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Characterization of peptidoglycan-recovering enzymes of Clostridium acetobutylicum

Characterization of peptidoglycan-recovering enzymes of Clostridium acetobutylicum

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REITH, Jan, 2012. Characterization of peptidoglycan-recovering enzymes of Clostridium acetobutylicum [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Reith2012Chara-19290, title={Characterization of peptidoglycan-recovering enzymes of Clostridium acetobutylicum}, year={2012}, author={Reith, Jan}, address={Konstanz}, school={Universität Konstanz} }

The cell wall is the essential exoskeleton of most bacteria that maintains cell shape and protects the cell against osmotic lysis. The major component of the bacterial cell wall is the peptidoglycan, a heteropolymer consisting of glycan strands cross-linked by short peptides. Despite its rigidity, the peptidoglycan is a highly dynamic structure, which is continuously undergoing turnover and remodeling during cell growth and division. In Gram-negative bacteria like Escherichia coli, peptidoglycan fragments released during this turnover process are reutilized (recycled) efficiently. However, it is still unknown whether recycling also proceeds in Gram-positive bacteria. Recently, a pathway for the recovery of peptidoglycan fragments, which involves the sequential processing of N-acetylglucosamine-N-acetylmuramic acid-peptides (muropeptides) by the secreted enzymes N-acetylglucosaminidase (NagZBs) and N-acetylmuramic acid-L-alanine amidase (AmiEBs), has been proposed for the Gram-positive bacterium Bacillus subtilis. Orthologs of muropeptide recycling enzymes of B. subtilis are also present in Clostridium acetobutylicum, an anerobic Gram-positive solvent producer that is closely related to Bacillus. We recognized that orthologs of NagZ (CA_C0182) and AmiE (CA_C0181) of C. acetobutylicum are apparently not secreted as in B. subtilis, indicating that catabolism of muropeptides may proceed different.<br /><br />This thesis presented here investigates the peptidoglycan recycling pathway of C. acetobutylicum and focuses on two enzymes showing a novel specificity for the amino sugars of the bacterial peptidoglycan, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). We identified a kinase of C. acetobutylicum, named MurK (formerly CA_C0183), which is encoded by a gene that is clustered with putative recycling genes. MurK catalyzes the phosphorylation of both GlcNAc and MurNAc at the 6-position, yielding GlcNAc-6-phosphate and MurNAc-6-phosphate, respectively. The biochemical characterization of MurK revealed a 1.5-fold higher catalytic activity for GlcNAc than for MurNAc. Applying phosphorylation assays using radioactive ATP indicated that structurally related cell wall sugars, i.e., epimers (N-acetylgalactosamine and N-acetylmannosamine), non-N-acetylated derivatives (glucosamine and muramic acid) and an anhydro form of MurNAc (1,6-anhydroMurNAc) are no substrates for this enzyme. MurK displays no amino acid sequence similarity with the amino sugar kinases AnmK and NagK of E. coli but with human GlcNAc kinase. We showed that MurK kinase can be used for a sensitive detection of the cell wall sugars at amounts as low as 5 fmol. Within the same gene cluster downstream of murK on the genome of C. acetobutylicum, we identified a second encoded enzyme, a novel acetyltransferase, named GlmA (formerly CA_C0184). It catalyzes the formation of GlcNAc by the transfer of an acetyl group from acetyl coenzyme A to glucosamine (GlcN) and terminal nonreducing GlcN residues, respectively. We determined a 3- to 4-fold higher catalytic efficiency of GlmA for linear di- and trisaccharides composed of β-1,4-glycosidically linked GlcN residues, compared to GlcN. However, the lactic acid derivative of GlcN, muramic acid, did not serve as an acetyl acceptor of GlmA. Acetylation assays with muropeptides derived from peptidoglycan of B. subtilis revealed that non-N-acetylated GlcN residues had to be N-acetylated by GlmA prior to the release of terminal GlcNAc by the N-acetylglucosaminidase NagZBs, whereas peptidoglycan of E. coli lacking these N-deacetylated modifications could not be acetylated by GlmA.<br /><br />In conclusion, the results presented in this thesis indicate that MurK, the GlcNAc/MurNAc kinase, and GlmA, the glucosamine/glucosaminide N-acetyltransferase, both are involved in a putative recycling pathway in C. acetobutylicum, which proceeds distinct to the known pathways of B. subtilis and E. coli. Our model of the C. acetobutylicum peptidoglycan recycling proposes that de-N-acetylated muropeptides liberated during cell wall turnover are transported into the cell by the putative ABC transporter AppABCDF. In the cytoplasm, recovery of GlcN residues requires N-acetylation at the nonreducing end by GlmA. The generated terminal GlcNAc can then be released from muropeptides by NagZ and MurNAc from the peptide moiety by AmiE. Both amino sugars can now be used for phosphorylation by the MurK kinase, yielding GlcNAc- and MurNac-6-phosphate, respectively. Further processing involves the conversion of MurNAc-6-phosphate to GlcNAc-6-phosphate by the MurQ etherase, which enters, after deacetylation, either glycolysis or the peptidoglycan biosynthesis pathway. deposit-license Reith, Jan 2012 eng Characterization of peptidoglycan-recovering enzymes of Clostridium acetobutylicum Reith, Jan 2012-05-31T13:51:46Z 2012-05-31T13:51:46Z

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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