Identification of a Novel Arabinofuranosyltransferase AftB Involved in a Terminal Step of Cell Wall Arabinan Biosynthesis in Corynebacterianeae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis

• Published in: The Journal of biological chemistry Vol. 282; no. 20; pp. 14729 - 14740
• Main Authors: Seidel, Mathias, Alderwick, Luke J., Birch, Helen L., Sahm, Hermann, Eggeling, Lothar, Besra, Gurdyal S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.05.2007; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Antitubercular Agents - pharmacology; Arabinose - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cell Wall - genetics; Cell Wall - metabolism; Corynebacterium glutamicum; Corynebacterium glutamicum - enzymology; Corynebacterium glutamicum - genetics; Drug Resistance, Bacterial - drug effects; Drug Resistance, Bacterial - genetics; Ethambutol - pharmacology; Gene Deletion; Genetic Complementation Test; Mutation, Missense; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - genetics; Mycolic Acids - metabolism; Pentosyltransferases - genetics; Pentosyltransferases - metabolism; Polysaccharides - biosynthesis; Polysaccharides - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M700271200

Arabinofuranosyltransferase enzymes, such as EmbA, EmbB, and AftA, play pivotal roles in the biosynthesis of arabinogalactan, and the anti-tuberculosis agent ethambutol (EMB) targets arabinogalactan biosynthesis through inhibition of Mt-EmbA and Mt-EmbB. Herein, we describe the identification and characterization of a novel arabinofuranosyltransferase, now termed AftB (Rv3805c), which is essential in Mycobacterium tuberculosis. Deletion of its orthologue NCgl2780 in the closely related species Corynebacterium glutamicum resulted in a viable mutant. Analysis of the cell wall-associated lipids from the deletion mutant revealed a decreased abundance of cell wall-bound mycolic acids, consistent with a partial loss of mycolylation sites. Subsequent glycosyl linkage analysis of arabinogalactan also revealed the complete absence of terminal β(1 → 2)-linked arabinofuranosyl residues. The deletion mutant biochemical phenotype was fully complemented by either Mt-AftB or Cg-AftB, but not with muteins of Mt-AftB, where the two adjacent aspartic acid residues, which have been suggested to be involved in glycosyltransferase activity, were replaced by alanine. In addition, the use of C. glutamicum and C. glutamicumΔaftB in an in vitro assay utilizing the sugar donor β-d-arabinofuranosyl-1-monophosphoryl-decaprenol together with the neoglycolipid acceptor α-d-Araf-(1 → 5)-α-d-Araf-O-C8 as a substrate confirmed AftB as a terminal β(1 → 2) arabinofuranosyltransferase, which was also insensitive to EMB. Altogether, these studies have shed further light on the complexities of Corynebacterianeae cell wall biosynthesis, and Mt-AftB represents a potential new drug target.