Open Access Senior Thesis
Bachelor of Arts
Matthew H. Sazinsky
2017 Arjun S Nanda
Toluene / o-xylene monooxyenase (ToMO) from Pseudomonas sp. OX1 is a multimeric metalloenzyme enzyme that efficiently catalyzes the hydroxylation of aromatic hydrocarbons with high specificity. Though included in a larger group of conserved bacterial multicomponent monooxygenases (BMMs) studied as potential biocatalysts for industrial hydrocarbon chemistry, the substrate specificity and oxygenated intermediates of ToMO differ greatly from its well-characterized, alkane-hydroxylating analog sMMO. Despite a shared global topology and near identical active sites, sMMO can cleave inert C-H bonds in alkanes while ToMO cannot - two seemingly similar structures give rise to vastly different chemistries. This work seeks to determine a structural basis for this difference by mutational analysis of residues thought to conformationally constrain the active site in ToMO, with the goal of replicating the terminal alkane hydroxylating activity of sMMO.
To this end, a library of potential alkane-hydroxylating mutants was generated and kinetically characterized, revealing a range of novel behaviors including significant reaction rate enhancements. In combination with low-level computational modeling to quantify the bulk and local rigidity of both sMMOH and ToMOH, we propose a broader strategy for BMM scaffolds to achieve a variety of specific and efficient hydrocarbon chemistries.
Nanda, Arjun, "Engineering an Alkane-Hydroxylating Bacterial Monooxygenase: A Tale of Two Chemistries" (2017). Pomona Senior Theses. 186.