Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. Here we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed a compact, globular shape with two α/β-sandwich domains. The core fold of GlpX is structurally similar to that of Li+-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr90 is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases. Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. Here we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed a compact, globular shape with two α/β-sandwich domains. The core fold of GlpX is structurally similar to that of Li+-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr90 is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases. Fructose-1,6-bisphosphatase (FBPase, 2The abbreviations used are: FBPase, fructose-1,6-bisphosphatase; FBP, fructose 1,6-bisphosphate; IMPase, inositol monophosphatase; PAPase, 3′-phosphoadenosine 5′-phosphatase; PIPase, enzyme acting on both inositol-1,4-bisphosphate and 3′-phosphoadenosine 5′-phosphate; CHES, 2-(cyclohexylamino)ethanesulfonic acid; PDB, Protein Data Bank. EC 3.1.3.11), a key enzyme of gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. It is the reverse of the reaction catalyzed by phosphofructokinase in glycolysis, and the product, fructose 6-phosphate, is an important precursor in various biosynthetic pathways (1Horecker B.L. Melloni E. Pontremoli S. Adv. Enzymol. Relat. Areas Mol. Biol.. 1975; 42: 193-226Google Scholar). In all organisms, gluconeogenesis is an important metabolic pathway that allows the cells to synthesize glucose from noncarbohydrate precursors, such as organic acids, amino acids, and glycerol. FBPases are members of the large superfamily of lithium-sensitive phosphatases, which includes three families of inositol phosphatases and FBPases (the phosphoesterase clan CL0171, 3167 sequences, Pfam data base). These enzymes show metal-dependent and lithium-sensitive phosphomonoesterase activity and include inositol polyphosphate 1-phosphatases, inositol monophosphatases (IMPases), 3′-phosphoadenosine 5′-phosphatases (PAPases), and enzymes acting on both inositol 1,4-bisphosphate and PAP (PIPases) (2York J.D. Ponder J.W. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A.. 1995; 92: 5149-5153Google Scholar). They possess a common structural core with the active site lying between α+β and α/β domains (3Patel S. Martinez-Ripoll M. Blundell T.L. Albert A. J. Mol. Biol.. 2002; 320: 1087-1094Google Scholar). Li+-sensitive phosphatases are putative targets for lithium therapy in the treatment of manic depressive patients (4Nahorski S.R. Ragan C.I. Challiss R.A. Trends Pharmacol. Sci.. 1991; 12: 297-303Google Scholar), whereas FBPases are targets for the development of drugs for the treatment of noninsulin-dependent diabetes (5Wright S.W. Carlo A.A. Carty M.D. Danley D.E. Hageman D.L. Karam G.A. Levy C.B. Mansour M.N. Mathiowetz A.M. McClure L.D. Nestor N.B. McPherson R.K. Pandit J. Pustilnik L.R. Schulte G.K. Soeller W.C. Treadway J.L. Wang I.K. Bauer P.H. J. Med. Chem.. 2002; 45: 3865-3877Google Scholar, 6Sassetti C.M. Rubin E.J. Proc. Natl. Acad. Sci. U. S. A.. 2003; 100: 12989-12994Google Scholar). In addition, FBPase is required for virulence in Mycobacterium tuberculosis and Leishmania major and plays an important role in the production of lysine and glutamate by Corynebacterium glutamicum (7Naderer T. Ellis M.A. Sernee M.F. De Souza D.P. Curtis J. Handman E. McConville M.J. Proc. Natl. Acad. Sci. U. S. A.. 2006; 103: 5502-5507Google Scholar, 8Becker J. Klopprogge C. Zelder O. Heinzle E. Wittmann C. Appl. Environ. Microbiol.. 2005; 71: 8587-8596Google Scholar). Presently, five different classes of FBPases have been proposed based on their amino acid sequences (FBPases I to V) (9Donahue J.L. Bownas J.L. Niehaus W.G. Larson T.J. J. Bacteriol.. 2000; 182: 5624-5627Google Scholar, 10Nishimasu H. Fushinobu S. Shoun H. Wakagi T. Structure (Lond.).. 2004; 12: 949-959Google Scholar, 11Hines J.K. Fromm H.J. Honzatko R.B. J. Biol. Chem.. 2006; 281: 18386-18393Google Scholar). Eukaryotes contain only the FBPase I-type enzyme, but all five types exist in various prokaryotes. Types I, II, and III are primarily in bacteria, type IV in archaea (a bifunctional FBPase/inositol monophosphatase), and type V in thermophilic prokaryotes from both domains (11Hines J.K. Fromm H.J. Honzatko R.B. J. Biol. Chem.. 2006; 281: 18386-18393Google Scholar). Many organisms have more than one FBPase, mostly the combination of types I + II or II + III, but no bacterial genome has a combination of types I and III FBPases (9Donahue J.L. Bownas J.L. Niehaus W.G. Larson T.J. J. Bacteriol.. 2000; 182: 5624-5627Google Scholar). The type I FBPase is the most widely distributed among living organisms and is the primary FBPase in Escherichia coli, most bacteria, a few archaea, and all eukaryotes (9Donahue J.L. Bownas J.L. Niehaus W.G. Larson T.J. J. Bacteriol.. 2000; 182: 5624-5627Google Scholar, 11Hines J.K. Fromm H.J. Honzatko R.B. J. Biol. Chem.. 2006; 281: 18386-18393Google Scholar, 12Fraenkel D.G. Horecker B.L. J. Bacteriol.. 1965; 90: 837-842Google Scholar, 13Fraenkel D.G. Pontremoli S. Horecker B.L. Arch. Biochem. Biophys.. 1966; 114: 4-12Google Scholar, 14Sedivy J.M. Daldal F. Fraenkel D.G. J. Bacteriol.. 1984; 158: 1048-1053Google Scholar, 15Sato T. Imanaka H. Rashid N. Fukui T. Atomi H. Imanaka T. J. Bacteriol.. 2004; 186: 5799-5807Google Scholar). The type II FBPases are represented by the E. coli GlpX and FBPase F-I from Synechocystis PCC6803 (9Donahue J.L. Bownas J.L. Niehaus W.G. Larson T.J. J. Bacteriol.. 2000; 182: 5624-5627Google Scholar, 16Tamoi M. Murakami A. Takeda T. Shigeoka S. Biochim. Biophys. Acta.. 1998; 1383: 232-244Google Scholar); type III is represented by the Bacillus subtilis FBPase (17Fujita Y. Yoshida K. Miwa Y. Yanai N. Nagakawa E. Kasahara Y. J. Bacteriol.. 1998; 180: 4309-4313Google Scholar); type IV is represented by the dual activity FBPases/inosine monophosphatases FbpA from Pyrococcus furiosus (18Verhees C.H. Akerboom J. Schiltz E. de Vos W.M. van der Oost J. J. Bacteriol.. 2002; 184: 3401-3405Google Scholar), MJ0109 from Methanococcus jannaschii (19Stec B. Yang H. Johnson K.A. Chen L. Roberts M.F. Nat. Struct. Biol.. 2000; 7: 1046-1050Google Scholar), and AF2372 from Archaeoglobus fulgidus (20Stieglitz K.A. Johnson K.A. Yang H. Roberts M.F. Seaton B.A. Head J.F. Stec B. J. Biol. Chem.. 2002; 277: 22863-22874Google Scholar); and type V is represented by the FBPases TK2164 from Pyrococcus (Thermococcus) kodakaraensis and ST0318 from Sulfolobus tokodai (10Nishimasu H. Fushinobu S. Shoun H. Wakagi T. Structure (Lond.).. 2004; 12: 949-959Google Scholar, 21Rashid N. Imanaka H. Kanai T. Fukui T. Atomi H. Imanaka T. J. Biol. Chem.. 2002; 277: 30649-30655Google Scholar). Three-dimensional structures of the type I (from pig kidney, spinach chloroplasts, and E. coli), type IV (MJ0109 and AF2372), and type V (ST0318) FBPases have been solved (10Nishimasu H. Fushinobu S. Shoun H. Wakagi T. Structure (Lond.).. 2004; 12: 949-959Google Scholar, 11Hines J.K. Fromm H.J. Honzatko R.B. J. Biol. Chem.. 2006; 281: 18386-18393Google Scholar, 19Stec B. Yang H. Johnson K.A. Chen L. Roberts M.F. Nat. Struct. Biol.. 2000; 7: 1046-1050Google Scholar, 20Stieglitz K.A. Johnson K.A. Yang H. Roberts M.F. Seaton B.A. Head J.F. Stec B. J. Biol. Chem.. 2002; 277: 22863-22874Google Scholar, 22Xue Y. Huang S. Liang J.Y. Zhang Y. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A.. 1994; 91: 12482-12486Google Scholar, 23Villeret V. Huang S. Zhang Y. Xue Y. Lipscomb W.N. Biochemistry.. 1995; 34: 4299-4306Google Scholar). FBPases I and IV and inositol monophosphatases share a common sugar phosphatase fold organized in five layered interleaved α-helices and β-sheets (α-β-α-β-α) (2York J.D. Ponder J.W. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A.. 1995; 92: 5149-5153Google Scholar, 19Stec B. Yang H. Johnson K.A. Chen L. Roberts M.F. Nat. Struct. Biol.. 2000; 7: 1046-1050Google Scholar, 24Choe J.Y. Fromm H.J. Honzatko R.B. Biochemistry.. 2000; 39: 8565-8574Google Scholar). ST0318 (an FBPase V enzyme) is composed of one domain with a completely different four-layer α-β-β-α fold (10Nishimasu H. Fushinobu S. Shoun H. Wakagi T. Structure (Lond.).. 2004; 12: 949-959Google Scholar). The FBPases from these three classes (I, IV, and V) require for activity or and their structures have revealed the of three or metal ions in the active site. E. coli has five Li+-sensitive phosphatases as (a (an (a FBPase I GlpX (a FBPase and (an the Pfam data base). is a 3′-phosphoadenosine in the pathway S. K. T. D.E. J. Bacteriol.. Scholar, C. S. K. Appl. Environ. Microbiol.. Scholar), whereas is an inositol that is as a of in E. coli A. N. T. K. J. Bacteriol.. 1995; Scholar, Y. K.A. M. D.L. Stec B. Roberts M.F. J. Biol. Chem.. Scholar). is required for on and the FBPase D.G. Horecker B.L. J. Bacteriol.. 1965; 90: 837-842Google Scholar). enzyme has been both and structurally and to inhibited by of (11Hines J.K. Fromm H.J. Honzatko R.B. J. Biol. Chem.. 2006; 281: 18386-18393Google Scholar, J. V. Arch. Biochem. Biophys.. Scholar, N. M.J. Biochim. Biophys. Acta.. 2002; Scholar). The E. coli a class II enzyme FBPase, has been to possess a FBPase activity (9Donahue J.L. Bownas J.L. Niehaus W.G. Larson T.J. J. Bacteriol.. 2000; 182: 5624-5627Google Scholar). The of from a the the on or (9Donahue J.L. Bownas J.L. Niehaus W.G. Larson T.J. J. Bacteriol.. 2000; 182: 5624-5627Google Scholar). In we present the first structure of a class II FBPase, the E. coli in a free state and in the complex with + or We have demonstrated that the fold of GlpX is similar to that of the lithium-sensitive We have identified the GlpX residues important for activity and proposed a catalytic mechanism. We have that is a third FBPase in E. coli, which has catalytic and is more than GlpX to the by lithium or and of GlpX and GlpX of the E. coli and the the for and and into the in which the site the and a from the site T. S. A. S. C.H. S. A. A. A. Structure (Lond.).. Scholar). The into the E. coli The from the M. T. M. T. E. H. H. 2005; 12: Scholar). GlpX and in E. coli and on with a and as T. S. A. S. C.H. S. A. A. A. Structure (Lond.).. Scholar). a as in and of the state of GlpX and with a with and The with and activity fructose 1,6-bisphosphate or other and fructose all from the A.A. as M. E. M. H. A. J. Biol. Chem.. 2004; Scholar). The reaction or fructose and of of the reaction by the of or A.A. Scholar), and the production of of the and the of determined by from the for The of phosphate on FBPase activity of GlpX and the with and (9Donahue J.L. Bownas J.L. Niehaus W.G. Larson T.J. J. Bacteriol.. 2000; 182: 5624-5627Google Scholar). of mutagenesis the mutagenesis to the The amino to all to GlpX into the used as a for The of and of The with and of reaction used to from the the and all by the into the E. coli and the GlpX and in the as the Protein and Data of GlpX by the with of with an of the as F. S. A. T. E. S. A. C.H. M. M. A.M. 2003; Scholar). The of the GlpX in the of and The of the GlpX complex with fructose 1,6-bisphosphate by in the of and fructose whereas the complex of the GlpX with phosphate in and the with the with as a and in Structure first of GlpX solved data on a crystal on the of the the K. Zhang N. J. M.J. L. J. L. M.A. E. A. J. 2006; the and of the as as a Data and with Scholar). The found with J. Biol. Scholar), by by and to an Biol. 2000; Scholar, Biol. 2003; Scholar). data for of in GlpX phosphate and GlpX on a with and on a The data and Scholar), and the structures solved molecular replacement the from the first GlpX structure replacement A. A. J. Appl. for the and complex and Biol. 2005; for the with several of K. Biol. 2004; and a A.A. E.J. Biol. the Biol. 1994; Scholar), with of the as an data and in are for the type + + phosphate to to to of of in a Protein Data and structure have been with and + and + phosphate E. coli the II the E. coli the an with (a and (a is one of five of the in the of E. coli on (9Donahue J.L. Bownas J.L. Niehaus W.G. Larson T.J. J. Bacteriol.. 2000; 182: 5624-5627Google Scholar). of the E. coli genome the GlpX amino as a identified the amino which with The is of a large and which a and a a and a putative The role of in E. coli is GlpX is to in gluconeogenesis on (9Donahue J.L. Bownas J.L. Niehaus W.G. Larson T.J. J. Bacteriol.. 2000; 182: 5624-5627Google Scholar). of the with the E. coli GlpX as a revealed that the type II FBPases are conserved enzymes with sequences or E. coli, two a type II FBPase found in of and of GlpX and with the type II FBPases from M. tuberculosis F. Larson T.J. 2004; Scholar), C. glutamicum S. H. Arch. Microbiol.. 2003; 180: Scholar), and Synechocystis M. Murakami A. Takeda T. Shigeoka S. Biochim. Biophys. Acta.. 1998; 1383: 232-244Google identified conserved residues These sequences show the of of conserved residues and The is a of the Li+-sensitive phosphatase that has been by and to and in catalysis (2York J.D. Ponder J.W. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A.. 1995; 92: 5149-5153Google Scholar, H. C.M. Seaton B.A. F. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A.. Scholar). has been that the of in is to members (2York J.D. Ponder J.W. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A.. 1995; 92: 5149-5153Google Scholar). the FBPase II sequences revealed no of the Li+-sensitive phosphatase with the conserved of or (2York J.D. Ponder J.W. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A.. 1995; 92: 5149-5153Google that the class II FBPases are from the of Li+-sensitive of GlpX and GlpX and a fructose-1,6-bisphosphatase a activity glucose 1,6-bisphosphate and no activity fructose or fructose fructose 1,6-bisphosphate as both activity and required a metal for with a of and only activity of both is in to FBPases from other which by or N. Imanaka H. Kanai T. Fukui T. Atomi H. Imanaka T. J. Biol. Chem.. 2002; 277: 30649-30655Google Scholar, F. J. T. 90: Scholar). GlpX a to than GlpX activity and to fructose 1,6-bisphosphate in three catalytic both with with a in substrate with other E. coli phosphatases, both GlpX and substrate and catalytic than of but they more in hydrolysis than and which to the superfamily of the E. coli YggF, and are from are from are from data from are from data from Data are from N. M.J. Biochim. Biophys. Acta.. 2002; Data are from data from E. M. L. H. C.H. A.M. J. Biol. Chem.. 2006; 281: in a The activity of by the of whereas activity by other and no on the activity of both and GlpX in the in J.K. Fromm H.J. Honzatko R.B. J. Biol. Chem.. Scholar). These are similar to that for the class FBPase from M. tuberculosis F. Larson T.J. 2004; Scholar). activity of GlpX but a on of the Li+-sensitive phosphatases are strongly inhibited by of (2York J.D. Ponder J.W. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A.. 1995; 92: 5149-5153Google Scholar, S. Martinez-Ripoll M. Blundell T.L. Albert A. J. Mol. Biol.. 2002; 320: 1087-1094Google Scholar, S. H. Arch. Microbiol.. 2003; 180: Scholar, A. L. S. Martinez-Ripoll M. Blundell T.L. J. Mol. Biol.. 2000; Scholar, Ragan C.I. Biochem. Scholar, Majerus P.W. J. Biol. Chem.. Scholar). We found that both GlpX and to with an of for GlpX and for the dual specificity enzymes AF2372 and MJ0109 (20Stieglitz K.A. Johnson K.A. Yang H. Roberts M.F. Seaton B.A. Head J.F. Stec B. J. Biol. Chem.. 2002; 277: 22863-22874Google Scholar, K.A. Chen L. Yang H. Roberts M.F. Stec B. Biochemistry.. Scholar), the E. coli class II FBPases to the of more Li+-sensitive In addition, FBPase activity of both GlpX and inhibited by of phosphate and in E. coli both type II FBPases are from FBPase I, which is to by and for activity J. V. Arch. Biochem. Biophys.. Scholar). Structure of the crystal structure of E. coli GlpX solved to in on a crystal the first structure as several to contain and the structure of solved in and structure more solved a similar The structure demonstrated that two of GlpX an The shape of the GlpX is a for the molecular of GlpX in of of the state of revealed that is to exist as a in of GlpX are by between the a The is by these and by a between and from the first of two The GlpX has a compact, globular shape with two domains as a The domain (the domain on includes the sequences from the and of GlpX and is by the with which is two α-helices (a The domain (the domain on includes the sequences from the of GlpX and a with the a mostly and are by two located and and residues is present between the which accommodates conserved residues and a putative active site of GlpX and identified the as the has found that the fructose-1,6-bisphosphatase and inositol share a similar structure their sequences show Y. Liang J.Y. Lipscomb W.N. Biochem. Biophys. Scholar). enzymes have a layered structure and five of the structures of inositol monophosphatases E. coli and from and three classes of FBPases (I, E. coli II, E. coli and IV, that they share the structure In these the first has two and or three whereas the of mostly β-sheets of or The two α-helices and or five are to the whereas the three to five α-helices of the have different The of the GlpX is the of an which is to the of the first and FBPase classes I, II, and IV have a their structural that they have a common evolutionary origin and catalytic mechanism. of the GlpX conserved residues are located or to the large located between two which a putative active site the active site residues in we have 12 conserved and residues of GlpX to 12 a FBPase activity that they are in substrate and or catalysis The both activity and substrate The GlpX structure that the of is in the with the conserved The of the conserved in the and FBPase activity the of substrate whereas activity to three the of the and The GlpX structure that is located on the of the putative active site and with substrate and the of the the catalytic of the of substrate and the reaction the and to that they are in substrate The of is to that of but is in substrate the demonstrated the activity and substrate Structure of the GlpX with or GlpX in the of FBP, and and structure revealed the of three metal and in the active site and In the active site of GlpX is than that of the dual activity inositol AF2372), with the substrate specificity of The molecule is bound in a form and by the with and of several conserved residues The 6-phosphate of with the of and of the and residues are present in the structures of other FBPases and in but are in inositol that these residues the substrate specificity of FBPases to The is by the of and the of the by the and the of and the by the of and the of The of the of (a are to the of and the of and Thr90 The crystal structure of the complex revealed the of three metal in the active which identified as or both present in the ions with identified as The is by the 6 and is located from the The other two and are located to other and to the of the substrate for and for is bound to the of and and whereas is to the of and the residues of GlpX are similar to that of S. L. Blundell T.L. J. Mol. Biol.. 2002; and (3Patel S. Martinez-Ripoll M. Blundell T.L. Albert A. J. Mol. Biol.. 2002; 320: 1087-1094Google that GlpX have a similar catalytic mechanism. on the E. coli GlpX demonstrated that phosphate FBPase activity with a of (9Donahue J.L. Bownas J.L. Niehaus W.G. Larson T.J. J. Bacteriol.. 2000; 182: 5624-5627Google Scholar). The structure of the complex revealed the of phosphate bound to the enzyme active site The phosphate molecule the of the phosphate 6 of fructose-1,6-bisphosphatase and is by the with the of and and and and the of to the GlpX active site with substrate the of for the of the II catalytic of the lithium-sensitive phosphatases has been the of for several (3Patel S. Martinez-Ripoll M. Blundell T.L. Albert A. J. Mol. Biol.. 2002; 320: 1087-1094Google Scholar, 10Nishimasu H. Fushinobu S. Shoun H. Wakagi T. Structure (Lond.).. 2004; 12: 949-959Google Scholar, 20Stieglitz K.A. Johnson K.A. Yang H. Roberts M.F. Seaton B.A. Head J.F. Stec B. J. Biol. Chem.. 2002; 277: 22863-22874Google Scholar, K.A. Chen L. Yang H. Roberts M.F. Stec B. Biochemistry.. Scholar, S. L. Blundell T.L. J. Mol. Biol.. 2002; Scholar). The phosphatase reaction of Li+-sensitive phosphatases has an for metal ions and has been suggested to the of a molecule and on the J.K. M.A. J. Scholar, Ragan C.I. S.R. Proc. Natl. Acad. Sci. U. S. A.. 1994; 91: Scholar). It is metal ions are required for the of the and two catalytic have been The on the structure of the that one of the two is for coordinating the and the on the whereas the metal ion the (3Patel S. Martinez-Ripoll M. Blundell T.L. Albert A. J. Mol. Biol.. 2002; 320: 1087-1094Google Scholar, Proc. Natl. Acad. Sci. U. S. A.. Scholar, L. Biochemistry.. 1994; Scholar). The with the and or residues the with three proposed for the catalytic of Li+-sensitive phosphatases (20Stieglitz K.A. Johnson K.A. Yang H. Roberts M.F. Seaton B.A. Head J.F. Stec B. J. Biol. Chem.. 2002; 277: 22863-22874Google Scholar, K.A. Chen L. Yang H. Roberts M.F. Stec B. Biochemistry.. Scholar, S. L. Blundell T.L. J. Mol. Biol.. 2002; Scholar). In metal ions and are to in substrate and of the whereas metal ions and are to for the of the In the are three bound in the active but the to phosphate to in catalysis that GlpX and other class II FBPases are to a for the hydrolysis of similar to that for the (3Patel S. Martinez-Ripoll M. Blundell T.L. Albert A. J. Mol. Biol.. 2002; 320: 1087-1094Google Scholar). In the proposed GlpX the ion is to in the as as in phosphate and the The molecule by the and by the of the conserved Thr90 in which is by a with the conserved in are two in the of the and which are to the and Thr90 and as a in the hydrolysis of the The of the of the conserved and and in to for the reaction of Li+-sensitive phosphatases (3Patel S. Martinez-Ripoll M. Blundell T.L. Albert A. J. Mol. Biol.. 2002; 320: 1087-1094Google Scholar). the of the and in the of the The of Thr90 in the GlpX catalysis is by the of The ion the phosphate of a by the with the the crystal structure of GlpX has demonstrated that core fold is to that of Li+-sensitive phosphatases and important structural for the substrate and catalytic of class II We all members of the for in for their in these with

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