Department or Program
Because of their ability to catalyze the stereo- and regioselective terminal oxidation of diverse alkanes into alcohols, alkane monooxygenase enzymes (AlkBs) have tremendous potential as templates for biomimetic catalysts for use in bioremediation, pharmaceutical synthesis, and bioreactors. Thus far, research into these non-heme diiron enzymes has focused upon two mesophilic γ-proteobacteria and has yielded no three-dimensional structures. This thesis seeks to characterize three recently discovered AlkBs, which represent more of the extant diversity of this enzyme, sharing less than 50% amino acid sequence identity with prototypical AlkB of Pseudomonas putida. Both the wild-type enzyme and a catalytically active fragment of the AlkB produced by Mycobacterium tuberculosis were studied, as was the fusion protein of Dietzia cinnamea, which contains an AlkB domain and a rubredoxin domain. This thesis also examined the AlkB of Thermomonospora curvata, a thermophile known to produce thermostable enzymes. Synthetic gene products encoding the protein of interest and affinity tags to facilitate purification were obtained and cloned into E. coli. These recombinant cells were then cultured, and their AlkBs purified. Detergent assays and assays of enzymatic activity were conducted. Ongoing crystallization trials may eventually yield the structural resolution necessary to understand and replicate the catalytic mechanism of AlkB enzymes.
Level of Access
Restricted: Embargoed [Bates Community After Expiration]
Date of Graduation
Bachelor of Science
Hamilton, Grace Elizabeth, "The diversity of AlkBs: a structural and mechanistic comparison of three little-known alkane monooxygenase enzymes" (2015). Honors Theses. 136.
Number of Pages
Components of Thesis