PMID:8366125
| Citation |
Kelly, TM, Stachula, SA, Raetz, CR and Anderson, MS (1993) The firA gene of Escherichia coli encodes UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase. The third step of endotoxin biosynthesis. J. Biol. Chem. 268:19866-74 |
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| Abstract |
The possibility that the firA gene of Escherichia coli (Dicker, I. B., and Seetharam, S. (1991) Mol. Microbiol. 6, 817-823) might function in lipid A biosynthesis was examined based on its homology to the lpxA gene, which encodes UDP-N-acetylglucosamine O-acyl-transferase, the first enzyme in lipid A formation. Extracts of a temperature-sensitive firA mutant, RL-25, were assayed for their ability to acylate UDP-GlcNAc, using a coupled assay. The results suggested that extracts of RL-25 might be defective in the third enzyme of this pathway, the UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase. Living cells of RL-25 also displayed a 5-fold decreased rate of lipid A biosynthesis at the nonpermissive temperature as judged by a 32Pi incorporation assay. In order to examine N-acyltransferase activity directly, the substrate [alpha-32P]UDP-3-O-(R-3-hydroxymyristoyl)-GlcN was synthesized enzymatically. N-Acyltransferase specific activity in RL-25 extracts was reduced to less than 10% of wild-type. When the wild-type firA gene was cloned into a T7-based expression vector, N-acyltransferase specific activity increased almost 360-fold relative to wild-type extracts, demonstrating that firA is the structural gene for the enzyme. The N-acyltransferase displays absolute specificity for the R-3-OH moiety of R-3-hydroxymyristoyl-ACP, as does the O-acyltransferase, consistent with the placement of R-3-hydroxymyristate in E. coli lipid A. |
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| Keywords |
Acyltransferases/biosynthesis; Acyltransferases/genetics; Acyltransferases/metabolism; Amino Acid Sequence; Bacterial Proteins/biosynthesis; Bacterial Proteins/genetics; Base Sequence; Chromosome Mapping; Chromosomes, Bacterial; Endotoxins/biosynthesis; Escherichia coli/enzymology; Escherichia coli/genetics; Gene Expression; Genes, Bacterial; Genotype; Kinetics; Lipid A/biosynthesis; Molecular Sequence Data; Oligodeoxyribonucleotides; Phosphorus Radioisotopes; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Substrate Specificity; Uridine Diphosphate N-Acetylglucosamine/metabolism |
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