title: Development of a plate-based scintillation proximity assay for the mycobacterial AftB enzyme involved in cell wall arabinan biosynthesis.
abstract: A number of mycobacterial arabinosyltransferases, such as the Emb proteins, AftA, AftB, AftC, and AftD have been characterized and implicated to be involved in the cell wall arabinan assembly. These arabinosyltransferases are essential for the viability of the organism and are logically valid targets for developing new anti-tuberculosis agents. For instance, Ethambutol, a first line anti-tuberculosis drug, targets the Emb proteins involved in the formation of the arabinan of cell wall arabinogalactan. Among these arabinosyltransferases, the terminal β-(1→2) arabinosyltransferase activity has been associated with AftB. The predicted topology of AftB in Mycobacterium tuberculosis has 10 N terminal transmembrane domains and a C terminal hydrophilic domain similar to the Emb proteins. It has a conserved GT-C motif and is difficult to express. In a cell free assay, synthetic disaccharide, α-D-Araf-(1→5)-α-D-Araf-octyl, has been used as a substrate to explore the function of AftB. In our work, the disaccharide was synthesized in its pentenylated and biotinylated form, and the enzymatic product formed was identified as the β-(1→2) arabinofuranose adduct. When synthetic tri- and tetra-saccharides were used as substrates, a mixture of products containing both β-(1→2) and α-(1→5) linkages were formed. Therefore, the biotinylated disaccharide was selected to develop a scintillation proximity assay.
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA. jianz@adatech.com
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Anita G
authors.initials: AG
authors.lastname: Amin
authors.forename: Alexandra
authors.initials: A
authors.lastname: Hölemann
authors.forename: Peter H
authors.initials: PH
authors.lastname: Seeberger
authors.forename: Delphi
authors.initials: D
authors.lastname: Chatterjee
mesh_terms: D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism*; D004977:Ethambutol / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000201:enzymology*; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D012588:Scintillation Counting
chemical_list: D000995:Antitubercular Agents; D011134:Polysaccharides; C030080:araban; D004977:Ethambutol; D010430:Pentosyltransferases; C101967:arabinosyltransferase