Functional analysis of thermostable proteins involved in carbohydrate metabolism

Thermostable proteins can resist temperature stress whilst keeping their integrity and functionality. In many cases, thermostable proteins originate from hyperthermophilic microorganisms that thrive in extreme environments. These systems are generally located close to geothermal (volcanic) activity, such as in smoking vents on the bottom of the ocean or hot springs at the earth’s surface. Thermostable proteins, especially enzymes, have found extensive use in various biotechnological processes as their stability makes them ideal catalysts at a variety of temperatures and in harsh solvents. Carbohydrates are one of the main sources of energy for many living organisms. To gain insight into the carbohydrate metabolism of hyperthermophilic organisms, as well as the potential applications of thermostable proteins, several proteins involved in different aspects of carbohydrate metabolism were selected for detailed molecular genetic and biochemical analysis, as described below. The glycolytic enzyme phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus was overproduced, purified, crystallized and biochemically characterized. Several structures together with electron paramagnetic resonance data, nuclear magnetic resonance spectroscopy data and enzyme inhibition analysis were used to elucidate the reaction mechanism of this enzyme. Computational analysis of the cupin superfamily, to which this protein belongs, allowed the essential residues for this protein’s integrity to be predicted. Subsequently, these predictions were verified by testing the activity of several generated mutants. To study regulation of carbohydrate metabolism in two related hyperthermophilic organisms; P. furiosus and Thermococcus kodakaraensis, the transcription initiation sites of glycolytic genes in P. furiosus were mapped and analysed. The obtained promoters were compared computationally to upstream regions of non-glycolytic genes. This allowed for the identification of a potential palindromic transcription factor binding site present in many genes involved in glycolysis and starch metabolism (Thermococcales-Glycolytic-Motif, TGM). Subsequently, two putative regulators of this regulon were selected for further analysis. One of them, the sugar fermentation stimulation protein SfsA, appeared to be an endonuclease. SfsA from both the thermophilic archaeon P. furiosus and the mesophilic bacterium E. coli were further characterized. Both SfsAs appeared to bind nucleotides with a distinct specificity with the P. furiosus SfsA appearing to bind RNA while the E. coli SfsA appeared to bind DNA. The P. furiosus SfsA showed hydrolytic activity on dsDNA. Genomic context analysis together with Histidine-tag pull-downs of E. coli SfsA indicated a possible role in RNA/DNA/nucleotide turnover. The other putative regulator of glycolytic genes in P. furiosus and T. kodakaraensis, Tgr, was over-expressed, purified and binding to the Thermococcales glycolytic promoter motif (TGM) by gel retardation assays was established. Furthermore, whole-genome DNA microarray analysis of T. kodakaraensis and an isogenic tgr knockout of this organism revealed that transcriptional regulation of genes containing a TGM in their promoter region in the knockout of tgr was severely affected, indicating that Tgr was regulating these genes in vivo. Co-factor analysis with several maltooligosaccharides proved that maltotriose was the effector of Tgr. To study the applicability of thermostable proteins, a biosensor was constructed based on the maltotriose binding protein from the ABC-transporter of P. furiosus. Several amino acids of this binding protein were mutated to cysteines and tagged with a fluorescent probe. Addition of maltotriose resulted in a change in fluorescence intensity in some of the biosensors. Tests at 20 and 60 degrees Celsius indicated that the affinity of the sensor was dependent on the temperature of the environment, and proved that the protein could be used at higher temperatures. The work described in this thesis is a contribution to the understanding of how thermostable proteins function in vivo, and how these proteins can be employed in a practical setup. The presented studies have revealed molecular details on enzymatic performance, transcription factor dependent regulation of glycolysis and the use of a thermostable protein in a biosensor setup.