Arylsulfatase fromKlebsiella pneumoniaeCarries a Formylglycine Generated from a Serine

Claudia Miech, Thomas Dierks, Thorsten Selmer, Kurt von Figura, Bernhard Schmidt
1998 Journal of Biological Chemistry  
Eukaryotic sulfatases share an unusual posttranslational protein modification, which converts a cysteine into ␣-formylglycine. The ␣-formylglycine is essential for the catalytic activity. Klebsiella pneumoniae expresses an inducible arylsulfatase for which the DNA predicts a serine at the position occupied by the ␣-formylglycine residue in eukaryotic sulfatases. Structural analysis showed that the majority of the arylsulfatase polypeptides from K. pneumoniae carries the ␣-formylglycine, whereas
more » ... the remaining arylsulfatase polypeptides contain the predicted serine residue. This demonstrates the evolutionary conservation between prokaryotes and eukaryotes of this novel protein modification that so far has been found only in sulfatases. ␣-Formylglycine in Klebsiella is generated from a serine and not from a cysteine as in eukaryotes. Eukaryotic sulfatases share a cysteine residue, which is posttranslationally converted into ␣-formylglycine (FGly; 2-amino-3-oxopropanoic acid). 1 This novel posttranslational modification occurs in the endoplasmic reticulum and is directed by a linear sequence surrounding the cysteine to be modified (1-3). The FGly residue is critical for the catalytic activity of sulfatases. Crystallographic analysis of two sulfatases has shown that the FGly residue is part of the catalytic site (4, 5). The aldehyde is likely to be hydrated and to serve as an acceptor for sulfate during catalysis (5). 2 In prokaryotes expression of sulfatases is generally controlled by the sulfur or the carbon content of their environment. Under appropriate conditions such as the absence of SO 4 2Ϫ and the presence of alkyl sulfates, the sulfatases are expressed in the periplasmic space (for review see Ref. 7). The sequences of five prokaryotic sulfatases have been reported (8 -12), and they share sequence homology with eukaryotic sulfatases to a similar extent as the members of the eukaryotic sulfatase family among each other. Surprisingly, in the genes encoding the sulfatases from Klebsiella pneumoniae (8) and from Escherichia coli (9), a serine residue is predicted at a position where all other known sulfatase DNAs predict a cysteine that is known to be converted into FGly in eukaryotes. To examine whether the FGly residue is found also in prokaryotic sulfatases and whether it can be generated also from a serine residue, we purified arylsulfatase from K. pneumoniae and examined the protein for the presence of a FGly residue. EXPERIMENTAL PROCEDURES Materials-K. pneumoniae strain DSM 681 was obtained from DSM GmbH, Braunschweig. [ 3 H]NaBH 4 was from Amersham Buchler. Sulfatase Production and Purification-Bacterial culture and purification of arylsulfatase were performed as described by Okamura et al. (13) with some modifications: K. pneumoniae DSM 681 from blood agar plates was grown overnight in aliquots of 0.4 liters of medium containing methionine as sulfur source. At an A 600 of 1.5-2.5 cells were sedimented, washed, and disrupted for 20 min in aliquots of 30 ml using a Sonicator W 220-F, Heat Systems-Ultrasonics, Inc. For analysis of arylsulfatase activity 200 l of sample were incubated with 200 l of 20 mM p-nitrocatecholsulfate in 10 mM Tris/HCl, 150 mM NaCl, pH 7.4, for 10 min at 37°C. After addition of 1 ml of 1 M NaOH, absorbance at 515 nm was measured. The initial ammonium sulfate precipitation (13) was omitted, and Sephadex G-100 was substituted by Superdex 75 (120-ml volume, Pharmacia Biotech Inc.) equilibrated with 20 mM Tris/HCl, 150 mM NaCl, pH 7.2 on a fast protein liquid chromatograph. Arylsulfatase activity eluted after 66 ml. After concentration in an ultra thimble and dialysis against 20 mM Tris/HCl, pH 7.4 arylsulfatase was loaded on a MonoQ fast protein liquid chromatography column replacing the DEAE-Sephadex A-25 column (13). By this procedure arylsulfatase was purified about 224-fold to a specific activity of 123 units/mg protein yielding about 1 mg of enzyme from 30 liters of culture with a recovery of 7%. For further analysis the sulfatase was desalted by RP-HPLC on a SMART system (Pharmacia) using an Aquapore Butyl 7 micron (220 ϫ 2.1 mm) column (Applied Biosystems) equilibrated with 0.1% trifluoroacetic acid/H 2 O. Arylsulfatase A was eluted by an acetonitrile gradient from 0 to 90% in 36 min. Reduction with [ 3 H]NaBH 4 -30 g of arylsulfatase were lyophilized and solubilized in 4 M guanidinium hydrochloride, 25 mM Tris/HCl, 10 mM EDTA, pH 9. Reduction with [ 3 H]NaBH 4 , desalting, tryptic digestion, and purification of the peptides on RP-HPLC were performed as described, omitting reductive carboxymethylation with dithiothreitol and iodacetic acid (1, 3). Before digestion with trypsin an aliquot of the 3 H-labeled arylsulfatase was analyzed by SDS-PAGE using high Tris gels containing 10% acrylamide, 0.13% bis-acrylamide (14), followed by Coomassie Blue staining and phosphoimaging. Fractions from RP-HPLC were analyzed by liquid scintillation counting, mass spectrometry, amino acid sequencing, and radiosequencing (1, 3). Proteolytic In-gel Digestion-180 g of arylsulfatase were lyophilized and subjected to SDS-PAGE (see above). After staining with Coomassie Blue (0.195% Coomassie R-250, 0.005% Coomassie G-250, 0.5% acetic acid, 20% methanol) for 20 min and destaining with 30% methanol, the gel slice containing arylsulfatase was excised and cut into small pieces. The gel pieces were washed twice with 0.5 ml of 50% acetonitrile, 50% 100 mM ammonium carbonate (buffer A) and dried on air. 3.6 g of trypsin in 100 l of buffer A were added. After 30 min of incubation at room temperature buffer A was added to cover the gel pieces. Following incubation for 16 h at 37°C, peptides were extracted with 0.2 ml of 50% acetonitrile, 50% trifluoroacetic acid, 0.2 ml of 50% acetonitrile/H 2 O, and 0.2 ml of 75% acetonitrile/H 2 O, each at 60°C for 30 min. The supernatants were pooled, concentrated to 20 l, filled up to 100 l with 0.1% trifluoroacetic acid/H 2 O and subjected to RP-HPLC (see above). Fractions containing nonmodified and modified peptide 2, as identified by mass spectrometry (MALDI III, Shimadzu), were pooled, concentrated to 50% volume, and applied to a peak C2/C18 column (Pharmacia) equilibrated with 0.1% trifluoroacetic acid/H 2 O. The peptides
doi:10.1074/jbc.273.9.4835 pmid:9478923 fatcat:c425ii3ftvgx3bmbt7t5o6swhe