Evolutionary Cycles for Pericyclic Reactions – Or Why We Keep Mutating Mutases

Directed evolution strategies are being applied ever more frequently to develop novel and improved enzymes for many applications, including those contributing to 'white biotechnology'. In addition to engineering new biocatalysts, evolutionary strategies are equally suited to the elucidation of enzyme structure and function. Here, we illustrate with selected examples from our own work on chorismate mutases how such strategies can be employed to address a range of fundamental questions. Over the last decade, this model system, which was once considered to be a 'very simple' enzyme from the shikimate pathway, has afforded many – sometimes surprising – discoveries about biocatalysis. It has also taught us how to upgrade evolutionary approaches to overcome technical hurdles. Both the new insights and the methodological improvements should enhance our ability to tailor enzymes for novel uses

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

Efficient introduction of aryl bromide functionality into proteins in vivo

Artificial proteins can be engineered to exhibit interesting solid state, liquid crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials. The utility of protein-based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biological systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means. In this study, we incorporate para-bromophenylalanine (p-Br-phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl-tRNA synthetase (PheRS) variant with relaxed substrate specificity. Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p-Br-phe-supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p-Br-phe; variation in the relative amounts of phe and p-Br-phe in the medium allows control of the degree of substitution by the analog. Protein expression yields of 20–25 mg/l were obtained from cultures supplemented with p-Br-phe; this corresponds to about two-thirds of the expression levels characteristic of cultures supplemented with phe. The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post-translational derivatization of brominated proteins via metal-catalyzed coupling reactions. In addition, bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique

Consensus Protein Design without Phylogenetic Bias

Consensus design is an appealing strategy for the stabilization of proteins. It exploits amino acid conservation in sets of homologous proteins to identify likely beneficial mutations. Nevertheless, its success depends on the phylogenetic diversity of the sequence set available. Here, we show that randomization of a single protein represents a reliable alternative source of sequence diversity that is essentially free of phylogenetic bias. A small number of functional protein sequences selected from binary-patterned libraries suffice as input for the consensus design of active enzymes that are easier to produce and substantially more stable than individual members of the starting data set. Although catalytic activity correlates less consistently with sequence conservation in these extensively randomized proteins, less extreme mutagenesis strategies might be adopted in practice to augment stability while maintaining function

Iowa Farm and Rural Life Poll: 2010 Summary Report

The Iowa Farm and Rural Life Poll is an annual survey that collects and disseminates information on issues of importance to rural communities across Iowa and the Midwest. Conducted every year since its establishment in 1982, the Farm Poll is the longest-running survey of its kind in the nation. Iowa State University Extension, the Iowa Agriculture and Home Economics Experiment Station, the Iowa Department of Agriculture and Land Stewardship, and the Iowa Agricultural Statistics Service are all partners in the Farm Poll effort. The information gathered through the Farm Poll is used to inform the development and improvement of research and extension programs and is used by local, state, and national leaders in their decision-making processes. We thank the many farm families who responded to this year’s survey and appreciate their continued participation in the Farm Poll.https://lib.dr.iastate.edu/extension_communities_pubs/1020/thumbnail.jp