A novel, simple, and sensitive colorimetric method to determine aromatic amino acid aminotransferase activity using the Salkowski reagent

This study describes the development of a new colorimetric assay to determine aromatic amino acid aminotransferase (ArAT) activity. The assay is based on the transamination of l-tryptophan in the presence of 2-oxoglutarate, which yields indole-3-pyruvate (IPyA). The amount of IPyA formed was quantified by reaction with the Salkowski reagent. Optimized assay conditions are presented for ArAT isozymes isolated from Pseudomonas putida. For comparative purposes, ArAT activity was also determined by high-performance liquid chromatography. ArAT activity staining in polyacrylamide gels with the Salkowski reagent is also presented

1+1 = 3: A Fusion of 2 Enzymes in the Methionine Salvage Pathway of Tetrahymena thermophila Creates a Trifunctional Enzyme That Catalyzes 3 Steps in the Pathway

The methionine salvage pathway is responsible for regenerating methionine from its derivative, methylthioadenosine. The complete set of enzymes of the methionine pathway has been previously described in bacteria. Despite its importance, the pathway has only been fully described in one eukaryotic organism, yeast. Here we use a computational approach to identify the enzymes of the methionine salvage pathway in another eukaryote, Tetrahymena thermophila. In this organism, the pathway has two fused genes, MTNAK and MTNBD. Each of these fusions involves two different genes whose products catalyze two different single steps of the pathway in other organisms. One of the fusion proteins, mtnBD, is formed by enzymes that catalyze non-consecutive steps in the pathway, mtnB and mtnD. Interestingly the gene that codes for the intervening enzyme in the pathway, mtnC, is missing from the genome of Tetrahymena. We used complementation tests in yeast to show that the fusion of mtnB and mtnD from Tetrahymena is able to do in one step what yeast does in three, since it can rescue yeast knockouts of mtnB, mtnC, or mtnD. Fusion genes have proved to be very useful in aiding phylogenetic reconstructions and in the functional characterization of genes. Our results highlight another characteristic of fusion proteins, namely that these proteins can serve as biochemical shortcuts, allowing organisms to completely bypass steps in biochemical pathways

Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp PCC 6803

The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter P(trc) resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, P(A1lacO-1), induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density

Investigating the Connection Between Scale and Intermolecular Forces in the General Chemistry Laboratory

The American Association for the Advancement of Science (AAAS) has outlined four themes that define science literacy; these are systems, models, constancy and change, and scale. More recently, the National Research Council has released the framework for K-12 science education that includes “Scale, Proportion, and Quantity”. Our research has already shown that scale literacy is a better predictor for success in a general chemistry course than traditional measures and integrating scale as a theme in the undergraduate general chemistry curriculum has been accomplished through a variety of methods. Of particular interest was developing a laboratory sequence that would help students increase their knowledge of chemistry concepts through explicit inclusion of scale concepts.One particular activity was developed based on the difficulty students have in understanding the important role of intermolecular forces in chemistry. Given that intermolecular forces exist on the particle level (far below the threshold of sight) it is not surprising that students have difficulty when asked to describe how these forces can be used to explain the observed macroscopic properties of substances. This activity gave students the opportunity to think about and explore the molecular level on which these forces exist through model building and experimentation. The preliminary data collected from this activity will show how incorporating scale concepts into this activity helped students to not only better understand intermolecular forces but also how to use this understanding to develop explanations for observed trends in the properties of chemical substances

A complete inventory of all enzymes in the eukaryotic methionine salvage pathway

The methionine salvage pathway is universally used to regenerate methionine from 5’-methylthioadenosine (MTA), a by-product of some reactions involving S-adenosylmethionine. We have identified and verified genes encoding the enzymes of all steps in this cycle in a commonly used eukaryotic model system, the yeast Saccharomyces cerevisiae. The genes encoding 5’-methylthioribose-1-phosphate isomerase and 5’-methylthioribulose-1-phosphate dehydratase were hereby named MRI1 and MDE1, respectively. The MTA phosphorylase was verified as Meu1p, the 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase as, Utr4p and the aci-reductone dioxygenase as Adi1p. The homolog of the enolase/phosphatase gene, YNL010w, could be excluded from its candidate role in the cycle. The methodology used was auxotrophic growth tests and analysis of intracellular MTA in deletion mutants. The last step, a transamination of 4-methylthio-2-oxobutyrate (MOB) to yield methionine, was found to be a highly redundant step. It was catalysed by amino acid transaminases mainly coupled with aromatic and branched chain amino acids as amino donors but also with proline, lysine and glutamate/glutamine. The aromatic amino acid transaminases, Aro8p and Aro9p and the branched chain amino acid transaminases, Bat1p and Bat2p, seemed to be the main enzymes exhibiting the MOB transaminase activity. Bat2p was found to be less specific and used proline, lysine, tyrosine, and glutamate as amino donors beside the branched chain amino acids. Thus, for the first time, all enzymes of the methionine salvage pathway were identified in a eukaryote

A complete inventory of all enzymes in the eukaryotic methionine salvage pathway

The methionine salvage pathway is universally used to regenerate methionine from 5’-methylthioadenosine (MTA), a by-product of some reactions involving S-adenosylmethionine. We have identified and verified genes encoding the enzymes of all steps in this cycle in a commonly used eukaryotic model system, the yeast Saccharomyces cerevisiae. The genes encoding 5’-methylthioribose-1-phosphate isomerase and 5’-methylthioribulose-1-phosphate dehydratase were hereby named MRI1 and MDE1, respectively. The MTA phosphorylase was verified as Meu1p, the 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase as, Utr4p and the aci-reductone dioxygenase as Adi1p. The homolog of the enolase/phosphatase gene, YNL010w, could be excluded from its candidate role in the cycle. The methodology used was auxotrophic growth tests and analysis of intracellular MTA in deletion mutants. The last step, a transamination of 4-methylthio-2-oxobutyrate (MOB) to yield methionine, was found to be a highly redundant step. It was catalysed by amino acid transaminases mainly coupled with aromatic and branched chain amino acids as amino donors but also with proline, lysine and glutamate/glutamine. The aromatic amino acid transaminases, Aro8p and Aro9p and the branched chain amino acid transaminases, Bat1p and Bat2p, seemed to be the main enzymes exhibiting the MOB transaminase activity. Bat2p was found to be less specific and used proline, lysine, tyrosine, and glutamate as amino donors beside the branched chain amino acids. Thus, for the first time, all enzymes of the methionine salvage pathway were identified in a eukaryote