Supporting Information

Files in this Data Supplement:

Fig. 5. Further details of MS. (A) Positive ion electrospray (ES) MS of deutero-re-N-acetylated GlcNAc-GlcN-GlcNAc and GlcNAc-GlcN-GlcN. The ions at m/z 653 and 656 represent the [M+Na]⁺ ions of GlcNAcGlcNAc_DGlcNAc and GlcNAcGlcNAc_DGlcNAc_D, respectively. The ions at m/z 631 and 634 represent the [M+H]⁺ ions of GlcNAcGlcNAc_DGlcNAc and GlcNAcGlcNAc_DGlcNAc_D, respectively. (B) Positive ion ES-tandem MS (MS/MS) of deutero-re-N-acetylated GlcNAc-GlcN-GlcN. The ions at m/z 226, 229, 432, 435, and 656 represent the Y-series [M+Na]⁺ ions of GlcNAc, GlcNAc_D, GlcNAcGlcNAc_D, GlcNAc_DGlcNAc_D, and GlcNAcGlcNAc_DGlcNAc_D, respectively. The ions at m/z 244, 247, 450, and 453 represent the C-series [M+Na]⁺ ions of GlcNAc, GlcNAc_D, GlcNAcGlcNAc_D, and GlcNAc_DGlcNAc_D, respectively. The ions at m/z 211 and 638 represent the [M+Na-H₂O]⁺ ions of GlcNAc_D and GlcNAcGlcNAc_DGlcNAc_D, respectively.

Fig. 6. Sequence alignment of the CE-4 family. The sequences of four known de-N-acetylases from the CE-4 family of carbohydrate esterases are shown. PGDA_SPNEU = SpPgdA, PDAA_BACSU = BsPdaA, NODB_RHIME = Rhizobium meliloti NodB, CDA_MUCRO = Mucor rouxii chitin deacetylase. The five CE-4 motifs (MT1-5, gray), metal ligands (triangles), and catalytic residues (magenta) are also shown

Table 1. Details of data collection and structure refinement

Values in parentheses are for the highest-resolution shell. All measured data were included in structure refinement.

PEG, polyethylene glycol.

Supporting Materials and Methods

Cloning

Residues 38-463 of peptidoglycan GlcNAc deacetylase (PgdA) were amplified by PCR from Streptococcus pneumoniae American Type Culture Collection BAA-255D genomic DNA by using the Expand High Fidelity PCR system (Roche) and oligonucleotide primers ( forward: 5'-GGATCCATGAATAAAAGTAGACTAGGACG-3', reverse: 5'-GAATTCTTATTCATCACGACTATAGTACAG-3'). The resulting PCR product was ligated into the pCR 2.1-TOPO cloning vector (Invitrogen) and subcloned into the pGEX-6P-1 vector (Amersham Pharmacia Biosciences) by using the BamHI/EcoRI restriction sites. All plasmids were transformed into Escherichia coli DH5 cells and plated out on LB medium agar plates with 100 mg/ml ampicillin. Single amino acid substitutions were generated by using primers designed for the PgdA-pGEX-6P-1 construct and the QuickChange XL Site-Directed Mutagenesis Kit (Stratagene). The sequences of the constructs were verified by DNA sequencing.

The PgdA-pGEX-6P-1 construct was transformed into E. coli BL21-DE3 pLysS cells. Cultures (10 ml) were grown overnight in LB + ampicillin media (100 mg/ml) and used to inoculate 1 liter of LB + ampicillin media. The cells were grown at 37°C to OD₆₀₀ = 0.6, expression was initiated by the addition of 100 mM isopropyl-d-thiogalactopyranoside. The cells were then cultured for an additional 3 h at 37°C, harvested by centrifugation at 2,250 ´g for 30 min, and the cell pellet from each liter of culture was resuspended in 40 ml of LB media and subjected to another round of centrifugation. The resulting cell pellets were flash frozen in liquid nitrogen, thawed at 37°C in a water bath, and resuspended in 10 ml of lysis buffer (25 mM Tris·HCl/2 mM EDTA/150 mM NaCl/5 mM DTT, pH 7.5/0.5 tablet of Complete protease inhibitor mixture tablets (Roche). Cells were lysed by the addition of 10 mg of DNase-1 and a cell disrupter at 30 K psi. The lysate was centrifuged at 40,000 ×g for 30 min and filtered through a 0.45-mm filter. The supernatant was incubated for 2 h at 4°C on a rotating platform with glutathione-Sepharose (Amersham Pharmacia Biosciences) beads prewashed with lysis buffer in a ratio of 1 ml of beads per liter of bacterial culture. The beads were then incubated with PreScission protease (200 mg of protease per milliliter of beads) at 4°C for 18 h in protease inhibitor-free lysis buffer. The supernatant of the beads and a subsequent wash were passed over a Bio-Rad 20-ml disposable column to remove the beads. The presence of protein was determined by Bradford assay, and the resulting filtrate was then buffer exchanged into 12.5 mM NaCl/25 mM Tris, pH 7.5 using a 20-ml VivaScience 10,000 molecular weight cutoff concentrator and concentrated down to 4.5 ml. This was followed by ion exchange using a 5-ml HiTrapTM QFF column on an AKTA purifier (Amersham Pharmacia Biosciences) using a 0-500 mM NaCl gradient. The eluted fractions were verified by SDS/PAGE, pooled, concentrated down to 5 ml, and loaded onto a Superdex 75, 26/60 gel filtration column (Amersham Pharmacia Biosciences) preequilibrated in buffer (25 mM Tris·HCl/150 mM NaCl/2 mM EDTA, pH 7.5). The resulting fractions were verified by SDS/PAGE, pooled, and subjected to buffer exchange with 25 mM Tris·HCl, pH 8. This was concentrated to 50 mg/ml (»1 mM), as verified by a Bradford assay and absorbance at 280 nm.

For some of the mutants (D275N, H326A, L415F, and R364S), proper folding was verified by crystallization and structure solution under similar conditions (data not shown).

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