Enzymatic Hydroxylation vs Desaturation: Mechanistic Insights from the Case of Diiron Alkane Monooxygenase AlkB

Abstract

Abstract Understanding the enzymatic catalytic preferences for hydroxylation or desaturation could provide useful information for the potential regulation and engineering of enzymatic activity. AlkB, a non-heme diiron-dependent alkane oxygenase that catalyzes the hydroxylation of mid-chain alkanes and also exhibits the desaturase activity toward special substrates, serves as a representative system for studying the “hydroxylation versus desaturation” dichotomy. The molecular dynamics and density functional calculations reveal that the AlkB hydroxylation reaction requires not only the presence of dioxygen but also water as a co-substrate to form the Fe-oxyl active species. The mechanism involves the coordination of O2 with iron to form an Fe(III)–O–O–· complex as the initial step, followed by hydrogen transfer between Fe(III)–O–O–· and water, O–O bond cleavage forming Fe-oxyl assisted by the H+/e– addition, hydrogen abstraction by the Fe(III)–O–· species generated via state crossing, and hydroxyl rebound, ultimately resulting in the substrate hydroxylation. Additionally, the Fe-oxyl can facilitate a hydrogen transfer between Fe-bound oxygen atoms to produce a similar Fe-oxyl species on the other iron site, which may enhance the catalytic efficiency of the enzyme. The mechanisms of substrate hydrogen abstraction directly by Fe(III)–O–O–· or Fe(III)–O–OH were found to be unfavorable. The competitive desaturation is unfavorable for dodecane, which is thought to be related to the orientation and distance between the Fe-OH group and the substrate carbon radical. Furthermore, our study explored the effects of residue mutations, providing further understanding of alkane oxygenases and the non-heme diiron enzyme family.