SULFATASE ACTIVITY OF E. COLI ALKALINE PHOSPHATASE DEMONSTRATES A FUNCTIONAL LINK TO ARYLSULFATASES, AN EVOLUTIONARILY RELATED ENZYME FAMILY

Recent crystallographic analyses have revealed structural similarity between alkaline phosphatases and arylsulfatases, strongly suggesting that these distinct classes of enzymes are evolutionarily related.1,2 Superposition of 169 CR atoms from the central â-sheet of arylsulfatase B and E. coli alkaline phosphatase (AP) results in the alignment of the nucleophilic residues, the phosphoryl/sulfuryl moieties at the active site, and active site divalent metal ions (Figure 1).1 There is no significant overall sequence similarity, suggesting that these enzymes are only distantly related.1,2 Nevertheless, a recent sequence analysis that focused on conserved metal ligands suggested that AP’s and arylsulfatases are related within a larger superfamily.3 It was previously reported that AP has no sulfatase activity.4 However, even a low level of activity could facilitate divergence of an enzyme encoded by a duplicated gene by providing a selective advantage, which would then allow optimization via natural selection. We therefore investigated whether AP exhibits sulfatase activity. The results establish a functional relationship between these two evolutionarily related enzymes. Such relationships can yield insight into past evolutionary pathways and present evolutionary potential. A highly purified preparation of E. coli AP was found to catalyze hydrolysis of p-nitrophenyl sulfate (PNPS). Despite the activated leaving group, kcat/Km is low (0.01 M-1 s-1). Nevertheless, this represents considerable catalysis, corresponding to a rate enhancement of ∼109 relative to the nonenzymatic attack by water on PNPS.5 Several independent lines of evidence strongly suggest that the AP active site is responsible for this sulfatase activity. AP was overexpressed in E. coli strain SM547 (phoA-) from plasmid pEK48 and purified from the periplasm by osmotic shock, ammonium sulfate fractionation, heat treatment, and subsequent anion exchange and gel filtration chromatography.6 The sulfatase activity toward PNPS co-purifies with the phosphatase activity toward p-nitrophenyl phosphate (PNPP) upon anion exchange and gel filtration chromatography (Figure 2 and data not shown). No sulfatase activity was detectable in the periplasmic fraction from osmotic shock of E. coli lacking the AP-encoding plasmid. Inorganic phosphate, a strong competitive inhibitor of AP, inhibits the phosphatase and sulfatase activities with the same inhibition constant (Figure 3), suggesting that both reactions are catalyzed by the same active site. Finally, kcat/Km for the phosphatase and sulfatase activities followed the same pH dependence over the experimentally accessible range of pH 7-10.6 To characterize the sulfatase activity and compare this activity to the phosphatase activity, several previously described mutants of AP were overexpressed, purified, and assayed for sulfatase activity. The mutant S102C changes the identity of the nucleophile and greatly reduces the level of phosphatase activity;7,8 R166S removes an active site residue that interacts with the negatively charged oxygens of phosphomonoester substrates;9 and D327A removes a bidentate ligand of ZnI, disrupting the metal ion binding site.10 Each of these mutations substantially reduces both the phosphatase and sulfatase activity, with effects of 102-106-fold (1) Bond, C. S.; Clements, P. R.; Ashby, S. J.; Collyer, C. A.; Harrop, S. J.; Hopwood, J. J.; Guss, J. M. Structure 1997, 5, 277-289. (2) Lukatela, G.; Krauss, N.; Theis, K.; Selmer, T.; Gieselmann, V.; von Figura, K.; Saenger, W. Biochemistry 1998, 37, 3654-3664. (3) Galperin, M. Y.; Bairoch, A.; Koonin, E. V. Protein Sci. 1998, 7, 18291835. (4) Yount, R. G.; Simchuck, S.; Yu, I.; Kottke, M. Arch. Biochem. Biophys. 1966, 113, 288-295. (5) Rate enhancement ) (kcat/Km)/kw ) 0.01 M-1 s-1/ 9 × 10-12 M-1 s-1 ) 109; kw is the second-order rate constant for attack of water on PNPS (kw ) kobs/55 M). The nonenzymatic rate constant at 25 °C of kobs ) 5.1 × 10-10 s-1 was corrected from the value at 35 °C by using the reported temperature dependence. Benkovic, S. J.; Benkovic, P. A. J. Am. Chem. Soc. 1966, 88, 5504-5511. (6) See Supporting Information. (7) Stec, B.; Hehir, M. J.; Brennan, C.; Nolte, M.; Kantrowitz, E. R. J. Mol. Biol. 1998, 277, 647-662. (8) (a) Jones, S. R.; Kindman, L. A.; Knowles, J. R. Nature 1978, 275, 564-565. (b) Ghosh, S. S.; Block, S. C.; Rokita, S. E.; Kaiser, E. T. Science 1986, 231, 145-148. (c) Butler-Ransohoff, J. E.; Kendall, D. A.; Freeman, S.; Knowles, J. R.; Kaiser, E. T. Biochemistry 1988, 27, 4777-4780. (d) Han, R.; Coleman, J. E. Biochemistry 1995, 34, 4238-4245. (9) Chaidaroglou, A.; Brezinski, D. J.; Middleton, S. A.; Kantrowitz, E. R. Biochemistry 1988, 27, 8338-8343. (10) Xu, X.; Kantrowitz, E. R. J. Biol. Chem. 1992, 267, 16244-16251. Figure 1. Local superposition of homologous active site residues for AP and arylsulfatase B (AS), adapted from refs 1 and 2. Differences between the CR positions of D53, X91 (X ) formylglycine), and D300 in AS and D51, S102, and D369 in H331Q AP,21 respectively, were minimized by using Insight. For clarity, only selected active site residues are shown for AP (dark gray, stick representation) and AS (light gray, ball-and-stick representation). The position of the phosphorylated serine of AP (AP: S102-PO4) coincides with that of the sulfurylated formylglycine of AS (AS: X91-SO4), ZnII (AP) coincides with Ca (AS), ZnI (AP) with K318Ne (AS), Mg (AP) with K145Ne (AS), and the metal ligand H370 (AP) with the metal ligand N301 (AS). Coordinates were obtained from the PDB (1fsu and 1hjk for AS and H331Q AP, respectively).