Characterization of anN-Acetylmuramic Acid/N-Acetylglucosamine Kinase of Clostridium acetobutylicum

Abstract

ABSTRACTWe report here the cloning and characterization of a cytoplasmic kinase ofClostridium acetobutylicum, named MurK (formurein sugarkinase). The enzyme has a unique specificity for both amino sugars of the bacterial cell wall,N-acetylmuramic acid (MurNAc) andN-acetylglucosamine (GlcNAc), which are phosphorylated at the 6-hydroxyl group. Kinetic analyses revealedKmvalues of 190 and 127 μM for MurNAc and GlcNAc, respectively, and akcatvalue (65.0 s−1) that was 1.5-fold higher for the latter substrate. Neither the non-N-acetylated forms of the cell wall sugars, i.e., glucosamine and/or muramic acid, nor epimeric hexoses or 1,6-anhydro-MurNAc were substrates for the enzyme. MurK displays low overall amino acid sequence identity (24%) with human GlcNAc kinase and is the first characterized bacterial representative of the BcrAD/BadFG-like ATPase family. We propose a role of MurK in the recovery of muropeptides during cell wall rescue inC. acetobutylicum. The kinase was applied for high-sensitive detection of the amino sugars in cell wall preparations by radioactive phosphorylation.