L. Kremer et al., Galactan biosynthesis in Mycobacterium tuberculosis - Identification of a bifunctional UDP-galactofuranosyltransferase, J BIOL CHEM, 276(28), 2001, pp. 26430-26440
The cell wall of Mycobacterium tuberculosis and related genera is unique am
ong prokaryotes, consisting of a covalently bound complex of mycolic acids,
D-arabinan and D-galactan, which is linked to peptidoglycan via a special
linkage unit consisting of Rhap-(1 -->3)-GlcNAc-P. Information concerning t
he biosynthesis of this entire polymer is now emerging with the promise of
new drug targets against tuberculosis. Accordingly, we have developed a gal
actosyltransferase assay that utilizes the disaccharide neoglycolipid accep
ters beta -D-Galf-(1 -->5)-beta -Galf-O-C-10:1 and beta -D-Galf-(1 -->6)-be
ta -D-Galf-O-C-10:1, with UDP-Gal in conjunction with isolated membranes. C
hemical analysis of the subsequent reaction products established that the e
nzymatically synthesized products contained both beta -D-Galf linkages ((1
-->5) and (1 -->6)) found within the mycobacterial cell, as well as in an a
lternating (1 -->5) and (1 -->6) fashion consistent with the established st
ructure of the cell wall. Furthermore, through a detailed examination of th
e M. tuberculosis genome, we have shown that the gene product of Rv3808c, n
ow termed glfT is a novel UDP-galactofuranosyltransferase, This enzyme poss
esses dual functionality in per forming both (1 -->5) and (1 -->6) galactof
uranosyltransferase reactions with the above neoglycolipid accepters, using
membranes isolated from the heterologous host Escherichia coli expressing
Rv3808c, Thus, at a biochemical and genetic level, the polymerization of th
e galactan region of the mycolyl-arabinogalactan complex has been defined,
allowing the possibility of further studies toward substrate recognition an
d catalysis and assay development. Ultimately, this may also lead to a more
rational approach to drug design to be explored in the context of mycobact
erial infections.