Xylene monooxygenase, a membrane-spanning non-heme diiron enzyme that hydroxylates hydrocarbons via a substrate radical intermediate

摘要: The non-heme diiron enzyme xylene monooxygenase (XylM) has been shown to hydroxylate hydrocarbons via a hydrogen abstraction–carbon radical recombination mechanism (oxygen rebound). Using the clock bicyclo[4.1.0]heptane (norcarane) in whole-cell assay, and observing ratio of rearranged 3-(hydroxymethyl)cyclohexene unrearranged 2-norcaranol products, lifetime substrate was determined be approximately 0.2 ns. wild-type organism Pseudomonas putida mt-2 two separate Escherichia coli clones expressing xylMA genes gave similar results. One clone produced XylMA hydroxylase other Sphingomonas yanoikuyae B1 hydroxylase. Clones were constructed by inserting for reductase downstream from an IPTG-inducible T7 promoter. Mechanistic investigations using assays will facilitate more rapid screening structure–function relationships identification novel oxygenases. This approach should enable construction picture key metalloenzymes mechanisms they use selected parts global carbon cycle without requiring isolation every protein involved.