Characterization of the Arsenate Respiratory Reductase from Shewanella sp. Strain ANA-3

Microbial arsenate respiration contributes to the mobilization of arsenic from the solid to the soluble phase in various locales worldwide. To begin to predict the extent to which As(V) respiration impacts arsenic geochemical cycling, we characterized the expression and activity of the Shewanella sp. strain ANA-3 arsenate respiratory reductase (ARR), the key enzyme involved in this metabolism. ARR is expressed at the beginning of the exponential phase and persists throughout the stationary phase, at which point it is released from the cell. In intact cells, the enzyme localizes to the periplasm. To purify ARR, a heterologous expression system was developed in Escherichia coli. ARR requires anaerobic conditions and molybdenum for activity. ARR is a heterodimer of ~131 kDa, composed of one ArrA subunit (~95 kDa) and one ArrB subunit (~27 kDa). For ARR to be functional, the two subunits must be expressed together. Elemental analysis of pure protein indicates that one Mo atom, four S atoms associated with a bis-molybdopterin guanine dinucleotide cofactor, and four to five [4Fe-4S] are present per ARR. ARR has an apparent melting temperature of 41°C, a Km of 5 µM, and a Vmax of 11,111 µmol of As(V) reduced min–1 mg of protein–1 and shows no activity in the presence of alternative electron acceptors such as antimonite, nitrate, selenate, and sulfate. The development of a heterologous overexpression system for ARR will facilitate future structural and/or functional studies of this protein family

On the pulse of change: the new beat of special education in higher education

The roles and responsibilities of special educators in both special and regular school settings are changing rapidly. More than two decades ago the move towards more inclusive practices disrupted the traditional, niche separatism of special educators to the extent that they are now an integral part of the regular school teaching staff. Today the broad agenda to facilitate access and participation for all students in education, not just students with disabilities, influences the roles and responsibilities of special educators. This article briefly identifies some of the generic pulses that are moving the special education profession from a focus on low incidence disabilities towards a more comprehensive approach to inclusion, school responsiveness and individualised learning pathways. From the foundation of inclusive practice, this paper will describe how a qualitative study was used to understand the changing roles and responsibilities of special educators. A case study analysis of 17 schools formed the basis of the investigation. Principals, lead teachers in special education and special education teachers were asked to identify trends and priorities in special education and also to identify rewards and challenges in their jobs. Further cross referencing with teacher and special educator standards, a focus group, a stakeholder group and research in the field increased the opportunity for academics and special educators to critically reflect on the emerging demands placed on special educators and the attributes that are needed to be professionally effective

Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity

Computational protein design methods were used to predict five variants of monofunctional Escherichia coli chorismate mutase expected to maintain catalytic activity. The variants were tested experimentally and three active site mutants exhibited catalytic activity similar to or greater than the wild-type enzyme. One mutant, Ala32Ser, showed increased catalytic efficiency

Mapping Responsive Envelopes: Material culture evolution and climatically responsive building facades

This research paper proposes the transfer of biological evolutionary methods to the field of climatically responsive building facades. Through charting their trajectories and situating them across the physical and biological sciences, the paper will explore knowledge transfer, historical flows and the fate of information from a sustainable perspective. Despite similarities to biological evolutionary processes, the application of biological methods does not directly correlate to responsive building facades. Through a database of precedents and projects, a hybridized method of mapping will be developed as a means towards understanding the genus of ideas, mutation, and selection within this process so as to establish to what degree it is similar to that of biological systems as well as Gould and Elderedge’s (1972) theory of ‘punctuated equilibrium’. ‘Punctuated equilibrium’ states that evolution within biological systems occurs in short, sharp bursts after long periods of inactivity, contradicting orthodoxy of evolution as a slow, gradual process. This theory proves correct for climatically responsive facades due to the convergence of technology with cultural awareness for environmentally conscious architecture. Temkin and Eldredge (2007), in their application of phylogenetic analysis on musical instruments, identified lateral exchange of information as the most significant factor in material culture systems.Through developing a hybridized map of climatically responsive facades, this research will indicate characteristics, ideas, and historical flows of information as well as their subsequent fate. Within material culture there is considerable conservatism, pointing to selection by designers and manufacturers as an inhibitor of evolution. As a result, this process identifies new architectural connections and areas of focus where distinct innovation may occur

Exhaustive mutagenesis of six secondary active-site residues in Escherichia coli chorismate mutase shows the importance of hydrophobic side chains and a helix N-capping position for stability and catalysis

Secondary active-site residues in enzymes, including hydrophobic amino acids, may contribute to catalysis through critical interactions that position the reacting molecule, organize hydrogen-bonding residues, and define the electrostatic environment of the active site. To ascertain the tolerance of an important model enzyme to mutation of active-site residues that do not directly hydrogen bond with the reacting molecule, all 19 possible amino acid substitutions were investigated in six positions of the engineered chorismate mutase domain of the Escherichia coli chorismate mutase-prephenate dehydratase. The six secondary active-site residues were selected to clarify results of a previous test of computational enzyme design procedures. Five of the positions encode hydrophobic side chains in the wild-type enzyme, and one forms a helix N-capping interaction as well as a salt bridge with a catalytically essential residue. Each mutant was evaluated for its ability to complement an auxotrophic chorismate mutase deletion strain. Kinetic parameters and thermal stabilities were measured for variants with in vivo activity. Altogether, we find that the enzyme tolerated 34% of the 114 possible substitutions, with a few mutations leading to increases in the catalytic efficiency of the enzyme. The results show the importance of secondary amino acid residues in determining enzymatic activity, and they point to strengths and weaknesses in current computational enzyme design procedures