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

Hepatitis E virus (HEV) is globally the most common cause of acute viral hepatitis. In industrialized countries, HEV infection can be seen in travelers returning from hyperendemic countries or in individuals at risk for autochthonous infection due to zoonotic exposure. Hepatitis E virus infection is often unrecognized and at times misdiagnosed because of nonspecific findings that can overlap with other causes of hepatitis, including autoimmune hepatitis (AIH). Although most cases of acute HEV infection resolve spontaneously and do not require treatment, life-threatening acute liver failure may occur in some cases. We discuss the case of an 8-year-old boy returning from Bangladesh with progressive acute liver injury and a clinical profile suggestive of AIH, who showed a favorable response to corticosteroid treatment before the diagnosis of an acute HEV infection could be established.

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