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J. Biol. Chem., Vol. 279, Issue 5, 3354-3360, January 30, 2004

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The Prodrug Activator EtaA from Mycobacterium tuberculosis Is a Baeyer-Villiger Monooxygenase^*

Marco W. Fraaije, Nanne M. Kamerbeek, Annelies J. Heidekamp, Riccardo Fortin, and Dick B. Janssen

From the Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

EtaA is a newly identified FAD-containing monooxygenase that is responsible for activation of several thioamide prodrugs in Mycobacterium tuberculosis. It was found that purified EtaA displays a remarkably low activity with the antitubercular prodrug ethionamide. Hinted by the presence of a Baeyer-Villiger monooxygenase sequence motif in the EtaA sequence, we have been able to identify a large number of novel EtaA substrates. It was discovered that the enzyme converts a wide range of ketones to the corresponding esters or lactones via a Baeyer-Villiger reaction, indicating that EtaA represents a Baeyer-Villiger monooxygenase. With the exception of aromatic ketones (phenylacetone and benzylacetone), long-chain ketones (e.g. 2-hexanone and 2-dodecanone) also are converted. EtaA is also able to catalyze enantioselective sulfoxidation of methyl-p-tolylsulfide. Conversion of all of the identified substrates is relatively slow with typical k_cat values of around 0.02 s^–1. The best substrate identified so far is phenylacetone (K_m = 61 µM, k_cat = 0.017 s^–1). Redox monitoring of the flavin cofactor during turnover of phenylacetone indicates that a step in the reductive half-reaction is limiting the rate of catalysis. Intriguingly, EtaA activity could be increased by one order of magnitude by adding bovine serum albumin. This reactivity and substrate acceptance-profiling study provides valuable information concerning this newly identified prodrug activator from M. tuberculosis.

Received for publication, July 18, 2003 , and in revised form, September 22, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Federation of European Microbiological Societies fellowship.

To whom correspondence should be addressed. Tel.: 31-50-3634345; Fax: 31-50-3634165; E-mail: m.w.fraaije{at}chem.rug.nl.

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