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J. Biol. Chem., Vol. 281, Issue 23, 15653-15661, June 9, 2006

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Identification of a Novel Arabinofuranosyltransferase (AftA) Involved in Cell Wall Arabinan Biosynthesis in Mycobacterium tuberculosis^*

Luke J. Alderwick¹², Mathias Seidel¹, Hermann Sahm³, Gurdyal S. Besra⁴, and Lothar Eggeling³

From the School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom and Institute for Biotechnology 1, Research Centre Juelich, D-52425 Juelich, Germany

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several anti-tubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. Following a detailed bioinformatics analysis of genes surrounding the conserved emb locus, we present the identification and characterization of a novel arabinofuranosyltransferase AftA (Rv3792). The enzyme catalyzes the addition of the first key arabinofuranosyl residue from the sugar donor -D-arabinofuranosyl-1-monophosphoryldecaprenol to the galactan domain of the cell wall, thus "priming" the galactan for further elaboration by the arabinofuranosyltransferases. Because aftA is an essential gene in M. tuberculosis, we deleted its orthologue in Corynebacterium glutamicum to produce a slow growing but viable mutant. Analysis of its cell wall revealed the complete absence of arabinose resulting in a truncated cell wall structure possessing only a galactan core with a concomitant loss of cell wall-bound mycolates. Complementation of the mutant was fully restored to the wild type phenotype by Cg-aftA. In addition, by developing an in vitro assay using recombinant Escherichia coli expressing Mt-aftA and use of cell wall galactan as an acceptor, we demonstrated the transfer of arabinose from -D-arabinofuranosyl-1-monophosphoryldecaprenol to galactan, and unlike the Mt-Emb proteins, Mt-AftA was not inhibited by ethambutol. This newly discovered glycosyltransferase represents an attractive drug target for further exploitation by chemotherapeutic intervention.

Received for publication, January 3, 2006 , and in revised form, March 31, 2006.

^* 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.

¹ Both authors contributed equally to this work.

² Biotechnology and Biological Sciences Research Council Quota Student.

³ Supported by the Fonds der Chemischen Industrie.

⁴ Supported as a Lister Institute-Jenner Research Fellow and the Medical Research Council (UK). To whom correspondence should be addressed. Tel.: 121-415-8125; Fax: 121-414-5925; E-mail: g.besra{at}bham.ac.uk.

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