Biosynthetic origin of mycobacterial cell wall galactofuranosyl residues.
METHODS
Mycobacterial galactofuran is essential to the linking of the peptidoglycan and mycolic acid cell wall layers. Galactofuran biosynthesis should thus be essential for viability.


OBJECTIVE
The objective was to determine the pathway of galactofuranosyl biosynthesis and to clone a gene encoding an essential enzyme necessary for its formation.


METHODS
Specific enzymatic conversions involved in formation of galactopyranose and galactofuranose residues in other bacteria were tested for in Mycobacterium smegmatis. M. tuberculosis deoxyribonucleic acid (DNA) was identified by homology.


RESULTS
It was shown that the de novo synthesis of the galactose carbon skeleton occurred in M. smegmatis by the transformation of UDP-glucopyranose to UDP-galactopyranose via the enzyme UDP-glucose 4-epimerase (E.C. 5.1.3.2). The N-terminal sequence of this enzyme was obtained after purification. The galactose salvage pathway enzyme, UDP-glucose-galactose-1-phosphate uridylyltransferase (E.C. 2.7.7.12), was also shown to be present. The critical biosynthetic transformation of the galactopyranose to galactofuranose ring form was shown to occur at the sugar nucleotide level via the enzyme UDP-galactopyranose mutase (E.C. 5.4.99.9). The M. tuberculosis DNA encoding this enzyme was sequenced, the gene expressed in Escherichia coli, and the expected enzymatic activity demonstrated.


CONCLUSIONS
Galactofuranose biosynthesis can now be pursued as a potential drug target in M. tuberculosis.
Glaxo Wellcome Research and Development, Stevenage, Hertfordshire, UK.
A
A
Weston
R J
RJ
Stern
R E
RE
Lee
P M
PM
Nassau
D
D
Monsey
S L
SL
Martin
M S
MS
Scherman
G S
GS
Besra
K
K
Duncan
M R
MR
McNeil
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000201:enzymology / Q000378:metabolism; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005685:Galactans / Q000096:biosynthesis*; D019751:Intramolecular Transferases / Q000235:genetics; D007711:Klebsiella pneumoniae / Q000235:genetics; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D017386:Sequence Homology, Amino Acid; D014534:UDPglucose 4-Epimerase / Q000302:isolation & purification / Q000378:metabolism; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000302:isolation & purification
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D005685:Galactans; D011135:Polysaccharides, Bacterial; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase; D014534:UDPglucose 4-Epimerase; D019751:Intramolecular Transferases; C490824:Glf protein, E coli; C098051:UDP-galactopyranose mutase; C005653:arabinogalactan