Molecular mechanisms of alkane hydroxylase (AlkB) reactivity and selectivity - Abstract The class-III diiron proteins, a group of integral membrane proteins unified by a histidine-rich active site, catalyze a wide range of reactions including hydroxylations required for the production of sphingolipids (an essential component of the myelin sheath), desaturating fatty acids that regulate metabolism and cancer progress, and hydroxylating straight chain alkanes, enabling them to be biodegraded in oil-impacted environments. The diverse chemistry in this enzyme family is controlled, at least in part, by substrate channels well matched to the structure of the different substrates. The structure of the active site, only recently determined, is puzzling because no covalent bridge linking the two redox active iron ions is apparent. Electrons are required to activate these enzymes. Some class-III diiron proteins have their electron-transfer partners covalently bound while others do not. The functional significance of these different modalities is not known. This program will target structure/function relationships in alkane monooxygenases (AlkB), the most biochemically tractable member of the class-III diiron protein family, to understand reaction mechanisms and the factors that control reaction scope. We will combine mechanistic work on AlkB variants with spectroscopic characterization of the protein using a variety of techniques that can shed light on the three dimensional and electronic structure of the active site. We will mine our large library of functional AlkB enzymes (currently we can express more than 60 different AlkBs from different bacteria) to search for patterns in reactivity. We will utilize information obtained from the cryo-EM structure of AlkB we published in 2023, as well as deep mutational scanning, to develop a large library of variant to probe the structural factors that control reactivity. We will determine the mechanism of the recently discovered capability of AlkB to catalyze the defluorination of fluorinated alkanes. We will attempt to determine the structure of the new family of AlkBs whose existence we recently reported—a fusion protein containing two electron transfer partners not previously seen linked to AlkB—and characterize its reactivity. We will also explore whether archaea express AlkBs capable of participating in the global carbon cycle. We expect to generate new insights into mechanisms of selective C-H and C-F bond activation. By leveraging our unique expertise with this key member of the class-III diiron proteins, we expect to contribute to a fundamental understanding of how these metalloenzymes work, with implications for both human and environmental health.
