A polysaccharide deacetylase homologue, PdaA, in Bacillus subtilis acts as an N-acetylmuramic acid deacetylase in vitro

Abstract

A polysaccharide deacetylase homologue, PdaA, was determined to act as an N-acetylmuramic acid deacetylase in vitro. Histidine-tagged truncated PdaA (with the putative signal sequence removed) was overexpressed in Escherichia coli cells and purified. Measurement of deacetylase activity showed that PdaA could deacetylate peptidoglycan treated with N-acetylmuramoyl-L-alanine amidase CwlH but could not deacetylate peptidoglycan treated with or without DL-endopeptidase LytF (CwlE). Reverse-phase high-performance liquid chromatography and mass spectrometry (MS) and MS-MS analyses indicated that PdaA could deacetylate the N-acetylmuramic acid residues of purified glycan strands derived from Bacillus subtilis peptidoglycan.

FIG. 1.

SDS-14% PAGE of h-ΔPdaA. h-ΔPdaA was expressed in E. coli JM109 cells in the presence of 1 mM IPTG. Lanes 1 and 2, proteins extracted from whole cells without and with IPTG added, respectively; lane 3, purified h-ΔPdaA; lane M, protein standards (Bio-Rad).

FIG. 2.

Deacetylase activity of h-ΔPdaA with nontreated peptidoglycan (•), peptidoglycan treated with l-alanine amidase CwlH (▪), and peptidoglycan treated with dl-endopeptidase LytF (▴). One milligram of chitin oligomer (hexa-N-acetylchitohexaose) per ml was also used (○). Purified h-ΔPdaA (final concentration, 2 μg/ml) was added to the following buffers (final concentration, 50 mM) containing 1 mg of nontreated peptidoglycan per ml or peptidoglycan treated with CwlH or LytF as a substrate (see Materials and Methods): morpholineethanesulfonic acid (MES) buffer for pH 5.5, 6.0, and 6.5; HEPES buffer for pH 7.0, 7.5, 8.0, and 8.5; and CHES [2-(cyclohexylamino)-ethanesulfonic acid] buffer for pH 9.0, 9.5, and 10.0. The deacetylase reaction was carried out at 37°C for 4 h.

FIG. 3.

Lysozyme digestion of isolated N-acetylated peptidoglycan glycan strands treated with (A) or without (B) PdaA. Peptidoglycan from B. subtilis 168 was digested with l-alanine amidase CwlH, and then N-acetylated glycan strands were purified mainly by size exclusion chromatography (see Materials and Methods). After the N-acetylated glycan strands had been treated with (A) or without (B) PdaA, samples were treated with lysozyme, and this was followed by addition of NaBH₄. The samples were separated by RP-HPLC. Peak 1, disaccharide (GlcNAc-MurNAcr); peak 2, N-deacetylated tetrasaccharide (GlcNAc-Mur-GlcNAc-MurNAcr); peak 3, tetrasaccharide (GlcNAc-MurNAc-GlcNAc-MurNAcr); peak A, reaction buffer; peak B, NaN₃.

FIG. 4.

(A to E) ESI-MS analyses (A and B) and ESI-MS-MS analyses (C to E) of peak 2 (GlcNAc-Mur-GlcNAc-MurNAcr) in Fig. 3A. Panels A and B show ESI-MS analysis in the positive and negative modes, respectively. Panels C to E show ESI-MS-MS analyses of the [M+H]⁺ precursor ion (m/z 935.4), [M+Na]⁺ precursor ion (m/z 957.6), and [M-H]⁻ precursor ion (m/z 933.6), respectively. (F) Putative structure and calculated molecular weight of each fragment of peak 2 in Fig. 3A. Ion series b, y, a, and z correspond to the fragment peaks in panels C to E. Intens., intensity.

FIG. 4.

(A to E) ESI-MS analyses (A and B) and ESI-MS-MS analyses (C to E) of peak 2 (GlcNAc-Mur-GlcNAc-MurNAcr) in Fig. 3A. Panels A and B show ESI-MS analysis in the positive and negative modes, respectively. Panels C to E show ESI-MS-MS analyses of the [M+H]⁺ precursor ion (m/z 935.4), [M+Na]⁺ precursor ion (m/z 957.6), and [M-H]⁻ precursor ion (m/z 933.6), respectively. (F) Putative structure and calculated molecular weight of each fragment of peak 2 in Fig. 3A. Ion series b, y, a, and z correspond to the fragment peaks in panels C to E. Intens., intensity.