LmbE proteins from Bacillus cereus are de-N-acetylases with broad substrate specificity and are highly similar to proteins in Bacillus anthracis

Journal Article · Wed May 19 00:00:00 EDT 2010 · Federation of European Biochemical Societies (FEBS) Journal

DOI:https://doi.org/10.1111/j.1742-4658.2010.07691.x· OSTI ID:1153832

Deli, Alexandra; Koutsioulis, Dimitrios; Fadouloglou, Vasiliki E.; Spiliotopoulou, Panagiota; Balomenou, Stavroula; Arnaouteli, Sofia; Tzanodaskalaki, Maria; Mavromatis, Konstantinos; Kokkinidis, Michalis; Bouriotis, Vassilis

The genomes of Bacillus cereus and its closest relative Bacillus anthracis each contain two LmbE protein family homologs: BC1534 (BA1557) and BC3461 (BA3524). Only a few members of this family have been biochemically characterized including N-acetylglucosaminylphosphatidyl inositol (GlcNAc-PI), 1-_D-myo-inosityl-2-acetamido-2-deoxy-α-_D-glucopyranoside (GlcNAc-Ins), N,N'-diacetylchitobiose (GlcNAc₂) and lipoglycopeptide antibiotic de-N-acetylases. All these enzymes share a common feature in that they de-N-acetylate the N-acetyl-_D-glucosamine (GlcNAc) moiety of their substrates. The bc1534 gene has previously been cloned and expressed in Escherichia coli. The recombinant enzyme was purified and its 3D structure determined. In this study, the bc3461 gene from B. cereus ATCC14579 was cloned and expressed in E. coli. The recombinant enzymes BC1534 (EC 3.5.1.-) and BC3461 were biochemically characterized. The enzymes have different molecular masses, pH and temperature optima and broad substrate specificity, de-N-acetylating GlcNAc and N-acetylchito-oligomers (GlcNAc₂, GlcNAc₃ and GlcNAc₄), as well as GlcNAc-1P, N-acetyl-d-glucosamine-1 phosphate; GlcNAc-6P, N-acetyl-d-glucosamine-6 phosphate; GalNAc, N-acetyl-d-galactosamine; ManNAc, N-acetyl-d-mannosamine; UDP-GlcNAc, uridine 5'-diphosphate N-acetyl-d-glucosamine. However, the enzymes were not active on radiolabeled glycol chitin, peptidoglycan from B. cereus, N-acetyl-d-glucosaminyl-(β-1,4)-N-acetylmuramyl-l-alanyl-d-isoglutamine (GMDP) or N-acetyl-d-GlcN-Nα1-6-d-myo-inositol-1-HPO₄-octadecyl (GlcNAc-I-P-C₁₈). Kinetic analysis of the activity of BC1534 and BC3461 on GlcNAc and GlcNAc₂ revealed that GlcNAc₂ is the favored substrate for both native enzymes. Based on the recently determined crystal structure of BC1534, a mutational analysis identified functional key residues, highlighting their importance for the catalytic mechanism and the substrate specificity of the enzyme. Finally, the catalytic efficiencies of BC1534 variants were significantly decreased compared to the native enzyme. An alignment-based tree places both de-N-acetylases in functional categories that are different from those of other LmbE proteins.

• Research Organization: USDOE Joint Genome Institute (JGI), Berkeley, CA (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• OSTI ID: 1153832
• Journal Information: Federation of European Biochemical Societies (FEBS) Journal, Vol. 277, Issue 13; ISSN 1742-464X
• Publisher: Federation of European Biochemical Societies
• Country of Publication: United States
• Language: English