1887

Abstract

The mycobacterial cell wall core consists of an outer lipid layer of mycolic acids connected, via arabinogalactan polysaccharide, to an inner peptidoglycan layer. An α-L-rhamnopyranosyl residue has been shown to be a key component linking the mycolated arabinogalactan to the peptidoglycan and, therefore, the biosynthesis of L-rhamnose (Rha) in mycobacteria was investigated as the first step of developing inhibitors of its biosynthesis. Biochemical assays were used to show that dTDP-Rha was synthesized in from α-D-glucose 1-phosphate (α-D-Glc-1-P) and dTTP by the same four enzymic steps used by and other bacteria. PCR primers based on consensus regions of known sequences of the first enzyme in this series, α-D-Glc-1-P thymidylyltransferase (RfbA) were used to amplify DNA from . The entire gene was then cloned and sequenced. The deduced amino acid sequence revealed a 31362 Da putative protein product which showed similarity to RfbA proteins of other bacteria (59% identity to that found in ). Sequencing of DNA flanking the gene did not reveal any of the other genes required for dTDP-Rha biosynthesis. Therefore, the four Rha biosynthetic genes are not clustered in . The enzymic activity of the sequenced gene product was confirmed by transformation of with pBluescript KS(–) containing the gene from . Analysis of enzyme extracts prepared from this transformant revealed an 11-fold increase in α-D-Glc-1-P thymidylyltransferase activity.

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1997-03-01
2021-10-25
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