Scission of the lactyl ether bond of N-acetylmuramic acid by Escherichia coli "etherase".
The ubiquitous bacterial cell wall sugar N-acetylmuramic acid (MurNAc) carries a unique D-lactyl ether substituent at the C3 position. Recently, we proposed an etherase capable of cleaving this lactyl ether to be part of the novel bacterial MurNAc dissimilation pathway (Dahl, U., Jaeger, T., Nguyen, B. T., Sattler, J. M., Mayer, C. (2004) J. Bacteriol. 186, 2385-2392). Here, we report the identification of the first known MurNAc etherase. The encoding gene murQ is located at 55 min on the Escherichia coli chromosome adjacent to murP, the MurNAc-specific phosphotransferase system. A murQ deletion mutant could not grow on MurNAc as the sole source of carbon and energy but could be complemented by expressing murQ from a plasmid. The mutant had no obvious phenotype when grown on different carbon sources but accumulated MurNAc 6-phosphate at millimolar concentrations from externally supplied MurNAc. Purified MurQ-His6 fusion protein and extracts of cells expressing murQ both catalyze the cleavage of MurNAc 6-phosphate, with GlcNAc 6-phosphate and D-lactate being the primary products. The 18O label from enriched water is incorporated into the sugar molecule, showing that the C3-O bond is cleaved and reformed by the enzyme. Moreover, an intermediate was detected and identified as an unsaturated sugar molecule. Based on this observation, we suggested a lyase-type mechanism (beta-elimination/hydration) for the cleavage of the lactyl ether bond of MurNAc 6-phosphate. Close homologs of murQ were found on the chromosome of several bacteria, and amino acid sequence similarity with the N-terminal domain of human glucokinase-regulatory protein (GckR or GKRP) was recognized.
Fachbereich Biologie, Microbiology, University of Konstanz, 78457 Konstanz, Germany.
Tina
T
Jaeger
Momo
M
Arsic
Christoph
C
Mayer
D002244:Carbon / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry; D002855:Chromatography, Thin Layer; D002874:Chromosome Mapping; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D004987:Ethers; D017353:Gene Deletion; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000378:metabolism*; D007773:Lactates / Q000737:chemistry; D019344:Lactic Acid / Q000737:chemistry; D013058:Mass Spectrometry; D008956:Models, Chemical; D009112:Muramic Acids / Q000737:chemistry*; D009154:Mutation; D010641:Phenotype; D010710:Phosphates / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism; D013696:Temperature; D013997:Time Factors
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D004987:Ethers; D007773:Lactates; D009112:Muramic Acids; D010710:Phosphates; D011993:Recombinant Fusion Proteins; C080906:glucokinase regulatory protein; C031651:N-acetylmuramic acid; D019344:Lactic Acid; D002244:Carbon; C504246:MurP protein, E coli; D017853:Phosphotransferases (Alcohol Group Acceptor); D006026:Glycoside Hydrolases; C504247:MurQ protein, E coli