STRUCTURAL BASIS FOR THERMOSTABILITY AND THERMAL DEPENDENCE OF ACTIVITY IN ALPHA/BETA BARREL GLYCOSYL HYDROLASES
Open Access
- Author:
- Panasik, Nicholas
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 21, 2002
- Committee Members:
- Richard Koerner, Committee Member
James Gregory Ferry, Committee Member
Allen T Phillips, Committee Member
B Tracy Nixon, Committee Member
Jean Elnora Brenchley, Committee Chair/Co-Chair - Keywords:
- directed evolution
psychrophile
structure
thermostability
alpha/beta barrel - Abstract:
- A fundamental biochemical observation is that most enzymes have a limited temperature range for activity and stability. Many attempts have been made to understand the mechanisms that determine this range for a specific enzyme. Studies comparing the structural variations between mesophilic and thermophilic enzymes have led to speculation that different structural factors such as the number of ion pairs, the number of hydrogen bonds, the amount and type of solvent exposed surface area, and the amino acid composition are important. In order to determine whether any of these factors varied significantly between mesophilic enzymes alone, I performed a statistical analysis of the distributions of these factors in all mesophilic and thermophilic alpha/beta barrel glycosyl hydrolase structures. These data provide a spectrum of values against which researchers can compare findings from pairwise studies of naturally occurring homologues to evaluate whether the differences they observe are significant and worth further study. Only two significant differences were observed between the two groups studied: the number of glycine residues in turns at the bottom of the barrel was higher in mesophilic structures and the B factors were consistently higher in the thermophilic structures. These observations suggest differences in enzyme flexibility may play a key role in temperature adaptation. For the factors most commonly conjectured as imparting thermostability (ion pairs, hydrogen bonds, solvent accessible surface area, and amino acid composition) the degree of fluctuation within one temperature regime (mesophiles) was equal to the variability between the two regimes (mesophiles vs. thermophiles). Consequently, such comparisons of gross structural features between naturally occurring extremophilic homologs were found not to contain sufficient resolution to determine the molecular basis for thermostability. <p> I examined several approaches that would more specifically address the question of what structural changes, if any, could alter the temperature range for enzyme activity. I selected a directed evolution approach in which I used random mutagenesis to increase low temperature activity of one thermophilic and one psychrophilic family 42 beta-galactosidase. <p> In the first case, a low temperature enrichment strategy was developed to select for increased activity at low temperatures in the psychophilic enzyme, and the existence of a selective pressure was validated. Two mutations, K206G and L423F, were found to double the specific activity of the beta-galactosidase toward ONPG substrate. The effects of these two mutations on enzyme kinetics were examined with lactose as a substrate. The Km values decreased by a factor of two while the kcat value remained unchanged. <p> In directed evolution of the thermophilic enzyme, the low temperature limit of the mutant enzyme was extended 25ºC below that of its parent while maintaining stability at high temperatures (60ºC). The individual mutations responsible for this phenotype were identified as V3E, L4I, F187I, and F258S. The effects of these four mutations on enzyme kinetics were examined. The Km value remained unchanged while the kcat value was increased. The effects of the individual mutations were found to be additive. Titration experiments using dithiobisnitrobenoic acid (DTNB) to determine the number of cysteine residues accessible in the soluble enzyme indicated that molecular flexibility is increased. This work demonstrates that the temperature range of enzyme activity may be broadened without the loss of thermostability. <p> Although there is not sufficient data to identify the location of mutations F187I and F258S in relation to structure or the active site, the localization of the other two mutations (V3E and L4I) to the N-terminus of the protein and their resultant effects on enzyme flexibility suggests that the N-terminus of an eight-stranded alpha/beta barrel strongly influences the flexibility and thermostability of these molecules and allowed us to propose this as a general mechanism for adaptation to low temperatures. Subsequent saturation mutagenesis in the N-terminal region of the gene for the thermophilic enzyme led to a high percentage (over 50%) of mutant enzymes that were significantly altered in their thermal characteristics. To determine if the same region was important in the thermal adaptation of a related psychrophilic enzyme, the N-terminal region of the psychrophilic gene was subjected to saturation mutagenesis and two variants were obtained that exhibited increased activity at low temperatures. A statistical comparison of the crystallographic structures of other thermophilic and mesophilic eight-stranded alpha/beta barrel glycosyl hydrolases was then performed, and results suggest this mechanism may be common to many eight-stranded alpha/beta barrels.<p> The unique structural role that the N-terminus plays in eight-stranded alpha/beta barrel architecture further suggests that some mechanisms leading to low temperature activity and thermostability are protein fold dependent. <p> Taken as a whole, this work shows that while the commonly undertaken comparisons of naturally occurring extremophilic homologues that are based on differences in the gross amounts of structural features may not reveal mechanisms of thermostability or thermal dependency of activity, strategies may be developed that can lead to the identification of general mechanisms that control an enzyme’s thermostat.