Biochemical Characterisation of Reconstructed Ancestral CM-DAH7PS Enzymes
McMillan, J. P. (2012). Biochemical Characterisation of Reconstructed Ancestral CM-DAH7PS Enzymes (Thesis, Master of Science (MSc)). University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/7568
Permanent Research Commons link: http://hdl.handle.net/10289/7568
Thermophily has been proposed to be a primitive trait (Stetter, 2006, Pace, 1991, Woese, 1987) which has led to suggestions that all contemporary thermophilic species are the direct descendants of ancient thermophilic organisms. Ancestral sequence reconstruction (ASR) is a modern molecular technique which has been used to study the evolution of thermophily, however these studies have produced conflicting results. Studies utilising ASR to investigate the evolution of thermophily with two different proteins, elongation factor Tu and thioredoxin, have suggested a primitive origin for thermophily. However a recent study by Hobbs et al. (2012), in which ancestral IPMDH enzymes were reconstructed and biochemically characterised in order to investigate the evolution of thermophily in the Bacillus genus, suggested that thermophily may have evolved at least twice within the Bacillus genus, indicating that thermophily may not be strictly a primitive trait. This finding needs to be supported by data from other reconstructed enzymes. Four ancestral Bacillus 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase enzymes with chorismate mutase fused to their N-termini (CM-DAH7PS) were reconstructed to investigate whether a similar trend would be observed with a different enzyme. The ages of these ancestral Precambrian enzymes were estimated to be between 570 and 1,079 million years old. The three most recent ancestral enzymes were characterised and deemed biologically feasible as evidenced by the kinetic properties they display, which are similar to those displayed by two extant CM-DAH7PS enzymes from Bacillus subtilis and Bacillus stearothermophilus. The X-ray crystal structure of the most recent CM-DAH7PS ancestor has been solved and found to be very similar to previously solved DAH7PS structures. The optimal temperatures for activity of the ancestral DAH7PS enzymes were used to infer how thermophily may have evolved in the Bacillus genus. Interestingly, the results conflict with the findings of Hobbs et al. (2012). This suggests that the biochemical characteristics of ancestral enzymes may be highly susceptible to the biochemical properties of the contemporary species used in the inference or that different enzymes may result in different inferences being drawn regarding the evolution of thermophily.
University of Waikato
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