Characterization of a Specific Arabinosyltransferase Activity Involved in Mycobacterial Arabinan Biosynthesis

Abstract

Mycobacterium smegmatis strains that contain inactivated EmbA or EmbB proteins are unable to synthesize terminal Arabeta1-->2Araalpha1-->5(Arabeta1-->2Araalpha1-->3)Araalpha1-->5Araalpha1-->(Ara(6)) motif in the cell wall polysaccharide arabinogalactan. Instead, the mutants contain a linear Arabeta1-->2Araalpha1-->5Araalpha1-->5Araalpha1-->(Ara(4)) motif, suggesting that these proteins are involved in the synthesis or transfer of the disaccharide Arabeta1-->2Araalpha1--> to an internal 5-linked Ara. Therefore, we synthesized a linear Arabeta1-->2Araalpha1-->5Araalpha1-->5Araalpha1-->5Araalpha1--> with an octyl aglycon as an arabinosyl acceptor in cell-free assays. A facile assay was developed using the chemically synthesized glycan, membrane, and particulate cell wall as the enzyme source, and 5-phosphoribose diphosphate pR[(14)C]pp as the arabinose donor. The results unequivocally show that two arabinofuranosyl residues were added at the tertiary -->5Araalpha1--> of the synthetic glycan. This activity was undetectable in strains of M. smegmatis where embB or embA had been genetically disrupted. Normal activity could be restored only in the presence of both EmbA and EmbB proteins.

Identification of a Novel Mycobacterial Arabinosyltransferase Activity Which Adds an Arabinosyl Residue to α-d-Mannosyl Residues

“…We and others have previously evaluated the effectiveness of several synthetic neoglycolipids as substrates for mycobacterial glycosyltransferase activities 11, 12 . Specifically, di- and trimannosides with an octyl aglycon chain (Fig.…”

Antitubercular Drugs Based on Carbohydrate Derivatives

“…Enzymes which incorporate and polymerize the arabinan, the arabinofuranosyltransferases (Aft), are potential targets for an anti-tuberculosis drug-development [32,[117][118][119]. The arabinofuranose (Araf ) motif, is a biologically rare sugar.…”

“…These studies elucidate the contributions of EmbA and EmbB in the formation of the terminal hexaarabinofuranosyl (Ara 6 ) (Fig. 18.9) assembly by catalyzing the transfer of the β-d-Araf-(1→2)-α-d-Araf disaccharide to the AG[119,129]. Analogous studies on the inactivation ofFigure 18.9 Terminating residue of arabinofuranosyl hexasaccharide.…”

“…The role played by EmbA and EmbB in the AG synthesis, and EmbC in the LAM synthesis, respectively, are explained via genetic disruption experiments in M. smegmatis[119,127,129,132]. These studies elucidate the contributions of EmbA and EmbB in the formation of the terminal hexaarabinofuranosyl (Ara 6 ) (Fig.…”

The cell envelope glycoconjugates ofMycobacterium tuberculosis

“…It is synthesized from 5-phosphoribose-1-pyrophosphate (Scherman et al ., 1995; Scherman et al ., 1996) - the product of the phosphoribosyl-pyrophosphate synthetase PrsA (Alderwick et al ., 2011a) - through four reaction steps involving the Dec-P 5-phosphoribosyltransferase, UbiA (Rv3806c) (Huang et al ., 2005; Huang et al ., 2008; Alderwick et al ., 2005), the phosphoribosyl-monophosphodecaprenol phosphatase Rv3807c (Jiang et al ., 2011), and DprE1 (Rv3790) and DprE2 (Rv3791) responsible for the epimerization of decaprenyl-phosphate ribose to Dec-P-Ara (Mikušová et al ., 2005). The AraTs involved in the formation of the arabinan domain of AG identified to date include AftA (Rv3792), responsible for the transfer of the very first Ara f residues to the galactan domain of AG (Alderwick et al ., 2006a), the terminal β–(1,2)-capping AraT AftB (Rv3805c) (Seidel et al ., 2007), AftC (Rv2673) involved in the internal α–(1,3)-branching of AG (Birch et al ., 2008) and the EmbA (Rv3794) and EmbB (Rv3795) proteins involved in the formation of the Ara 6 motif of AG (Escuyer et al ., 2001; Khasnobis et al ., 2006). We successfully overexpressed and purified a soluble form of AftC from M. smegmatis and showed that it retains α–(1,3)-branching AraT activity in vitro upon reconstitution into proteoliposomes containing mycobacterial lipids (Zhang et al ., 2011).…”