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Glycobiology Advance Access published online on November 6, 2006
Glycobiology, doi:10.1093/glycob/cwl066
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© 2006 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received September 14, 2006
Revised October 23, 2006
Accepted October 26, 2006

Article

Topology and mutational analysis of the single Emb arabinofuranosyltransferase of Corynebacterium glutamicum as a model of Emb proteins of Mycobacterium tuberculosis

Mathias Seidel 1, Luke J. Alderwick 2, Hermann Sahm 1, Gurdyal S. Besra 2, and Lothar Eggeling 1 *

1 Institute for Biotechnology 1, Research Centre Juelich, D-52425 Juelich, Germany
2 School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

* To whom correspondence should be addressed.
Lothar Eggeling, E-mail: l.eggeling{at}fz-juelich.de


  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 (EMB) targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB, as well as the single Emb from Corynebacterium glutamicum. Here we present for the first time an experimental analysis of the membrane topology of Emb. The domain organisation clearly positions highly conserved loop regions, like the recognised glycosyltransferase C (GT-C) motif and the hydrophilic C-terminus towards the periplasmic side of the cell. Moreover, the assignment and orientation of hydrophobic segments identified a loop region, which might dip into the membrane and could possibly line a transportation channel for the emerging substrate. Site-directed mutations introduced into plasmid encoded Cg-emb were analysed in a C. glutamicum{Delta}emb strain for their arabinogalactan glycosyl composition and linkage analysis. Mutations analysed did not perturb galactan synthesis, however D297A produced a dramatically reduced arabinan content and prevented growth, indicating an inactive Emb. A second D298A mutation also drastically reduced arabinan content, however growth of the corresponding mutant was not altered, indicating a certain tolerance of this mutation in terms of Emb function. A W659L-P667A-Q674E triple mutation in the chain length regulation motif (Pro-motif) resulted in a reduced arabinose deposition in arabinogalactan but retained all arabinofuranosyl linkages. Taken together, the data clearly define important residues of Emb involved in arabinan domain formation, and for the first time shed new light on the topology of this important enzyme.

Keywords: Glycosyltransferase; Arabinosyltransferase; Arabinogalactan; Cell wall; Ethambutol.
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