Characterization of a Glucosamine/Glucosaminide N -Acetyltransferase of Clostridium acetobutylicum

Abstract

Many bacteria, in particular Gram-positive bacteria, contain high proportions of non-N-acetylated amino sugars, i.e., glucosamine (GlcN) and/or muramic acid, in the peptidoglycan of their cell wall, thereby acquiring resistance to lysozyme. However, muramidases with specificity for non-N-acetylated peptidoglycan have been characterized as part of autolytic systems such as of Clostridium acetobutylicum. We aim to elucidate the recovery pathway for non-N-acetylated peptidoglycan fragments and present here the identification and characterization of an acetyltransferase of novel specificity from C. acetobutylicum, named GlmA (for glucosamine/glucosaminide N-acetyltransferase). The enzyme catalyzes the specific transfer of an acetyl group from acetyl coenzyme A to the primary amino group of GlcN, thereby generating N-acetylglucosamine. GlmA is also able to N-acetylate GlcN residues at the nonreducing end of glycosides such as (partially) non-Nacetylated peptidoglycan fragments and ␤-1,4-glycosidically linked chitosan oligomers. K m values of 114, 64, and 39 M were determined for GlcN, (GlcN) 2 , and (GlcN) 3 , respectively, and a 3-to 4-fold higher catalytic efficiency was determined for the di-and trisaccharides. GlmA is the first cloned and biochemically characterized glucosamine/glucosaminide N-acetyltransferase and a member of the large GCN5-related N-acetyltransferases (GNAT) superfamily of acetyltransferases. We suggest that GlmA is required for the recovery of non-N-acetylated muropeptides during cell wall rescue in C. acetobutylicum.The glycan chains of the peptidoglycan of the bacterial cell wall are composed of alternating, ␤-1,4-glycosidically linked amino sugars N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) (30). The two glycosidic bonds of these glycans are targeted by muramidases, such as lysozymes that hydrolyze the MurNAc-GlcNAc linkages, and by (endo-)Nacetylglucosaminidases that cleave the GlcNAc-MurNAc bonds. Many bacteria, in particular Gram-positive pathogens, have been reported to acquire resistance against cell lysis due to the action of lysozyme and N-acetylglucosaminidases by Ndeacetylation of a great portion of the amino sugars of the peptidoglycan of their cell wall (29). Araki et al. showed that the majority of glucosamine residues of the peptidoglycans of Bacillus cereus, B. subtilis, and B. megaterium have free amino groups and that non-N-acetylated glucosamine residues accounted for the resistance of these strains to lysozyme (1, 2, 12). The peptidoglycan of Streptococcus pneumoniae, which contains 40 to 80% glucosamine (GlcN) and up to 10% muramic acid (19), is N-deacetylated at the GlcNAc residues in the peptidoglycan by the GlcNAc deacetylase PgdA, the first characterized peptidoglycan deacetylase (31). Clostridium acetobutylicum, which is an endospore-forming, anaerobic firmicute that is closely related to bacilli, presumably also contains N-deacetylated peptidoglycan since orthologs of pgdA are present on its chromosome. Moreover, an autolytic muramidase has been i...

Transcriptional Profile during Deoxycholate-Induced Sporulation in a Clostridium perfringens Isolate Causing Foodborne Illness

“…As for AC7_0201, encoding sodium/solute symporter family proteins, a bacterial homolog of the apical sodium-dependent bile acid transporter was detected in Neisseria meningitides (51), implying that deoxycholate can be absorbed in the cytoplasm in NCTC8239 through this symporter. Gcn5-related N-acetyltransferase is required for the recovery of non-N-acetylated muropeptides during cell wall turnover in Clostridium acetobutylicum (52). dapA and dapB encode the precursor synthetic enzymes for dipicolinic acid and were activated during sporulation in Bacillus cereus (53,54).…”

Geobacillus thermoglucosidasius Endospores Function as Nuclei for the Formation of Single Calcite Crystals

“…Homology searching using MASCOT PMF indicated that the calcite-specific proteins are related to putative N-acetyltransferase YitH (GenBank accession no. CAB12939) (15,16,21), which is a member of the acetyltransferase family in B. subtilis. The enzyme N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl coenzyme A (acetyl-CoA) to glucosamine 6-phosphate, the precursor of N-acetylglucosamine, a constituent of the peptidoglycan cell wall of Gram-positive bacteria (21,22).…”

“…CAB12939) (15,16,21), which is a member of the acetyltransferase family in B. subtilis. The enzyme N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl coenzyme A (acetyl-CoA) to glucosamine 6-phosphate, the precursor of N-acetylglucosamine, a constituent of the peptidoglycan cell wall of Gram-positive bacteria (21,22). The thick peptidoglycan shell of endospores is known as the spore cortex and aids in survival during dormancy (23).…”

Structural Studies on a Glucosamine/Glucosaminide N-Acetyltransferase

“…Recently, an enzyme from Clostridium acetobutylicum , referred to as GlmA, has been characterized and has been proposed to play such a role in cell wall rescue. 12 Importantly, GlmA shows high specificity for both glucosamine and chitosan fragments ( β −1 → 4-linked glucosamine residues).…”