title: Identification of a novel arabinofuranosyltransferase (AftA) involved in cell wall arabinan biosynthesis in Mycobacterium tuberculosis.
abstract: 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 beta-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 beta-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.
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
authors.forename: Luke J
authors.initials: LJ
authors.lastname: Alderwick
authors.forename: Mathias
authors.initials: M
authors.lastname: Seidel
authors.forename: Hermann
authors.initials: H
authors.lastname: Sahm
authors.forename: Gurdyal S
authors.initials: GS
authors.lastname: Besra
authors.forename: Lothar
authors.initials: L
authors.lastname: Eggeling
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002473:Cell Wall / Q000378:metabolism*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis*; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D011134:Polysaccharides; C030080:araban; D014166:Transferases