Topology and mutational analysis of the single Emb arabinofuranosyltransferase of Corynebacterium glutamicum as a model of Emb proteins of Mycobacterium tuberculosis.
The cell wall mycolyl-arabinogalactan (AG)--peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several antitubercular drugs. For instance, ethambutol (EMB) targets AG 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 organization clearly positions highly conserved loop regions, like the recognized glycosyltransferase 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 analyzed in a C. glutamicumDeltaemb strain for their AG glycosyl composition and linkage analysis. Mutations analyzed 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 AG 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.
Seidel
Mathias
M
Institute for Biotechnology 1, Research Centre Juelich, D-52425 Juelich, Germany.
Alderwick
Luke J
LJ
Sahm
Hermann
H
Besra
Gurdyal S
GS
Eggeling
Lothar
L
D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D002462:Cell Membrane / Q000201:enzymology*; D002473:Cell Wall / Q000737:chemistry; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004252:DNA Mutational Analysis; D004977:Ethambutol / Q000494:pharmacology; D005685:Galactans / Q000032:analysis; D008055:Lipids / Q000032:analysis; D008954:Models, Biological; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics*; D010457:Peptidoglycan / Q000032:analysis; D017434:Protein Structure, Tertiary
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D005685:Galactans; D008055:Lipids; D010457:Peptidoglycan; C053856:arabinogalactan-peptidoglycan complex; D004977:Ethambutol; D010430:Pentosyltransferases; C101967:arabinosyltransferase; C005653:arabinogalactan