Genome-wide Analysis of Substrate Specificities of the Escherichia coli Haloacid Dehalogenase-like Phosphatase Family
2006; Elsevier BV; Volume: 281; Issue: 47 Linguagem: Inglês
10.1074/jbc.m605449200
ISSN1083-351X
AutoresEkaterina Kuznetsova, Michael Proudfoot, Claudio F. González, Greg Brown, Marina V. Omelchenko, Ivan Borozan, Liran Carmel, Yuri I. Wolf, Hirotada Mori, Alexei Savchenko, C.H. Arrowsmith, Eugene V. Koonin, A.M. Edwards, Alexander F. Yakunin,
Tópico(s)Enzyme Production and Characterization
ResumoHaloacid dehalogenase (HAD)-like hydrolases are a vast superfamily of largely uncharacterized enzymes, with a few members shown to possess phosphatase, β-phosphoglucomutase, phosphonatase, and dehalogenase activities. Using a representative set of 80 phosphorylated substrates, we characterized the substrate specificities of 23 soluble HADs encoded in the Escherichia coli genome. We identified small molecule phosphatase activity in 21 HADs and β-phosphoglucomutase activity in one protein. The E. coli HAD phosphatases show high catalytic efficiency and affinity to a wide range of phosphorylated metabolites that are intermediates of various metabolic reactions. Rather than following the classical “one enzyme-one substrate” model, most of the E. coli HADs show remarkably broad and overlapping substrate spectra. At least 12 reactions catalyzed by HADs currently have no EC numbers assigned in Enzyme Nomenclature. Surprisingly, most HADs hydrolyzed small phosphodonors (acetyl phosphate, carbamoyl phosphate, and phosphoramidate), which also serve as substrates for autophosphorylation of the receiver domains of the two-component signal transduction systems. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, YniC. Many of the secondary activities of HADs might have no immediate physiological function but could comprise a reservoir for evolution of novel phosphatases. Haloacid dehalogenase (HAD)-like hydrolases are a vast superfamily of largely uncharacterized enzymes, with a few members shown to possess phosphatase, β-phosphoglucomutase, phosphonatase, and dehalogenase activities. Using a representative set of 80 phosphorylated substrates, we characterized the substrate specificities of 23 soluble HADs encoded in the Escherichia coli genome. We identified small molecule phosphatase activity in 21 HADs and β-phosphoglucomutase activity in one protein. The E. coli HAD phosphatases show high catalytic efficiency and affinity to a wide range of phosphorylated metabolites that are intermediates of various metabolic reactions. Rather than following the classical “one enzyme-one substrate” model, most of the E. coli HADs show remarkably broad and overlapping substrate spectra. At least 12 reactions catalyzed by HADs currently have no EC numbers assigned in Enzyme Nomenclature. Surprisingly, most HADs hydrolyzed small phosphodonors (acetyl phosphate, carbamoyl phosphate, and phosphoramidate), which also serve as substrates for autophosphorylation of the receiver domains of the two-component signal transduction systems. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, YniC. Many of the secondary activities of HADs might have no immediate physiological function but could comprise a reservoir for evolution of novel phosphatases. Most enzymes form families of paralogs whose members are related by sequence and catalyze similar reactions but have evolved specific biological functions. Comprehensive determination of the substrate specificities and selectivities of all metabolic enzymes in an organism is an essential step toward understanding the relationship between the proteome and the metabolome. By the most recent estimate, Escherichia coli possesses at least 1186 metabolic enzymes and 1005 metabolites (1Keseler I.M. Collado-Vides J. Gama-Castro S. Ingraham J. Paley S. Paulsen I.T. Peralta-Gil M. Karp P.D. Nucleic Acids Res. 2005; 33: D334-D337Crossref PubMed Scopus (562) Google Scholar). The most common functional group in the metabolome is phosphate; 35-40% of the metabolites contain a phosphate group (2Nobeli I. Ponstingl H. Krissinel E.B. Thornton J.M. J. Mol. Biol. 2003; 334: 697-719Crossref PubMed Scopus (97) Google Scholar). The pool of phosphorylated metabolites is controlled by the activity of diverse kinases and phosphatases, of which there are hundreds in the E. coli genome.Haloacid dehalogenase (HAD) 4The abbreviations used are: HAD, haloacid dehalogenase-like hydrolase; CHES, 2-(cyclohexylamino)ethanesulfonic acid; PLP, pyridoxal 5′-phosphate; PEP, phosphoenolpyruvate; MOPS, 4-morpholinepropanesulfonic acid; pNPP, p-nitrophenyl phosphate. 4The abbreviations used are: HAD, haloacid dehalogenase-like hydrolase; CHES, 2-(cyclohexylamino)ethanesulfonic acid; PLP, pyridoxal 5′-phosphate; PEP, phosphoenolpyruvate; MOPS, 4-morpholinepropanesulfonic acid; pNPP, p-nitrophenyl phosphate.-like hydrolases (3Koonin E.V. Tatusov R.L. J. Mol. Biol. 1994; 244: 125-132Crossref PubMed Scopus (266) Google Scholar) represent the largest family of predicted small molecule phosphatases encoded in the genomes of bacteria, archaea, and eukaryotes, with 6,805 proteins in data bases. The great majority of these proteins have no known biochemical or biological function. In any individual genome, the number of HAD genes can range from 10 to 20 in different bacteria to 100 in humans and 115 in Arabidopsis thaliana (InterPro data base). HADs share little overall sequence similarity (15-30% identity), but they can be unequivocally identified by the presence of three short conserved sequence motifs (3Koonin E.V. Tatusov R.L. J. Mol. Biol. 1994; 244: 125-132Crossref PubMed Scopus (266) Google Scholar) (supplemental Fig. 1). Most of the characterized HADs have phosphatase activity (CO-P bond hydrolysis), and several also catalyze dehalogenase (C-halogen bond hydrolysis), phosphonatase (C-P bond hydrolysis), and β-phosphoglucomutase (CO-P bond hydrolysis and intramolecular phosphoryl transfer) reactions (3Koonin E.V. Tatusov R.L. J. Mol. Biol. 1994; 244: 125-132Crossref PubMed Scopus (266) Google Scholar, 4Allen K.N. Dunaway-Mariano D. Trends Biochem. Sci. 2004; 29: 495-503Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The biochemically and structurally studied HADs include phosphoserine phosphatase SerB from Methanococcus jannaschii (5Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure (Camb.). 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), phosphoglycolate phosphatase from Thermoplasma acidophilum (6Kim Y. Yakunin A.F. Kuznetsova E. Xu X. Pennycooke M. Gu J. Cheung F. Proudfoot M. Arrowsmith C.H. Joachimiak A. Edwards A.M. Christendat D. J. Biol. Chem. 2004; 279: 517-526Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), phosphonacetaldehyde hydrolase from Bacillus cereus (7Morais M.C. Zhang W. Baker A.S. Zhang G. Dunaway-Mariano D. Allen K.N. Biochemistry. 2000; 39: 10385-10396Crossref PubMed Scopus (123) Google Scholar), β-phosphoglucomutase from Lactococcus lactis (8Lahiri S.D. Zhang G. Dunaway-Mariano D. Allen K.N. Biochemistry. 2002; 41: 8351-8359Crossref PubMed Scopus (100) Google Scholar), haloacid dehalogenases from Pseudomonas sp. YL (9Hisano T. Hata Y. Fujii T. Liu J.Q. Kurihara T. Esaki N. Soda K. J. Biol. Chem. 1996; 271: 20322-20330Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), and Xanthobacter autotrophicus (10Ridder I.S. Rozeboom H.J. Kalk K.H. Janssen D.B. Dijkstra B.W. J. Biol. Chem. 1997; 272: 33015-33022Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and two E. coli phosphatases, YbiV and NagD (11Roberts A. Lee S.Y. McCullagh E. Silversmith R.E. Wemmer D.E. Proteins. 2005; 58: 790-801Crossref PubMed Scopus (29) Google Scholar, 12Tremblay L.W. Dunaway-Mariano D. Allen K.N. Biochemistry. 2006; 45: 1183-1193Crossref PubMed Scopus (51) Google Scholar). However, the vast majority of HADs remains uncharacterized. Since these enzymes generally show little sequence similarity, the catalyzed reaction and, especially, the substrate specificity are hard to predict on the basis of sequence conservation and have to be determined experimentally.The E. coli genome encodes five membrane-bound and 23 soluble HAD-like hydrolases, representing ∼40% of the E. coli proteins with known or predicted small molecule phosphatase activity (1Keseler I.M. Collado-Vides J. Gama-Castro S. Ingraham J. Paley S. Paulsen I.T. Peralta-Gil M. Karp P.D. Nucleic Acids Res. 2005; 33: D334-D337Crossref PubMed Scopus (562) Google Scholar). None of the E. coli HADs is essential for bacterial growth (13Gerdes S.Y. Scholle M.D. Campbell J.W. Balazsi G. Ravasz E. Daugherty M.D. Somera A.L. Kyrpides N.C. Anderson I. Gelfand M.S. Bhattacharya A. Kapatral V. D'Souza M. Baev M.V. Grechkin Y. Mseeh F. Fonstein M.Y. Overbeek R. Barabasi A.L. Oltvai Z.N. Osterman A.L. J. Bacteriol. 2003; 185: 5673-5684Crossref PubMed Scopus (586) Google Scholar). The physiological substrates have been experimentally identified for three soluble E. coli HADs, namely phosphoglycolate phosphatase Gph, 3-deoxy-d-manno-octulosonate 8-phosphate phosphatase YrbI, and trehalose 6-phosphatase OtsB (14Pellicer T.M. Felisa Nunez M. Aguilar J. Badia J. Baldoma L. J. Bacteriol. 2003; 185: 5815-5821Crossref PubMed Scopus (42) Google Scholar, 15Wu J. Woodard R.W. J. Biol. Chem. 2003; 278: 18117-18123Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 16Strom A.R. Kaasen I. Mol. Microbiol. 1993; 8: 205-210Crossref PubMed Scopus (279) Google Scholar) Only three enzymes (Gph, YrbI, and NagD) have been characterized biochemically (12Tremblay L.W. Dunaway-Mariano D. Allen K.N. Biochemistry. 2006; 45: 1183-1193Crossref PubMed Scopus (51) Google Scholar, 14Pellicer T.M. Felisa Nunez M. Aguilar J. Badia J. Baldoma L. J. Bacteriol. 2003; 185: 5815-5821Crossref PubMed Scopus (42) Google Scholar, 15Wu J. Woodard R.W. J. Biol. Chem. 2003; 278: 18117-18123Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and three-dimensional structures have been resolved for YbiV and NagD (11Roberts A. Lee S.Y. McCullagh E. Silversmith R.E. Wemmer D.E. Proteins. 2005; 58: 790-801Crossref PubMed Scopus (29) Google Scholar, 12Tremblay L.W. Dunaway-Mariano D. Allen K.N. Biochemistry. 2006; 45: 1183-1193Crossref PubMed Scopus (51) Google Scholar).Using a set of 80 representative phosphorylated metabolites, we characterized the substrate specificities of all 23 soluble E. coli HADs and found that they comprise a family of promiscuous phosphatases with overlapping substrate profiles and are capable of hydrolyzing a wide range of phosphorylated metabolites, including carbohydrates, nucleotides, organic acids, coenzymes, and small phosphodonors. Genetic analysis demonstrated that the activity of one of the HADs, YniC, toward its preferred substrate was biologically important. We further show that all E. coli HADs have phosphatase activity against small phosphate donors (acetyl phosphate, carbamoyl phosphate, phosphoramidate), which resembles the autophosphorylation reaction catalyzed by CheY fold receiver domains of the two-component regulatory systems. Together with the previously reported structural similarity, these results indicate that the HAD superfamily and the receiver domain originate from an ancestral low specificity phosphatase. Clustering of E. coli HADs on the basis of their phosphatase activities (kcat/Km) was incongruent with the sequence-based phylogeny. Thus, many of the secondary activities of HADs might be of no immediate functional importance but comprise a reservoir for evolution of phosphatases with novel specificities.EXPERIMENTAL PROCEDURESGene Cloning and Protein Purification—For most HADs analyzed in this work, the genes were amplified by PCR from the E. coli DH5α genomic DNA and cloned into a modified pET15b (Novagen) as previously described (17Zhang R.G. Skarina T. Katz J.E. Beasley S. Khachatryan A. Vyas S. Arrowsmith C.H. Clarke S. Edwards A. Joachimiak A. Savchenko A. Structure (Camb.). 2001; 9: 1095-1106Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Several HADs (YbjI, YaeD, and YrbI) were cloned from the E. coli K12 W3110 genomic DNA into the archive vector pCA24N (Genobase data base; available on the World Wide Web at ecoli.aist-nara.ac.jp). Purification of proteins for screening and biochemical characterization was performed as previously described (18Kuznetsova E. Proudfoot M. Sanders S.A. Reinking J. Savchenko A. Arrowsmith C.H. Edwards A.M. Yakunin A.F. FEMS Microbiol. Rev. 2005; 29: 263-279Crossref PubMed Google Scholar). NagD was expressed in an insoluble form and was partially refolded from the inclusion bodies by a buffer exchange method using the Spin-Column Protein Folding Screen kit (SFC01-10) and the column PFC02 from ProFoldin Protein Folding Services according to the manufacturer's instructions (available on the World Wide Web at www.profoldin.com).Enzymatic Screens and Assays—General phosphatase screens with p-nitrophenyl phosphate (pNPP) as substrate and natural substrate phosphatase screens with 80 phosphorylated compounds from Sigma (supplemental Table 1) were performed as previously described (18Kuznetsova E. Proudfoot M. Sanders S.A. Reinking J. Savchenko A. Arrowsmith C.H. Edwards A.M. Yakunin A.F. FEMS Microbiol. Rev. 2005; 29: 263-279Crossref PubMed Google Scholar). Acetyl-phosphatase activity was assayed by measuring the acetyl-phosphate concentration using the hydroxylamine protocol of Lipmann and Tuttle (19Lipmann F. Tuttle L.C. Biochim. Biophys. Acta. 1950; 4: 301-309Crossref PubMed Scopus (33) Google Scholar). The production of fructose in enzymatic reactions was determined using an enzyme-coupled assay with fructose dehydrogenase (F5152; Sigma), essentially as previously described (20Ameyama M. Shinagawa E. Matsushita K. Adachi O. J. Bacteriol. 1981; 145: 814-823Crossref PubMed Google Scholar). This assay was adapted for 96-well microplates (200-μl reaction mixtures). Haloacid dehalogenase activity was determined spectrophotometrically by measuring the release of halide ions using the mercuric thiocyanide method (21Iwasaki I. Utsumi S. Hagino K. Ozawa T. Bull Chem. Soc. Jpn. 1956; 29: 860-864Crossref Google Scholar). The assays were adapted to the 96-well format (125 μl) and contained 50 mm CHES buffer (pH 9.0), 10 mm substrate, and 5 μg of protein. Six compounds were used as substrates (R-chloropropionic acid, S-chloropropionic acid, bromoacetic acid, 4-chlorobenzoic acid, 2,2-dichloropropionic acid, and 2-bromopropionic acid), and l-2-haloacid dehalogenase from Pseudomonas sp. YL (22Kurihara T. Liu J.Q. Nardi-Dei V. Koshikawa H. Esaki N. Soda K. J Biochem. (Tokyo). 1995; 117: 1317-1322Crossref PubMed Scopus (76) Google Scholar) was used as a positive control. Phosphonatase activity was assayed using phosphonoacetate as a substrate by measuring the release of inorganic phosphate using the Malachite Green reagent as previously described (23Baykov A.A. Evtushenko O.A. Avaeva S.M. Anal. Biochem. 1988; 171: 266-270Crossref PubMed Scopus (683) Google Scholar). β-Phosphoglucomutase activity was determined using a glucose-6-phosphate dehydrogenase-coupled assay and 1 mm β-glucose 1-phosphate as substrate (24Zhang G. Dai J. Wang L. Dunaway-Mariano D. Tremblay L.W. Allen K.N. Biochemistry. 2005; 44: 9404-9416Crossref PubMed Scopus (48) Google Scholar).For Km and Vmax determination, the phosphatase assays contained substrates at concentrations 0.005-2.0 mm. Kinetic parameters were determined by nonlinear curve fitting from the Lineweaver-Burk plot using the GraphPad Prism software (version 4.00 for Windows, GraphPad Software, San Diego, CA).Mutagenesis and Growth Experiments—The yniC gene was deleted from the chromosome of the E. coli K-12 W3110 strain using a one-step inactivation method described by Datsenko and Wanner (25Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (10979) Google Scholar). The obtained ΔYniC strain contains an unmarked gene deletion, which was verified using PCR. The YniC-overexpressing strain was prepared by subcloning (PCR) of the wild-type yniC into the KpnI/HindIII sites of the arabinose-inducible vector pBAD-33 (26Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3905) Google Scholar). The resulting plasmid (pKC1) and the empty (no insert) vector pBAD-33 (a control) were transformed into the wild-type W3110 strain. Cells were grown aerobically at 37 °C on the MOPS-buffered minimal medium containing 0.2% of succinate as a carbon source, and the expression of YniC was induced by the addition of 0.02% arabinose. The culture growth (A600) was determined after 14 h of cultivation.Bioinformatic Analyses—Hierarchical clustering of HADs (based on their substrate profiles) and substrates (based on their HAD spectra) was calculated using cosine correlations, and groups were clustered using the average method (R Foundation for Statistical Computing; available on the World Wide Web at www.R-project.org). For hierarchical clustering of proteins across HAD substrates, we consider each protein as a point in the m-dimensional space, where m designates the total number of HAD substrates. Each protein can then be represented as a vector A = (s1,... sm) of length |A| and unit (normalized) vector A′(s1′ = s1/|A|,.. .sm′ = sm/|A|). We use the cosine angle between normalized vectors as a similarity measure between proteins across substrates. The protein profiles across substrates are clustered into groups using the average linkage method (R Foundation for Statistical Computing; available on the World Wide Web at www.R-project.org). For hierarchical clustering of substrates across proteins, we consider each protein as a point in the n-dimensional space, where n designates the total number of proteins. Each substrate can then be represented as a vector B = (p1,... pn) of length |B| and unit (normalized) vector B′(p1′ = p1/|A|,... pn′ = pn/|B|). We use the cosine angle between normalized vectors B′ as a similarity measure between substrates across proteins. The substrate profiles across proteins are clustered into groups using the average linkage method (R Foundation for Statistical Computing; available on the World Wide Web at www.R-project.org).The distance between the catalytic efficiency (kcat/Km) profiles of 18 enzymes acting on 26 different substrates (see Table 2) was computed using the square distance approach (see supplemental materials and “Experimental Procedures”). A neighbor-joining tree was constructed from the distance matrix using the NEIGHBOR program of the PHYLIP package (28Felsenstein J. Methods Enzymol. 1996; 266: 418-427Crossref PubMed Google Scholar). Multiple alignments of amino acid sequences were constructed using the MUSCLE program (29Edgar R.C. Nucleic Acids Res. 2004; 32: 1792-1797Crossref PubMed Scopus (29371) Google Scholar) and optimized manually to ensure the correct alignment of known sequence motifs of the HAD superfamily (3Koonin E.V. Tatusov R.L. J. Mol. Biol. 1994; 244: 125-132Crossref PubMed Scopus (266) Google Scholar). Sequence-based phylogenetic trees were constructed using the following methods: (i) neighbor-joining method, as implemented in the NEIGHBOR program of the PHYLIP package (28Felsenstein J. Methods Enzymol. 1996; 266: 418-427Crossref PubMed Google Scholar), (ii) least squares method as implemented in the FITCH program of the PHYLIP package (28Felsenstein J. Methods Enzymol. 1996; 266: 418-427Crossref PubMed Google Scholar), (iii) local maximum likelihood optimization of the least squares tree using the ProtML program of the MOLPHY package (30Adachi J. Hasegawa M. Comput. Sci. Monogr. 1992; 27: 1-77Google Scholar), (iv) quartet puzzling as implemented in the TREE-PUZZLE program (31Schmidt H.A. Strimmer K. Vingron M. von Haeseler A. Bioinformatics. 2002; 18: 502-504Crossref PubMed Scopus (2158) Google Scholar), and (v) Markov chain Monte Carlo Bayesian estimation using the MrBayes program (32Huelsenbeck J.P. Ronquist F. Bioinformatics. 2001; 17: 754-755Crossref PubMed Scopus (18920) Google Scholar, 33Ronquist F. Huelsenbeck J.P. Bioinformatics. 2003; 19: 1572-1574Crossref PubMed Scopus (24708) Google Scholar).TABLE 2Kinetic parameters of the E. coli HADs with natural substratesProtein and variable substrateMetalaThe saturating metal concentrations used in the assays were 0.63–5.0 mm Mg2+, 0.1–1.25 mm Mn2+, 0.3–0.75 mm Co2+, or 1 mm Zn2+KmKnown intracellular concentrations of the variable substratesbExperimentally determined intracellular (in vivo) concentrations of the variable substrates. References are given in parentheseskcatkcat/Kmmmmms–1m–1 s–1HAD1 (YniC)2-Deoxyglucose-6-PMn2+0.61 ± 0.0533 ± 0.85.4 × 104Mannose-6-PZn2+4.7 ± 0.411 ± 0.42.2 × 1032-Deoxyribose-5-PZn2+2.5 ± 0.19.4 ± 0.13.7 × 103Ribose-5-PZn2+2.6 ± 0.30.15–0.63 (61Buchholz A. Hurlebaus J. Wandrey C. Takors R. Biomol. Eng. 2002; 19: 5-15Crossref PubMed Scopus (186) Google Scholar)2.8 ± 0.11.1 × 103Glucose-6-PZn2+3.6 ± 0.50.18–2.0 (55Lowry O.H. Carter J. Ward J.B. Glaser L. J. Biol. Chem. 1971; 246: 6511-6521Abstract Full Text PDF PubMed Google Scholar)25 ± 1.76.7 × 103HAD2 (YfbT)Fructose-1-PMg2+1.0 ± 0.23.7 ± 0.243.5 × 103Ribose-5-PMg2+2.2 ± 0.20.15–0.63 (61Buchholz A. Hurlebaus J. Wandrey C. Takors R. Biomol. Eng. 2002; 19: 5-15Crossref PubMed Scopus (186) Google Scholar)2.7 ± 0.091.2 × 103Glucose-6-PCo2+1.8 ± 0.20.18–2.0 (55Lowry O.H. Carter J. Ward J.B. Glaser L. J. Biol. Chem. 1971; 246: 6511-6521Abstract Full Text PDF PubMed Google Scholar)13 ± 0.67.1 × 103Fructose-6-PMg2+1.3 ± 0.20.54–0.91 (60Bhattacharya M. Fuhrman L. Ingram A. Nickerson K.W. Conway T. Anal. Biochem. 1995; 232: 98-106Crossref PubMed Scopus (75) Google Scholar)3.1 ± 0.22.3 × 103β-Glucose-1-PMn2+7.0 ± 0.37.5 ± 0.21.1 × 103HAD3 (YieH)6-P-gluconateMg2+2.2 ± 0.20.1–15 (56de Silva A.O. Fraenkel D.G. J. Biol. Chem. 1979; 254: 10237-10242Abstract Full Text PDF PubMed Google Scholar)16 ± 0.67.3 × 103PhosphoenolpyruvateMn2+2.9 ± 0.70.15–8.6 (55Lowry O.H. Carter J. Ward J.B. Glaser L. J. Biol. Chem. 1971; 246: 6511-6521Abstract Full Text PDF PubMed Google Scholar, 60Bhattacharya M. Fuhrman L. Ingram A. Nickerson K.W. Conway T. Anal. Biochem. 1995; 232: 98-106Crossref PubMed Scopus (75) Google Scholar, 61Buchholz A. Hurlebaus J. Wandrey C. Takors R. Biomol. Eng. 2002; 19: 5-15Crossref PubMed Scopus (186) Google Scholar)0.75 ± 0.092.6 × 102HAD4 (YihX)α-Glucose-1-PMg2+0.24 ± 0.021.4 ± 0.15.9 × 103Fructose-1-PMn2+1.6 ± 0.10.10 ± 0.0040.61 × 102Acetyl-PMg2+3.6 ± 0.70.04–1.3 (66McCleary W.R. Stock J.B. J. Biol. Chem. 1994; 269: 31567-31572Abstract Full Text PDF PubMed Google Scholar)2.7 ± 0.20.73 × 103Imido-diPMg2+0.028 ± 0.0020.56 ± 0.0220 × 103HAD5 (YjjG)UMPMg2+2.4 ± 0.125 ± 0.51.0 × 104UMPMn2+1.0 ± 0.04.4 ± 0.054.3 × 103dTMPMg2+2.8 ± 0.231 ± 1.11.1 × 104dTMPMn2+0.64 ± 0.0316 ± 0.22.5 × 104AMPMn2+1.8 ± 0.10.31–0.54 (55Lowry O.H. Carter J. Ward J.B. Glaser L. J. Biol. Chem. 1971; 246: 6511-6521Abstract Full Text PDF PubMed Google Scholar, 60Bhattacharya M. Fuhrman L. Ingram A. Nickerson K.W. Conway T. Anal. Biochem. 1995; 232: 98-106Crossref PubMed Scopus (75) Google Scholar)1.3 ± 0.037.1 × 102dGMPMg2+2.0 ± 0.192.0 ± 0.091.0 × 103dGMPMn2+2.8 ± 0.322.1 ± 0.137.5 × 102Fructose-6-PMn2+2.1 ± 0.190.54–0.91 (60Bhattacharya M. Fuhrman L. Ingram A. Nickerson K.W. Conway T. Anal. Biochem. 1995; 232: 98-106Crossref PubMed Scopus (75) Google Scholar)0.18 ± 0.010.88 × 1026-P-gluconateMg2+5.3 ± 0.490.1–15.0 (56de Silva A.O. Fraenkel D.G. J. Biol. Chem. 1979; 254: 10237-10242Abstract Full Text PDF PubMed Google Scholar)0.88 ± 0.031.7 × 102HAD6 (YqaB)Fructose-1-PMg2+1.7 ± 0.219.5 ± 1.12.0 × 1046-P-gluconateMg2+3.9 ± 0.50.1–15.0 (56de Silva A.O. Fraenkel D.G. J. Biol. Chem. 1979; 254: 10237-10242Abstract Full Text PDF PubMed Google Scholar)7.1 ± 0.31.8 × 103HAD7 (YigB)FMNMg2+1.00 ± 0.19.4 ± 0.60.94 × 104HAD8 (YrfG)GMPMg2+1.9 ± 0.25.5 ± 0.22.9 × 103IMPMg2+1.2 ± 0.12.6 ± 0.12.3 × 103HAD9 (SerB)PhosphoserineMg2+0.097 ± 0.00666 ± 168 × 104Acetyl-PMg2+6.2 ± 1.10.04–1.3 (66McCleary W.R. Stock J.B. J. Biol. Chem. 1994; 269: 31567-31572Abstract Full Text PDF PubMed Google Scholar)76 ± 612 × 103Imido-di-PMg2+0.07 ± 0.0070.11 ± 0.011.6 × 103HAD10 (Gph)Acetyl-PMg2+8.9 ± 1.70.04–1.3 (66McCleary W.R. Stock J.B. J. Biol. Chem. 1994; 269: 31567-31572Abstract Full Text PDF PubMed Google Scholar)8.9 ± 0.81.0 × 103Imido-di-PMg2+0.13 ± 0.0221 ± 216 × 104HAD12 (YbiV)Fructose-1-PMg2+1.4 ± 0.3111 ± 6.48.0 × 104Ribose-5-PMg2+2.4 ± 0.20.15–0.63 (61Buchholz A. Hurlebaus J. Wandrey C. Takors R. Biomol. Eng. 2002; 19: 5-15Crossref PubMed Scopus (186) Google Scholar)28 ± 0.91.2 × 104Glucose-6-PMg2+3.1 ± 0.30.18–2.0 (55Lowry O.H. Carter J. Ward J.B. Glaser L. J. Biol. Chem. 1971; 246: 6511-6521Abstract Full Text PDF PubMed Google Scholar)22 ± 0.76.9 × 103Acetyl-PMg2+4.9 ± 0.70.04–1.3 (66McCleary W.R. Stock J.B. J. Biol. Chem. 1994; 269: 31567-31572Abstract Full Text PDF PubMed Google Scholar)101 ± 421 × 103Imido-di-PMg2+0.12 ± 0.027.5 ± 0.662 × 103HAD13 (YidA)Erythrose-4-PMg2+0.019 ± 0.00119 ± 0.410 × 105Mannose-1-PMg2+0.53 ± 0.0321 ± 0.43.8 × 104α-Glucose-1-PMg2+0.21 ± 0.0320 ± 1.39.4 × 104Ribose-5-PMg2+0.45 ± 0.050.15–0.63 (61Buchholz A. Hurlebaus J. Wandrey C. Takors R. Biomol. Eng. 2002; 19: 5-15Crossref PubMed Scopus (186) Google Scholar)9.2 ± 0.52.1 × 104Fructose-1-PMg2+0.39 ± 0.0311 ± 0.62.7 × 104Fructose-6-PMg2+0.44 ± 0.070.54–0.91 (60Bhattacharya M. Fuhrman L. Ingram A. Nickerson K.W. Conway T. Anal. Biochem. 1995; 232: 98-106Crossref PubMed Scopus (75) Google Scholar)5.4 ± 0.51.2 × 104Glucose-6-PMg2+0.81 ± 0.090.18–2.0 (55Lowry O.H. Carter J. Ward J.B. Glaser L. J. Biol. Chem. 1971; 246: 6511-6521Abstract Full Text PDF PubMed Google Scholar)11 ± 0.61.3 × 104Acetyl-PMg2+3.9 ± 0.30.04–1.3 (66McCleary W.R. Stock J.B. J. Biol. Chem. 1994; 269: 31567-31572Abstract Full Text PDF PubMed Google Scholar)24 ± 16.2 × 103Imido-di-PMg2+0.033 ± 0.00214 ± 042 × 104HAD14 (YbhA)PLPMg2+0.37 ± 0.051.0 ± 0.042.8 × 103Fructose-1,6-bis-PMg2+2.4 ± 0.23.29–6.0 (55Lowry O.H. Carter J. Ward J.B. Glaser L. J. Biol. Chem. 1971; 246: 6511-6521Abstract Full Text PDF PubMed Google Scholar, 60Bhattacharya M. Fuhrman L. Ingram A. Nickerson K.W. Conway T. Anal. Biochem. 1995; 232: 98-106Crossref PubMed Scopus (75) Google Scholar, 61Buchholz A. Hurlebaus J. Wandrey C. Takors R. Biomol. Eng. 2002; 19: 5-15Crossref PubMed Scopus (186) Google Scholar)5.3 ± 0.12.2 × 103HAD15 (YbjI)FMNMg2+2.3 ± 0.36.5 ± 0.42.8 × 103Erythrose-4-PMg2+6.8 ± 1.33.3 ± 0.44.9 × 102HAD16 (YigL)PLPMg2+1.5 ± 0.112 ± 0.57.7 × 1032-Deoxyglucose-6-PMg2+7.5 ± 0.713 ± 0.51.8 × 103β-Glucose-6-PMg2+5.9 ± 0.714 ± 0.72.4 × 103HAD17 (OtsB)Trehalose-6-PMg2+0.61 ± 0.070.76 (62Rimmele M. Boos W. J. Bacteriol. 1994; 176: 5654-5664Crossref PubMed Google Scholar)9.1 ± 0.51.5 × 104HAD18 (Cof)PLPMg2+0.68 ± 0.070.58 ± 0.028.5 × 1022-Deoxyglucose-6-PMg2+2.5 ± 0.30.50 ± 0.022.0 × 102HAD20 (YaeD)Fructose-1,6-bis-PMg2+0.42 ± 0.063.3–6.0 (55Lowry O.H. Carter J. Ward J.B. Glaser L. J. Biol. Chem. 1971; 246: 6511-6521Abstract Full Text PDF PubMed Google Scholar, 60Bhattacharya M. Fuhrman L. Ingram A. Nickerson K.W. Conway T. Anal. Biochem. 1995; 232: 98-106Crossref PubMed Scopus (75) Google Scholar, 61Buchholz A. Hurlebaus J. Wandrey C. Takors R. Biomol. Eng. 2002; 19: 5-15Crossref PubMed Scopus (186) Google Scholar)0.19 ± 0.014.5 × 102a The saturating metal concentrations used in the assays were 0.63–5.0 mm Mg2+, 0.1–1.25 mm Mn2+, 0.3–0.75 mm Co2+, or 1 mm Zn2+b Experimentally determined intracellular (in vivo) concentrations of the variable substrates. References are given in parentheses Open table in a new tab RESULTSThe Enzymatic Activities of the E. coli HADs—HADs share relatively little overall sequence similarity (∼15 to 30% identity), but they can be identified by the presence of three short conserved sequence motifs (3Koonin E.V. Tatusov R.L. J. Mol. Biol. 1994; 244: 125-132Crossref PubMed Scopus (266) Google Scholar) (supplemental Fig. 1). 22 soluble HADs encoded by the E. coli genome were purified to homogeneity (supplemental Fig. 2) and screened for phosphatase, dehalogenase, phosphonatase, and β-phosphoglucomutase activities. 16 previously uncharacterized HADs showed detectable activity with pNPP, a general phosphatase substrate (18Kuznetsova E. Proudfoot M. Sanders S.A. Reinking J. Savchenko A. Arrowsmith C.H. Edwards A.M. Yakunin A.F. FEMS Microbiol. Rev. 2005; 29: 263-279Crossref PubMed Google Scholar) (Table 1). All of these HADs required the presence of a divalent metal cation for activity (Mg2+ > Co2+ > Mn2+ > Ni2+) and showed a broad range of activities and affinities to pNPP (Vmax 0.03-4.95 μmol/min mg of protein; Km = 0.92-17.8 mm). Several proteins, YcjU (HAD11), YigB (HAD7), OtsB (HAD17), YedP (HAD19), YaeD (HAD20), and HisB (HAD21), had no activity against pNPP. YcjU (HAD11) has been annotated in sequence data bases as a putative β-phosphoglucomutase, and indeed, we detected this activity using a specific assay with β-glucose-1-P as a substrate (3.81 ± 0.13 μmol/min/mg of protein). Low β-phosphoglucomutase activity (0.02-0.15 μmol/min/mg of protein) was also detected in YihX (HAD4), YqaB (HAD6), YbhA (HAD14), Cof (HAD18), Gph (HAD10), and YaeD (HAD20). Screening of purified E. coli HADs for hydrolytic activity against phosphonoacetate (phosphonatase substrate) or several haloacid dehalogenase substrates (chloroacetate, bromoacetate, chloropropionate, dichloropropionate, bromopropionate, and chlorobenzoate) produced no positive results (data not shown). Thus, most of the soluble E. coli HADs are phosphatases.TABLE 1Phosphatase activity of purified E. coli HADs against the model substrate pNPP and small phosphodonors Phosphatase activity is shown in μmol/min/mg of protein (the mean values are shown; S.D. values for all substrates were 10.95 ± 2.99% of the mean value). Assays were performed in the presence of 2.5 mm Mg2+ and the following concentrations of substrates: 10 mm pNPP, 10 mm acetyl-P, 0.13 mm carbamoyl-P, or 0.13 mm imidodi-P. These substrate concentrations were saturating for most enzymes (except for carbamoyl-P, which showed no satur
Referência(s)