huge potential in biotechnology and medicine, which brings important ethical and moral issues to address, offering learning opportunities about the wider impact of microbiological research. Synthetic biology projects have developed into wide-ranging training and educational experiences through iGEM, the International Genetically Engineered Machines competition. Elements of the competition are judged against specific criteria and teams can win medals and prizes across several categories. Collaboration is an important element of iGEM, and all DNA constructs synthesized by iGEM teams are made available to all researchers through the Registry for Standard Biological Parts. An overview of microbiological developments in the iGEM competition is provided. This review is targeted at educators that focus on microbiology and synthetic biology, but will also be of value to undergraduate and postgraduate students with an interest in this exciting subject area.

Antimicrobial stewardship: the role of scientists?

We continue to be warned about the risk of antibiotic resistance. This campaign has targeted medicine and agriculture, asking these industries to pay attention to the risks of widespread resistance and to cut the use of antibiotics wherever possible. However, there has been little to no mention of the widespread use of antibiotics in the scientific research community. As scientists we use antibiotics and antibiotic resistance as fundamental tools for our research; almost all conventional plasmids use an antibiotic resistance gene as a selectable marker, offering us an easy method of screening. With molecular biology and genetics at the heart of many research disciplines, these tools are ubiquitous. Scientists have a responsibility to monitor and reduce our use of antibiotics. With the growth and fast advancement of synthetic biology, it is timely for us to consider other options and to teach the next generation of researchers by example how to truly value antibiotics by using them more responsibly.

Inspiring STEM undergraduates to tackle the AMR crisis

To address the growing problem of antimicrobial resistance (AMR), it is necessary to invest in, inspire and attract future generations of scientists to this research area. Undergraduate education should be a focus for attention and efforts should be made to ensure that students are afforded opportunities to actively engage with AMR. We illustrate how as a topic AMR provides opportunities to deliver effective research-led teaching in addition to traditional teaching methods. We have used a selection of case studies to illustrate how students can be engaged with AMR using a variety of research-led approaches to develop the required skills for biology-centric students. In addition, we indicate how these skills map to the UK Quality Assurance Framework and the Vision and Change report developed by the American Association for the Advancement of Science.

Twelve tips to teaching (legal and ethical aspects of) research ethics/responsible conduct of research.

Teaching research ethics is a requirement within modern health science, nursing and medical curricula. We have drawn on our experience of designing, developing and integrating the teaching of research ethics in a new, fully integrated medical school curriculum, delivered using Problem Based Learning and the recent literature relating to the teaching of research ethics to produce the following 12 Top Tips designed to encourage readers to seek opportunities to embed this teaching within a variety of curricula.

SAD at home: solving the structure of oxalate decarboxylase with the anomalous signal from manganese using X-ray data collected on a home source

Oxalate decarboxylase (OxdC) from Bacillus subtilis is a hexamer containing two manganese ions per 43.6 kDa subunit. A single highly redundant data set collected at a medium resolution of 2 A on an in-house X-ray source was sufficient to solve the structure by the single-wavelength anomalous diffraction (SAD) method using the anomalous signal from the manganese ions. The experimentally phased electron-density map was of high quality, enabling 96% of the amino-acid sequence to be automatically traced using ARP/wARP. Further analysis showed that only half of the original raw data were required for successful structure solution. Manganese currently occurs in approximately 2% of PDB entries. A brief survey suggests that several of these structures could also have been determined using manganese SAD. Moreover, the ability of manganese to substitute for other more commonly occurring divalent metal ions may indicate that the use of Mn SAD could have much wider application.

Development and Evaluation of an Undergraduate Science Communication Module

Abstract This paper describes the design and evaluation of an undergraduate final year science communication module for the Science Faculty at the University of East Anglia. The module focuses specifically on science communication and aims to bring an understanding of how science is disseminated to the public. Students on the module are made aware of the models surrounding science communication and investigate how the science culture interfaces with the public. During the module they learn how to adapt science concepts for different audiences and how to talk confidently about science to a lay-audience. Student motivation for module choice centres on the acquisition of transferable skills and students develop these skills through designing, running and evaluating a public outreach event at a school or in a public area. These transferable skills acquired include communication, interaction with different organisations such as museums and science centres, developing understanding of both the needs of different audiences and the importance of time management. They also develop skills relating to self-reflection and how to use this as a tool for future self development. The majority of students completing the module go on to further study, either a PhD, MSc or teacher training. The module can be sustained in its present formed if capped at 40 students, however it is recognised that to increase cohort size, further investment of faculty time and resources would be required.